Assessing phagotrophy in the mixotrophic ciliate Paramecium bursaria using GFP-expressing yeast cells.

PubMed

Miura, Takashi; Moriya, Hisao; Iwai, Sosuke

2017-07-03

We used cells of the yeast Saccharomyces cerevisiae expressing green fluorescent protein (GFP) as fluorescently labelled prey to assess the phagocytic activities of the mixotrophic ciliate Paramecium bursaria, which harbours symbiotic Chlorella-like algae. Because of different fluorescence spectra of GFP and algal chlorophyll, ingested GFP-expressing yeast cells can be distinguished from endosymbiotic algal cells and directly counted in individual P. bursaria cells using fluorescence microscopy. By using GFP-expressing yeast cells, we found that P. bursaria altered ingestion activities under different physiological conditions, such as different growth phases or the presence/absence of endosymbionts. Use of GFP-expressing yeast cells allowed us to estimate the digestion rates of live prey of the ciliate. In contrast to the ingestion activities, the digestion rate within food vacuoles was not affected by the presence of endosymbionts, consistent with previous findings that food and perialgal vacuoles are spatially and functionally separated in P. bursaria. Thus, GFP-expressing yeast may provide a valuable tool to assess both ingestion and digestion activities of ciliates that feed on eukaryotic organisms. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Genetic barcoding with fluorescent proteins for multiplexed applications.

PubMed

Smurthwaite, Cameron A; Williams, Wesley; Fetsko, Alexandra; Abbadessa, Darin; Stolp, Zachary D; Reed, Connor W; Dharmawan, Andre; Wolkowicz, Roland

2015-04-14

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded 'indefinitely'. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

STANDARDIZATION OF A FLUORESCENT-BASED QUANTITATIVE ADHESION ASSAY TO STUDY ATTACHMENT OF Taenia solium ONCOSPHERE TO EPITHELIAL CELLS In Vitro

PubMed Central

Chile, Nancy; Evangelista, Julio; Gilman, Robert H.; Arana, Yanina; Palma, Sandra; Sterling, Charles R; Garcia, Hector H.; Gonzalez, Armando; Verastegui, Manuela

2012-01-01

To fully understand the preliminary stages of Taenia solium oncosphere attachment in the gut, adequate tools and assays are necessary to observe and quantify this event that leads to infection. A fluorescent-based quantitative adhesion assay, using biotinylated activated-oncospheres and monolayers of Chinese hamster ovary cells (CHO-K1) or human intestinal monolayer cells (INT-407, HCT-8 or HT-29), was developed to study initial events during the infection of target cells and to rapidly quantify the in vitro adhesion of T. solium oncospheres. Fluorescein streptavidin was used to identify biotinylated activated-oncospheres adhered to cells. This adherence was quantified using an automated fluorescence plate reader, and the results were expressed as fluorescence intensity values. A series of three assays were performed. The first was to identify the optimum number of biotinylated activated-oncospheres to be used in the adhesion assay. The goal of the second assay was to validate this novel method with the established oncosphere-binding system using the immunofluorescent-antibody assay (IFA) method to quantify oncosphere adhesion. A total of 10,000 biotinylated activated-oncospheres were utilized to assess the role of sera and laminin (LM) in oncosphere adherence to a CHO-K1 cell monolayer. The findings that sera and LM increase the adhesion of oncospheres to monolayer cells were similar to results that were previously obtained using the IFA method. The third assay compared the adherence of biotinylated activated-oncospheres to different types of human intestinal monolayer cells. In this case, the fluorescence intensity was greatest when using the INT-407 cell monolayer. We believe this new method of quantification offers the potential for rapid, large-scale screening to study and elucidate specific molecules and mechanisms involved in oncosphere-host cell attachment. PMID:22178422

Tracking the engraftment and regenerative capabilities of transplanted lung stem cells using fluorescent nanodiamonds

NASA Astrophysics Data System (ADS)

Wu, Tsai-Jung; Tzeng, Yan-Kai; Chang, Wei-Wei; Cheng, Chi-An; Kuo, Yung; Chien, Chin-Hsiang; Chang, Huan-Cheng; Yu, John

2013-09-01

Lung stem/progenitor cells are potentially useful for regenerative therapy, for example in repairing damaged or lost lung tissue in patients. Several optical imaging methods and probes have been used to track how stem cells incorporate and regenerate themselves in vivo over time. However, these approaches are limited by photobleaching, toxicity and interference from background tissue autofluorescence. Here we show that fluorescent nanodiamonds, in combination with fluorescence-activated cell sorting, fluorescence lifetime imaging microscopy and immunostaining, can identify transplanted CD45-CD54+CD157+ lung stem/progenitor cells in vivo, and track their engraftment and regenerative capabilities with single-cell resolution. Fluorescent nanodiamond labelling did not eliminate the cells' properties of self-renewal and differentiation into type I and type II pneumocytes. Time-gated fluorescence imaging of tissue sections of naphthalene-injured mice indicates that the fluorescent nanodiamond-labelled lung stem/progenitor cells preferentially reside at terminal bronchioles of the lungs for 7 days after intravenous transplantation.

Dual fluorescent molecular substrates selectively report the activation, sustainability and reversibility of cellular PKB/Akt activity.

PubMed

Shen, Duanwen; Bai, Mingfeng; Tang, Rui; Xu, Baogang; Ju, Xiaoming; Pestell, Richard G; Achilefu, Samuel

2013-01-01

Using a newly developed near-infrared (NIR) dye that fluoresces at two different wavelengths (dichromic fluorescence, DCF), we discovered a new fluorescent substrate for Akt, also known as protein kinase B, and a method to quantitatively report this enzyme's activity in real time. Upon insulin activation of cellular Akt, the enzyme multi-phosphorylated a single serine residue of a diserine DCF substrate in a time-dependent manner, culminating in monophospho- to triphospho-serine products. The NIR DCF probe was highly selective for the Akt1 isoform, which was demonstrated using Akt1 knockout cells derived from MMTV-ErbB2 transgenic mice. The DCF mechanism provides unparalleled potential to assess the stimulation, sustainability, and reversibility of Akt activation longitudinally. Importantly, NIR fluorescence provides a pathway to translate findings from cells to living organisms, a condition that could eventually facilitate the use of these probes in humans.

Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

PubMed Central

Kuzma-Kuzniarska, Maria; Yapp, Clarence; Pearson-Jones, Thomas W.; Jones, Andrew K.; Hulley, Philippa A.

2014-01-01

Abstract. Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 β-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures. PMID:24390370

Real-time single cell analysis of molecular mechanism of apoptosis and proliferation using FRET technique

NASA Astrophysics Data System (ADS)

Chen, Tongsheng; Xing, Da; Gao, Xuejuan; Wang, Fang

2006-09-01

Bcl-2 family proteins (such as Bid and Bak/Bax) and 14-3-3 proteins play a key role in the mitochondria-mediated cell apoptosis induced by cell death factors such as TNF-α and lower power laser irradiation (LPLI). In this report, fluorescence resonance energy transfer (FRET) has been used to study the molecular mechanism of apoptosis in living cells on a fluorescence scanning confocal microscope. Based on the genetic code technique and the green fluorescent proteins (GFPs), single-cell dynamic analysis of caspase3 activation, caspase8 activation, and PKCs activation are performed during apoptosis induced by laser irradiation in real-time. To investigate the cellular effect and mechanism of laser irradiation, human lung adenocarcinoma cells (ASTC-a-1) transfected with plasmid SCAT3 (pSCAT3)/ CKAR FRET reporter, were irradiated and monitored noninvasively with both FRET imaging. Our results show that high fluence lower power laser irradiation (HFLPLI) can induce an increase of caspase3 activation and a decrease of PKCs activation, and that LPLI induces the ASTC-a-1 cell proliferation by specifically activating PKCs.

Imaging of protein kinase C activation by FRET during proliferation induced by low-energy laser irradiation in living cells

NASA Astrophysics Data System (ADS)

Gao, Xuejuan; Chen, Tongsheng; Xing, Da; Wang, Fang

2005-01-01

Protein kinase Cs (PKCs) play an important role in cellular proliferation, and low-energy laser irradiation (LELI) can enhance cellular proliferation. The present work contributes to the understanding of the mechanisms of action by studying effects of LELI at the dose of 0.8 J/cm2 on PKCs activities in the single lung adenocarcinoma cell (ASTC-a-1) and in real time by fluorescence resonance energy transfer (FRET) technique. C-kinase activity reporter (CKAR), consisting of a cyan fluorescent protein (CFP), the FHA2 phosphothreonine-binding domain, a PKC substrate sequence, and a yellow fluorescent protein (YFP), was utilized. The living cell imaging showed a decrease in FRET in the cytosol and nucleus after the cells were treated with LELI. These results suggest that PKCs could be activated by LELI throughout the cell, and the proliferation of ASTC-a-1 cells could be modulated by the activated PKCs.

In vivo near-infrared fluorescence imaging of FAP-expressing tumors with activatable FAP-targeted, single-chain Fv-immunoliposomes.

PubMed

Rüger, Ronny; Tansi, Felista L; Rabenhold, Markus; Steiniger, Frank; Kontermann, Roland E; Fahr, Alfred; Hilger, Ingrid

2014-07-28

Molecular and cellular changes that precede the invasive growth of solid tumors include the release of proteolytic enzymes and peptides in the tumor stroma, the recruitment of phagocytic and lymphoid infiltrates and alteration of the extracellular matrix. The reactive tumor stroma consists of a large number of myofibroblasts, characterized by high expression of fibroblast activation protein alpha (FAP). FAP, a type-II transmembrane sialoglycoprotein is an attractive target in diagnosis and therapy of several pathologic disorders especially cancer. In the underlying work, a fluorescence-activatable liposome (fluorescence-quenched during circulation and fluorescence activation upon cellular uptake), bearing specific single-chain Fv fragments directed against FAP (scFv'FAP) was developed, and its potential for use in fluorescence diagnostic imaging of FAP-expressing tumor cells was evaluated by whole body fluorescence imaging. The liposomes termed anti-FAP-IL were prepared via post-insertion of ligand-phospholipid-conjugates into preformed DY-676-COOH-containing liposomes. The anti-FAP-IL revealed a homogeneous size distribution and showed specific interaction and binding with FAP-expressing cells in vitro. The high level of fluorescence quenching of the near-infrared fluorescent dye sequestered in the aqueous interior of the liposomes enables fluorescence imaging exclusively upon uptake and degradation by cells, which results in fluorescence activation. Only FAP-expressing cells were able to take up and activate fluorescence of anti-FAP-IL in vitro. Furthermore, anti-FAP-IL accumulated selectively in FAP-expressing xenograft models in vivo, as demonstrated by blocking experiments using free scFv'FAP. The local tumor fluorescence intensities were in agreement with the intrinsic degree of FAP-expression in different xenograft models. Thus, anti-FAP-IL can serve as a suitable in vivo diagnostic tool for pathological disorders accompanied by high FAP-expression. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparison between immunofluorescence and immunomagnetic techniques of cytometry

NASA Astrophysics Data System (ADS)

Tchikov, V.; Schütze, S.; Krönke, M.

1999-04-01

Magnetophoresis and fluorescence activated cell sorting were used for evaluation of immunochemical properties of magnetic particles and fluorescent probes. The HLA-Bw6 antigen on surfaces of REH cells was detected with a primary monoclonal antibody and a secondary antibody coupled with fluorescent molecules or magnetic particles. Magnetophoresis can find applications in biology and medicine for measuring percentages of cell subpopulations.

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

PubMed Central

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M.; Specht, Christian G.; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-01

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling. PMID:26711992

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

PubMed

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M; Specht, Christian G; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-19

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

Non-fused phospholes as fluorescent probes for imaging of lipid droplets in living cells

NASA Astrophysics Data System (ADS)

Öberg, Elisabet; Appelqvist, Hanna; Nilsson, K. Peter R.

2017-04-01

Molecular tools for fluorescent imaging of specific compartments in cells are essential for understanding the function and activity of cells. Here, we report the synthesis of a series of pyridyl- and thienyl-substituted phospholes and the evaluation of these dyes for fluorescent imaging of cells. The thienyl-substituted phospholes proved to be successful for staining of cultured normal and malignant cells due to their fluorescent properties and low toxicity. Co-staining experiments demonstrated that these probes target lipid droplets, which are, lipid-storage organelles found in the cytosol of nearly all cell types. Our findings confirm that thienyl-substituted phospholes can be utilized as fluorescent tools for vital staining of cells, and we foresee that these fluorescent dyes might be used in studies to unravel the roles that lipid droplets play in cellular physiology and their role in diseases.

Naphthol AS-BI (7-bromo-3-hydroxy-2-naphtho-o-anisidine) phosphatase and naphthol AS-BI. beta. -D-glucuronidase in Chinese hamster ovary cells: biochemical and flow cytometric studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dolbeare, F.A.; Phares, W.

1979-01-01

Conditions for the biochemical and flow cytometric assay of 7-bromo-3-hydroxy-2-naphtho-o-anisidine phosphatase and ..beta..-D-glucuronidase activities in Chinese hamster ovary cells were studied. In the biochemical assays, the pH optimum for the phosphatase activity was pH 4.6 with a Km of 10/sup -5/ M; the pH optimum for ..beta..-D-glucuronidase activity was pH 5.0 with a Km of 2 x 10/sup -5/ M. For intact cells the derived constants were 3 to 10 times higher. The rate of hydrolysis of both substrates was also examined by flow cytometry. Cellular fluorescence increased linearly for only about 15 min. Diffusion of the fluorescent product probablymore » caused nonlinearity of the fluorescence increase and was demonstrated by mixing cells incubated with substrate with those that had not been incubated. After 15 min, cells that had not been exposed previously to product or substrate contained the fluorescent product. Cells fractionated into size classes by centrifugal elutriation also were analyzed by flow cytometry for ..beta..-D-glucuronidase activity. The activity increased linearly with the increase in cell size corresponding to the progression from G/sub 1/ through S and into G/sub 2/-M phases of the cell cycle.« less

A multiplexed fluorescent assay for independent second-messenger systems: decoding GPCR activation in living cells.

PubMed

Tewson, Paul H; Quinn, Anne Marie; Hughes, Thomas E

2013-08-01

There is a growing need in drug discovery and basic research to measure multiple second-messenger components of cell signaling pathways in real time and in relevant tissues and cell types. Many G-protein-coupled receptors activate the heterotrimeric protein, Gq, which in turn activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce two second messengers: diacylglycerol (DAG), which remains in the plasma membrane, and inositol triphosphate (IP3), which diffuses through the cytosol to release stores of intracellular calcium ions (Ca(2+)). Our goal was to create a series of multiplex sensors that would make it possible to simultaneously measure two different components of the Gq pathway in living cells. Here we describe new fluorescent sensors for DAG and PIP2 that produce robust changes in green or red fluorescence and can be combined with one another, or with existing Ca(2+) sensors, in a live-cell assay. These assays can detect multiple components of Gq signaling, simultaneously in real time, on standard fluorescent plate readers or live-cell imaging systems.

Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy.

PubMed

Indovina, Paola; Collini, Maddalena; Chirico, Giuseppe; Santini, Maria Teresa

2007-02-20

Hypoxia through HRE (hypoxia-responsive element) activity in MG-63 human osteosarcoma cells grown in monolayer and as very small, three-dimensional tumor spheroids was investigated using molecular imaging techniques. MG-63 cells were stably transfected with a vector constructed with multiple copies of the HRE sequence of the human vascular endothelial growth factor (VEGF) gene and with the enhanced green fluorescent protein (EGFP) coding sequence. During hypoxia when HIF-1alpha (hypoxia-inducible factor-1alpha) is stabilized, the binding of HIF-1 to the HRE sequences of the vector allows the transcription of EGFP and the appearance of fluorescence. Transfected monolayer cells were characterized by flow cytometric analysis in response to various hypoxic conditions and HIF-1alpha expression in these cells was assessed by Western blotting. Two-photon excitation (TPE) microscopy was then used to examine both MG-63-transfected monolayer cells and spheroids at 2 and 5 days of growth in normoxic conditions. Monolayer cells reveal almost no fluorescence, whereas even very small spheroids (<100 microm) after 2 days of growth contain regions of high fluorescence. For the first time in the literature, at least to our knowledge, it is demonstrated, using highly sensitive and non-perturbing molecular imaging techniques, that three-dimensional cell organization leads to almost immediate HRE activation. This activation of the HRE sequences, which control a wide variety of genes, suggests that monolayer cells and spheroids of the MG-63 cell line have different genes activated and thus diverse functional activities.

A Thiazole Coumarin (TC) Turn-On Fluorescence Probe for AT-Base Pair Detection and Multipurpose Applications in Different Biological Systems

NASA Astrophysics Data System (ADS)

Narayanaswamy, Nagarjun; Kumar, Manoj; Das, Sadhan; Sharma, Rahul; Samanta, Pralok K.; Pati, Swapan K.; Dhar, Suman K.; Kundu, Tapas K.; Govindaraju, T.

2014-09-01

Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based thiazole coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-activated cell sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology.

A Thiazole Coumarin (TC) Turn-On Fluorescence Probe for AT-Base Pair Detection and Multipurpose Applications in Different Biological Systems

PubMed Central

Narayanaswamy, Nagarjun; Kumar, Manoj; Das, Sadhan; Sharma, Rahul; Samanta, Pralok K.; Pati, Swapan K.; Dhar, Suman K.; Kundu, Tapas K.; Govindaraju, T.

2014-01-01

Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based thiazole coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-activated cell sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology. PMID:25252596

Single-cell analysis of dihydroartemisinin-induced apoptosis through reactive oxygen species-mediated caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells using fluorescence imaging techniques

NASA Astrophysics Data System (ADS)

Lu, Ying-Ying; Chen, Tong-Sheng; Wang, Xiao-Ping; Li, Li

2010-07-01

Dihydroartemisinin (DHA), a front-line antimalarial herbal compound, has been shown to possess promising anticancer activity with low toxicity. We have previously reported that DHA induced caspase-3-dependent apoptosis in human lung adenocarcinoma cells. However, the cellular target and molecular mechanism of DHA-induced apoptosis is still poorly defined. We use confocal fluorescence microscopy imaging, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching techniques to explore the roles of DHA-elicited reactive oxygen species (ROS) in the DHA-induced Bcl-2 family proteins activation, mitochondrial dysfunction, caspase cascade, and cell death. Cell Counting Kit-8 assay and flow cytometry analysis showed that DHA induced ROS-mediated apoptosis. Confocal imaging analysis in a single living cell and Western blot assay showed that DHA triggered ROS-dependent Bax translocation, mitochondrial membrane depolarization, alteration of mitochondrial morphology, cytochrome c release, caspase-9, caspase-8, and caspase-3 activation, indicating the coexistence of ROS-mediated mitochondrial and death receptor pathway. Collectively, our findings demonstrate for the first time that DHA induces cell apoptosis by triggering ROS-mediated caspase-8/Bid activation and the mitochondrial pathway, which provides some novel insights into the application of DHA as a potential anticancer drug and a new therapeutic strategy by targeting ROS signaling in lung adenocarcinoma therapy in the future.

Tumor cell differentiation by label-free microscopy

NASA Astrophysics Data System (ADS)

Schneckenburger, Herbert; Weber, Petra; Wagner, Michael

2013-05-01

Autofluorescence and Raman measurements of U251-MG glioblastoma cells prior and subsequent to activation of tumor suppressor genes are compared. While phase contrast images and fluorescence intensity patterns of the tumor (control) cells and the less malignant cells are similar, differences can be deduced from fluorescence spectra and nanosecond decay times. In particular, upon excitation around 375nm, the fluorescence ratio of the protein bound and the free coenzyme NADH depends on the state of malignancy and reflects different cytoplasmic (including lysosomal) and mitochondrial contributions. Slight differences are also observed in the Raman spectra of these cell lines, mainly originating from small granules (lysosomes) surrounding the cell nucleus. While larger numbers of fluorescence and Raman spectra are evaluated by multivariate statistical methods, additional information is obtained from spectral images and fluorescence lifetime images (FLIM).

In situ fluorescence activation of DNA-silver nanoclusters as a label-free and general strategy for cell nucleus imaging.

PubMed

Li, Duo; Qiao, Zhenzhen; Yu, Yanru; Tang, Jinlu; He, Xiaoxiao; Shi, Hui; Ye, Xiaosheng; Lei, Yanli; Wang, Kemin

2018-01-25

A facile, general and turn-on nucleus imaging strategy was first developed based on in situ fluorescence activation of C-rich dark silver nanoclusters by G-rich telomeres. After a simple incubation without washing, nanoclusters could selectively stain the nucleus with intense red luminescence, which was confirmed using fixed/living cells and several cell lines.

Traffic Lights in Trichodesmium. Regulation of Photosynthesis for Nitrogen Fixation Studied by Chlorophyll Fluorescence Kinetic Microscopy1

PubMed Central

Küpper, Hendrik; Ferimazova, Naila; Šetlík, Ivan; Berman-Frank, Ilana

2004-01-01

We investigated interactions between photosynthesis and nitrogen fixation in the non-heterocystous marine cyanobacterium Trichodesmium IMS101 at the single-cell level by two-dimensional (imaging) microscopic measurements of chlorophyll fluorescence kinetics. Nitrogen fixation was closely associated with the appearance of cells with high basic fluorescence yield (F0), termed bright cells. In cultures aerated with normal air, both nitrogen fixation and bright cells appeared in the middle of the light phase. In cultures aerated with 5% oxygen, both processes occurred at a low level throughout most of the day. Under 50% oxygen, nitrogen fixation commenced at the beginning of the light phase but declined soon afterwards. Rapid reversible switches between fluorescence levels were observed, which indicated that the elevated F0 of the bright cells originates from reversible uncoupling of the photosystem II (PSII) antenna from the PSII reaction center. Two physiologically distinct types of bright cells were observed. Type I had about double F0 compared to the normal F0 in the dark phase and a PSII activity, measured as variable fluorescence (Fv = Fm − F0), similar to normal non-diazotrophic cells. Correlation of type I cells with nitrogen fixation, oxygen concentration, and light suggests that this physiological state is connected to an up-regulation of the Mehler reaction, resulting in oxygen consumption despite functional PSII. Type II cells had more than three times the normal F0 and hardly any PSII activity measurable by variable fluorescence. They did not occur under low-oxygen concentrations, but appeared under high-oxygen levels outside the diazotrophic period, suggesting that this state represents a reaction to oxidative stress not necessarily connected to nitrogen fixation. In addition to the two high-fluorescence states, cells were observed to reversibly enter a low-fluorescence state. This occurred mainly after a cell went through its bright phase and may represent a fluorescence-quenching recovery phase. PMID:15299119

Fluorescein diacetate (FDA) and its analogue as substrates for Pi-class glutathione S-transferase (GSTP1) and their biological application.

PubMed

Fujikawa, Yuuta; Nampo, Taiki; Mori, Masaya; Kikkawa, Manami; Inoue, Hideshi

2018-03-01

Pi class glutathione S-transferase (GSTP1) is highly expressed in various cancerous cells and pre-neoplastic legions, where it is involved in apoptotic resistance or metabolism of several anti-tumour chemotherapeutics. Therefore, GSTP1 is a marker of malignant and pre-malignant cells and is a promising target for visualization and drug development. Here we demonstrate that fluorescein diacetate (FDA), a fluorescent probe used for vital staining, is a fluorescently activated by esterolytic activity of human GSTP1 (hGSTP1) selectively among various cytosolic GSTs. Fluorescence activation of FDA susceptible to GST inhibitors was observed in MCF7 cells exogenously overexpressing hGSTP1, but not in cells overexpressing hGSTA1 or hGSTM1. Inhibitor-sensitive fluorescence activation was also observed in several cancer cell lines endogenously expressing GSTP1, suggesting that GSTP1 is involved in FDA esterolysis in these cells. Among the FDA derivatives examined, FOMe-Ac, the acetyl ester of fluorescein O-methyl ether, was found to be a potential reporter for GSH-dependent GSTP1 activity as well as for carboxylesterase activity. Since GSTP1 is highly expressed in various types of cancer cells compared to their normal counterparts, improving the fluorogenic substrates to be more selective to the esterolysis activity of GSTP1 rather than carboxylesterases should lead to development of tools for detecting GSTP1-overexpressing cancer cells and investigating the biological functions of GSTP1. Copyright © 2017 Elsevier B.V. All rights reserved.

Fluorescence and confocal imaging of mammalian cells using conjugated oligoelectrolytes with phenylenevinylene core

DOE Office of Scientific and Technical Information (OSTI.GOV)

Milczarek, Justyna; Pawlowska, Roza; Zurawinski, Remigiusz

Over the last few years, considerable efforts are taken, in order to find a molecular fluorescent probe fulfilling their applicability requirements. Due to a good optical properties and affinity to biological structures conjugated oligoelectrolytes (COEs) can be considered as a promising dyes for application in fluorescence-based bioimaging. In this work, we synthetized COEs with phenylenevinylene core (PV-COEs) and applied as fluorescent membranous-specific probes. Cytotoxicity effects of each COE were probed on cancerous and non-cancerous cell types and little to no toxicity effects were observed at the high range of concentrations. The intensity of cell fluorescence following the COE staining wasmore » determined by the photoluminescence analysis and fluorescence activated cell sorting method (FACS). Intercalation of tested COEs into mammalian cell membranes was revealed by fluorescent and confocal microscopy colocalization with commercial dyes specific for cellular structures including mitochondria, Golgi apparatus and endoplasmic reticulum. The phenylenevinylene conjugated oligoelectrolytes have been found to be suitable for fluorescent bioimaging of mammalian cells and membrane-rich organelles. Due to their water solubility coupled with spontaneous intercalation into cells, favorable photophysical features, ease of cell staining, low cytotoxicity and selectivity for membranous structures, PV-COEs can be applied as markers for fluorescence imaging of a variety of cell types.« less

Increased metabolic activity detected by FLIM in human breast cancer cells with desmoplastic reaction: a pilot study

NASA Astrophysics Data System (ADS)

Natal, Rodrigo de Andrade; Pelegati, Vitor B.; Bondarik, Caroline; Mendonça, Guilherme R.; Derchain, Sophie F.; Lima, Carmen P.; Cesar, Carlos L.; Sarian, Luís. O.; Vassallo, José

2015-07-01

Introduction: In breast cancer (BC), desmoplastic reaction, assembled primarily by fibroblasts, is associated with unfavorable prognosis, but the reason of this fact remains still unclear. In this context, nonlinear optics microscopy, including Fluorescence Lifetime Imaging Microscopy (FLIM), has provided advancement in cellular metabolism research. In this paper, our purpose is to differentiate BC cells metabolism with or without contact to desmoplastic reaction. Formalin fixed, paraffin embedded samples were used at different points of hematoxylin stained sections. Methodology: Sections from 14 patients with invasive ductal breast carcinoma were analyzed with FLIM methodology to NAD(P)H and FAD fluorescence lifetime on a Confocal Upright LSM780 NLO device (Carl Zeiss AG, Germany). Quantification of the fluorescence lifetime and fluorescence intensity was evaluated by SPC Image software (Becker &Hickl) and ImageJ (NIH), respectively. Optical redox ratio was calculated by dividing the FAD fluorescence intensity by NAD(P)H fluorescence intensity. Data value for FLIM measurements and fluorescence intensities were calculated using Wilcoxon test; p< 0.05 was considered significant. Results: BC cells in contact with desmoplastic reaction presented a significantly lower NAD(P)H and FAD fluorescence lifetime. Furthermore, optical redox ratio was also lower in these tumor cells. Conclusion: Our results suggest that contact of BC cells with desmoplastic reaction increase their metabolic activity, which might explain the adverse prognosis of cases associated with higher peritumoral desmoplastic reaction.

Isolation of hair follicle bulge stem cells from YFP-expressing reporter mice.

PubMed

Nakrieko, Kerry-Ann; Irvine, Timothy S; Dagnino, Lina

2013-01-01

In this article we provide a method to isolate hair follicle stem cells that have undergone targeted gene inactivation. The mice from which these cells are isolated are bred into a Rosa26-yellow fluorescent protein (YFP) reporter background, which results in YFP expression in the targeted stem cell population. These cells are isolated and purified by fluorescence-activated cell sorting, using epidermal stem cell-specific markers in conjunction with YFP fluorescence. The purified cells can be used for gene expression studies, clonogenic experiments, and biological assays, such as viability and capacity for directional migration.

Segmentation and classification of cell cycle phases in fluorescence imaging.

PubMed

Ersoy, Ilker; Bunyak, Filiz; Chagin, Vadim; Cardoso, M Christina; Palaniappan, Kannappan

2009-01-01

Current chemical biology methods for studying spatiotemporal correlation between biochemical networks and cell cycle phase progression in live-cells typically use fluorescence-based imaging of fusion proteins. Stable cell lines expressing fluorescently tagged protein GFP-PCNA produce rich, dynamically varying sub-cellular foci patterns characterizing the cell cycle phases, including the progress during the S-phase. Variable fluorescence patterns, drastic changes in SNR, shape and position changes and abundance of touching cells require sophisticated algorithms for reliable automatic segmentation and cell cycle classification. We extend the recently proposed graph partitioning active contours (GPAC) for fluorescence-based nucleus segmentation using regional density functions and dramatically improve its efficiency, making it scalable for high content microscopy imaging. We utilize surface shape properties of GFP-PCNA intensity field to obtain descriptors of foci patterns and perform automated cell cycle phase classification, and give quantitative performance by comparing our results to manually labeled data.

Antimicrobial Penetration and Efficacy in an In Vitro Oral Biofilm Model ▿ †

PubMed Central

Corbin, Audrey; Pitts, Betsey; Parker, Albert; Stewart, Philip S.

2011-01-01

The penetration and overall efficacy of six mouthrinse actives was evaluated by using an in vitro flow cell oral biofilm model. The technique involved preloading biofilm cells with a green fluorescent dye that leaked out as the cells were permeabilized by a treatment. The loss of green color, and of biomass, was observed by time-lapse microscopy during 60 min of treatment under continuous flow conditions. The six actives analyzed were ethanol, sodium lauryl sulfate, triclosan, chlorhexidine digluconate (CHX), cetylpyridinium chloride, and nisin. Each of these agents effected loss of green fluorescence throughout biofilm cell clusters, with faster action at the edge of a cell cluster and slower action in the cluster center. The time to reach half of the initial fluorescent intensity at the center of a cell cluster, which can be viewed as a combined penetration and biological action time, ranged from 0.6 to 19 min for the various agents. These times are much longer than the predicted penetration time based on diffusion alone, suggesting that anti-biofilm action was controlled more by the biological action time than by the penetration time of the active. None of the agents tested caused any removal of the biofilm. The extent of fluorescence loss after 1 h of exposure to an active ranged from 87 to 99.5%, with CHX being the most effective. The extent of fluorescence loss in vitro, but not penetration and action time, correlated well with the relative efficacy data from published clinical trials. PMID:21537022

The induction of apoptosis by methotrexate in activated lymphocytes as indicated by fluorescence hyperpolarization: a possible model for predicting methotrexate therapy for rheumatoid arthritis patients.

PubMed

Herman, Shoshy; Zurgil, Naomi; Langevitz, Pnina; Ehrenfeld, Michael; Deutsch, Mordechai

2003-04-01

The objectives of this study were to test the in vitro response of healthy non-activated, activated, and rheumatoid arthritis (RA) lymphocytes to methotrexate (MTX), and design an in vitro model for predicting the efficiency of MTX treatment for RA patients. Considering the RA profile of clonal-expanded CD4(+) T cells, phytohemagglutinin-activated mononuclear cells taken from healthy donors were incubated with different concentrations of MTX. The MTX-immunosuppressive effect was tested by fluorescence intensity measurements, including PI assay and annexin V assay. For simple detection, we used the Individual Cell Scanner (IC-S), which enables the measurement of early events in individual cells. Healthy mononuclear cells (MNC), and MNC derived from RA patients, were tested by the IC-S while utilizing fluorescence polarization (FP) measurements of fluorescein diacetate (FDA) as an established marker of activation or suppression. In healthy activated MNC, we found that MTX, through its early incubation period, interferes with the activation signal obtained by PHA and exerts an apoptotic signal, which is noted by increases in the FP. Comparing our model to six long-standing RA patients and five newly-diagnosed patients revealed significant differences in the FP measurements, including fluorescence depolarization as an early established measurement of lymphocyte activation, and hyperpolarization as a measurement of an early immunosuppressive effect. We conclude that MTX, an effective therapy for RA patients, could easily be tested by fluorescence polarization measurements of FDA before (or during) clinical use in order to predict its efficiency on a specific RA patient. Moreover, the FP measurements can be used for the diagnosis, and making timing and dosage decisions.

Specific binding of tubeimoside-2 with proteins in hepatocarcinoma HepG2 cells: investigation by molecular spectroscopy

NASA Astrophysics Data System (ADS)

Yang, Sun; Shi-Sheng, Sun; Ying-Yong, Zhao; Jun, Fan

2012-07-01

In this study, we compared different binding interactions of TBMS2 with proteins both in hepatocarcinoma HepG2 cells and in normal embryo hepatic L02 cells by using fluorescence, absorption, and CD spectroscopy. The fluorescence data revealed that the fluorescence intensity of proteins in the HepG2 and L02 cells decreased in the presence of TBMS2 by 30.79% and 12.01%, respectively. Binding constants and thermodynamic parameters were obtained for systems of TBMS2 with the two kinds of cell proteins. The results indicated that HepG2 cell proteins had a higher TBMS2 binding activity than those in the L02 cells. Analysis of the TBMS2 cytotoxic activities showed that TBMS2 could selectively induce apoptosis of HepG2 cells by binding to them, while its apoptotic effect on L02 cells was relatively weaker.

Separation of periportal and perivenous rat hepatocytes by fluorescence-activated cell sorting: confirmation with colloidal gold as an exogenous marker.

PubMed

Braakman, I; Keij, J; Hardonk, M J; Meijer, D K; Groothuis, G M

1991-01-01

Periportal and perivenous hepatocytes are known to display various functional differences. In this study we present a new method to separate periportal and perivenous cells: after selectively loading zone 1 or zone 3 with the fluorescent label acridine orange in an antegrade or retrograde perfusion, respectively, we separated the isolated hepatocytes on a fluorescence-activated cell sorter. The common way to check on proper separation is to estimate activities of enzymes known to exhibit a heterogeneous acinar distribution. Using enzyme histochemistry, however, we found that already on short collagenase perfusion, some enzymes displayed a more shallow gradient than in vivo, making enzyme activities less suitable as zonal markers. We therefore used colloidal gold granules (17 nm) injected intravenously (2.5 mg) into the rat 2 to 3 hr before cell isolation. The gold is taken up predominantly by perivenous hepatocytes, probably because of the efficient removal of gold granules in zone 1 by competing Kupffer cells. We compared acridine orange fluorescence, presence of gold particles and activities of six marker enzymes, three biochemically and three histochemically determined. Acridine orange and gold both pointed to a high enrichment of the fractions, whereas most enzyme activities were more randomly distributed among the cells as a result of the isolation procedure. Our separation procedure yielded fractions highly enriched in either viable periportal or perivenous cells, both from one liver. The use of colloidal gold as a marker to monitor separation is a valuable alternative to the more risky estimation of enzyme activities.

Quantitative FRET imaging of leptin receptor oligomerization kinetics in single cells.

PubMed

Biener, Eva; Charlier, Madia; Ramanujan, V Krishnan; Daniel, Nathalie; Eisenberg, Avital; Bjørbaek, Christian; Herman, Brian; Gertler, Arieh; Djiane, Jean

2005-12-01

Leptin, an adipocyte-secreted hormone, signals through activation of its membrane-embedded receptor (LEPR). To study the leptin-induced events occurring in short (LEPRa) and long (LEPRb) LEPRs in the cell membrane, by FRET (fluorescence resonance energy transfer) methodology, the respective receptors, tagged at their C-terminal with CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein), were prepared. The constructs encoding mLEPRa (mouse LEPRa)-YFP and mLEPRa-CFP, mLEPRb-YFP and mLEPRb-CFP were tested for biological activity in transiently transfected CHO cells (Chinese-hamster ovary cells) and HEK-293T cells (human embryonic kidney 293 T cells) for activation of STAT3 (signal transduction and activators of transcription 3)-mediated LUC (luciferase) activity and binding of radiolabelled leptin. All four constructs were biologically active and were as potent as their untagged counterparts. The localization pattern of the fused protein appeared to be confined almost entirely to the cell membrane. The leptin-dependent interaction between various types of receptors in fixed cells were studied by measuring FRET, using fluorescence lifetime imaging microscopy and acceptor photobleaching methods. Both methods yielded similar results, indicating that (1) leptin receptors expressed in the cell membrane exist mostly as preformed LEPRa/LEPRa or LEPRb/LEPRb homo-oligomers but not as LEPRb/LEPRa hetero-oligomers; (2) the appearance of transient leptin-induced FRET in cells transfected with LEPRb/LEPRb reflects both a conformational change that leads to closer interaction in the cytosolic part and a higher FRET signal, as well as de novo homo-oligomerization; (3) in LEPRa/LEPRa, exposure to leptin does not lead to any increase in FRET signalling as the proximity of CFP and YFP fluorophores in space already gives maximal FRET efficiency of the preoligomerized receptors.

Design of Organic Solar Cells as a Function of Radiative Quantum Efficiency

NASA Astrophysics Data System (ADS)

Godefroid, Blaise; Kozyreff, Gregory

2017-09-01

We study the radiative decay, or fluorescence, of excitons in organic solar cells as a function of its geometrical parameters. Contrary to their nonradiative counterpart, fluorescence losses strongly depend on the environment. By properly tuning the thicknesses of the buffer layers between the active regions of the cell and the electrodes, the exciton lifetime and, hence, the exciton diffusion length can be increased. The importance of this phenomenon depends on the radiative quantum efficiency, which is the fraction of the exciton decay that is intrinsically due to fluorescence. Besides this effect, interferences within the cell control the efficiency of sunlight injection into the active layers. The optimal cell design must rely on a consideration of these two aspects. By properly managing fluorescence losses, one can significantly improve the cell performance. To demonstrate this fact, we use realistic material parameters inspired from literature data and obtain an increase of power-conversion efficiency from 11.3% to 12.7%. Conversely, not to take into account the strong dependence of fluorescence on the environment may lead to a suboptimal cell design and a degradation of cell performance. The presence of radiative losses, however small, significantly changes the optimal set of thicknesses. We illustrate the latter situation with experimental material data.

Single-cell analysis and sorting using droplet-based microfluidics.

PubMed

Mazutis, Linas; Gilbert, John; Ung, W Lloyd; Weitz, David A; Griffiths, Andrew D; Heyman, John A

2013-05-01

We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ∼200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ∼1 million cells, the microfluidic operations require 2-6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5-7 d.

Single-cell analysis and sorting using droplet-based microfluidics

PubMed Central

Mazutis, Linas; Gilbert, John; Ung, W Lloyd; Weitz, David A; Griffiths, Andrew D; Heyman, John A

2014-01-01

We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. as an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. the beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ~200 Hz as well as cell enrichment. the microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ~1 million cells, the microfluidic operations require 2–6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5–7 d. PMID:23558786

MEK inhibitor U0126 interferes with immunofluorescence analysis of apoptotic cell death.

PubMed

Blank, Norbert; Burger, Renate; Duerr, Birgit; Bakker, Frank; Wohlfarth, Anika; Dumitriu, Ingrid; Kalden, Joachim R; Herrmann, Martin

2002-08-01

Binding of extracellular growth factors to cell surface receptors often results in activation of the mitogen-activated protein kinase (MAPK). MAPK is regulated by MAPK kinase, also called MEK. Deprivation of growth factors during cell culture or intracellular MEK inhibition leads to inhibition of proliferation and apoptotic cell death. Besides other techniques, apoptotic cells can be identified by phosphatidylserine (PS) exposure and exclusion of membrane-impermeant propidium iodide (PI). We investigated the limitations of detection of apoptotic cell death and cytofluorometry in cells cultured in the presence of the MEK inhibitor U0126. Apoptotic cell death was induced in the plasmacytoma cell line INA-6, in peripheral blood mononuclear cells (PBMC), and in cultured T lymphoblasts by deprivation of interleukin-6 (IL-6) or by incubation with the MEK inhibitor U0126. Apoptotic cell death was quantified by flow cytometry using annexin V/propidium iodide (AxV/PI) double staining. U0126-treated cells dramatically changed their fluorescence pattern during cell culture. If AxV/PI staining is employed to detect apoptotic cell death, the background fluorescence mimicks PS exposure on viable cells. The compound itself has no intrinsic fluorescence in vitro but develops an intensive fluorescence during cell culture which can be observed in all fluorescence channels with a predominance in the FL1 channel (525 nm). We further demonstrate that at least some of the U0126-induced background fluorescence is dependent on cellular uptake and intracellular modifications or cellular responses. These results demonstrate that appropriate controls for every single time point are necessary if fluorescence analyses are performed in the presence of chemical enzyme inhibitors. In the case of MEK inhibitors, either the use of PD098059 or PD184352 as an alternative for U0126 or nonfluorometric methods for detection of apoptosis should be considered. Copyright 2002 Wiley-Liss, Inc.

Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate

NASA Technical Reports Server (NTRS)

Telford, W. G.; Cox, W. G.; Stiner, D.; Singer, V. L.; Doty, S. B.

1999-01-01

BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications. Copyright 1999 Wiley-Liss, Inc.

Characterisation of the nucleolar organising regions during the cell cycle in two varieties of Petunia hybrida as visualised by fluorescence in situ hybridisation and silver staining.

PubMed

Montijn, M B; ten Hoopen, R; Fransz, P F; Oud, J L; Nanninga, N

1998-05-01

The cell cycle-dependent spatial position, morphology and activity of the four nucleolar organising regions (NORs) of the Petunia hybrida cultivar Mitchell and the inbred line V26 have been analysed. Application of the silver staining technique and fluorescence in situ hybridisation on fixed root-tip material revealed that these interspecific hybrids possess four NORs of which only those of chromosome 2 are active during interphase, which implies that the NOR activity is not of parental origin. However, at the end of mitosis, activity of all NOR regions could be detected, suggesting that the high demand for ribosomes at this stage of the cell cycle requires temporal activity of all NORs. Using actin DNA probes as markers in fluorescence in situ hybridisation experiments enabled the identification of the individual petunia chromosomes.

Sodium chloride-induced volume changes of freshwater cyanobacterium Synechococcus sp. PCC 7942 cells can be probed by chlorophyll a fluorescence.

PubMed

Stamatakis, K; Ladas, N P; Alygizaki-Zorba, A; Papageorgiou, G C

1999-10-15

Freshwater species of the cyanobacterial genus Synechococcus import NaCl passively, and export Na(+) actively, by means of primary and secondary extrusion mechanisms. As a result of the ion and water fluxes, cell volumes are enlarged. We show in this paper that the NaCl-induced volume enlargement of Synechococcus sp. PCC 7942 cells is attended by a rapid (k = 0.39 s(-1)) increase in chlorophyll (Chl) a fluorescence. The cell turgor threshold (measured by osmotic titration of Chl a fluorescence) was lower in the absence of NaCl (0.195 Osm kg(-1)) than in the presence of 0.4 M NaCl (0.248 Osm kg(-1)) indicating NaCl uptake by the cells. Turgor thresholds of cells suspended in NaCl-containing medium were enlarged further by protonophoric uncouplers, P-type ATPase inhibitors, and light starvation, conditions that are known to interfere with the active extrusion of Na(+) ions. Cell swelling exerts probably a regulation on the distribution of phycobilisome (PBS) excitation between photosystem II (fluorescent Chl a) and photosystem I (nonfluorescent Chl a), since it affects PBS-sensitized Chl a fluorescence, but not directly excited Chl a fluorescence. The dependence of the Chl a fluorescence of cyanobacteria on cell volumes allows probing of bioenergetic phenomena that are related to dynamic osmotic volume changes, transmembrane solute and water fluxes, plasma membrane permeabilities, and internal osmotic conditions of cyanobacterial cells. Thus, cyanobacteria may serve as quite convenient models of aquatic microorganisms in experimental studies directed toward the elucidation of perception mechanisms and defense mechanisms of water and solute stresses. Copyright 1999 Academic Press.

A Caco-2 cell-based quantitative antioxidant activity assay for antioxidants.

PubMed

Wan, Hongxia; Liu, Dong; Yu, Xiangying; Sun, Haiyan; Li, Yan

2015-05-15

A Caco-2 cell-based antioxidant activity (CAA) assay for quantitative evaluation of antioxidants was developed by optimizing seeding density and culture time of Caco-2 cells, incubation time and concentration of fluorescent probe (2',7'-dichlorofluorescin diacetate, DCFH-DA), incubation way and incubation time of antioxidants (pure phytochemicals) and DCFH-DA with cells, and detection time of fluorescence. Results showed that the CAA assay was of good reproducibility and could be used to evaluate the antioxidant activity of antioxidants at the following conditions: seeding density of 5 × 10(4)/well, cell culture time of 24h, co-incubation of 60 μM DCFH-DA and pure phytochemicals with Caco-2 cells for 20 min and fluorescence recorded for 90 min. Additionally, a significant correlation was observed between CAA values and rat plasma ORAC values following the intake of antioxidants for selected pure phytochemicals (R(2) = 0.815, p < 0.01), demonstrating the good biological relevance of CAA assay. Copyright © 2014 Elsevier Ltd. All rights reserved.

Genetically encoded probe for fluorescence lifetime imaging of CaMKII activity

PubMed Central

Kwok, Showming; Lee, Claudia; Sánchez, Susana A.; Hazlett, Theodore L.; Gratton, Enrico; Hayashi, Yasunori

2008-01-01

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the central nervous system and is critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, due to lack of a specific method. Here, based on our previous work, we attempted to generate an optical probe for fluorescence lifetime imaging (FLIM) of CaMKII activity by fusing the protein with donor and acceptor fluorescent proteins at its amino- and carboxyl-termini. We first optimized the combinations of fluorescent proteins by taking advantage of expansion of fluorescent proteins towards longer wavelength in fluorospectrometric assay. Then using digital frequency domain FLIM (DFD-FLIM), we demonstrated that the resultant protein can indeed detect CaMKII activation in living cells. These FLIM versions of Camui could be useful for elucidating the function of CaMKII both in vitro and in vivo. PMID:18302935

Nitric oxide interferes with islet cell zinc homeostasis.

PubMed

Tartler, U; Kröncke, K D; Meyer, K L; Suschek, C V; Kolb-Bachofen, V

2000-12-01

Zinc is crucial for the biosynthesis, storage, and secretion of insulin in pancreatic islet cells. We have previously presented evidence that NO interferes with cellular Zn(2+) homeostasis and we therefore investigated the influence of chronic NO exposure on the labile islet cell Zn(2+) content. A strong fluorescence activity in a large islet cell subpopulation was found after staining with the Zn(2+)-specific fluorophore Zinquin. Culture for 24 h in the presence of nontoxic concentrations of the slow-releasing NO donor DETA/NO resulted in a significantly reduced Zn(2+)-dependent fluorescence. This appears to be islet specific as in endothelial cells DETA/NO exposure enhanced the Zn(2+)-dependent fluorescence activity in a concentration-dependent manner. These results suggest that NO interferes with cellular Zn(2+) homeostasis, which in islet cells is crucial for proper hormone delivery and thus special cell function. Copyright 2000 Academic Press.

Real-time detection of caspase-2 activation in a single living HeLa cell during cisplatin-induced apoptosis

NASA Astrophysics Data System (ADS)

Lin, Juqiang; Zhang, Zhihong; Yang, Jie; Zeng, Shaoqun; Liu, Bifeng; Luo, Qingming

2006-03-01

Caspase-2 is important for the mitochondrial apoptotic pathway, however, the mechanism by which caspase-2 executes apoptosis remains obscure. We carry out the first measurements of the dynamics of caspase-2 activation in a single living cell by a FRET (fluorescence resonance energy transfer) probe. Two FRET probes are constructed that each encoded a CRS (caspase-2 or caspase-3 recognition site) fused with a cyan fluorescent protein (CFP) and a red fluorescent protein (DsRed) (CFP-CRS-DsRed). Using these probes, we found that during cisplatin-induced apoptosis, caspase-2 activation occurred more slowly than did activation of caspase-3; additionally, caspase-2 activation was initiated much earlier than that of caspase-3.

Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis.

PubMed

Sergeeva, Tatiana F; Shirmanova, Marina V; Zlobovskaya, Olga A; Gavrina, Alena I; Dudenkova, Varvara V; Lukina, Maria M; Lukyanov, Konstantin A; Zagaynova, Elena V

2017-03-01

A complex cascade of molecular events occurs in apoptotic cells but cell-to-cell variability significantly complicates determination of the order and interconnections between different processes. For better understanding of the mechanisms of programmed cell death, dynamic simultaneous registration of several parameters is required. In this paper we used multiparameter fluorescence microscopy to analyze energy metabolism, intracellular pH and caspase-3 activation in living cancer cells in vitro during staurosporine-induced apoptosis. We performed metabolic imaging of two co-factors, NAD(P)H and FAD, and used the genetically encoded pH-indicator SypHer1 and the FRET-based sensor for caspase-3 activity, mKate2-DEVD-iRFP, to visualize these parameters by confocal fluorescence microscopy and two-photon fluorescence lifetime imaging microscopy. The correlation between energy metabolism, intracellular pH and caspase-3 activation and their dynamic changes were studied in CT26 cancer cells during apoptosis. Induction of apoptosis was accompanied by a switch to oxidative phosphorylation, cytosol acidification and caspase-3 activation. We showed that alterations in cytosolic pH and the activation of oxidative phosphorylation are relatively early events associated with the induction of apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Optimization of a cAMP response element signal pathway reporter system.

PubMed

Shan, Qiang; Storm, Daniel R

2010-08-15

A sensitive cAMP response element (CRE) reporter system is essential for studying the cAMP/protein kinase A/cAMP response element binding protein signal pathway. Here we have tested a few CRE promoters and found one with high sensitivity to external stimuli. Using this optimal CRE promoter and the enhanced green fluorescent protein as the reporter, we have established a CRE reporter cell line. This cell line can be used to study the signal pathway by fluorescent microscope, fluorescence-activated cell analysis and luciferase assay. This cell line's sensitivity to forskolin, using the technique of fluorescence-activated cell sorting, was increased to approximately seven times that of its parental HEK 293 cell line, which is currently the most commonly used cell line in the field for the signal pathway study. Therefore, this newly created cell line is potentially useful for studying the signal pathway's modulators, which generally have weaker effect than its mediators. Our research has also established a general procedure for optimizing transcription-based reporter cell lines, which might be useful in performing the same task when studying many other transcription-based signal pathways. (c) 2010 Elsevier B.V. All rights reserved.

[Development of a Fluorescence Probe for Live Cell Imaging].

PubMed

Shibata, Aya

2017-01-01

 Probes that detect specific biological materials are indispensable tools for deepening our understanding of various cellular phenomena. In live cell imaging, the probe must emit fluorescence only when a specific substance is detected. In this paper, we introduce a new probe we developed for live cell imaging. Glutathione S-transferase (GST) activity is higher in tumor cells than in normal cells and is involved in the development of resistance to various anticancer drugs. We previously reported the development of a general strategy for the synthesis of probes for detection of GST enzymes, including fluorogenic, bioluminogenic, and 19 F-NMR probes. Arylsulfonyl groups were used as caging groups during probe design. The fluorogenic probes were successfully used to quantitate very low levels of GST activity in cell extracts and were also successfully applied to the imaging of microsomal MGST1 activity in living cells. The bioluminogenic and 19 F-NMR probes were able to detect GST activity in Escherichia coli cells. Oligonucleotide-templated reactions are powerful tools for nucleic acid sensing. This strategy exploits the target strand as a template for two functionalized probes and provides a simple molecular mechanism for multiple turnover reactions. We developed a nucleophilic aromatic substitution reaction-triggered fluorescent probe. The probe completed its reaction within 30 s of initiation and amplified the fluorescence signal from 0.5 pM target oligonucleotide by 1500 fold under isothermal conditions. Additionally, we applied the oligonucleotide-templated reaction for molecular releasing and peptide detection.

Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems?

PubMed

Lebaron, P; Servais, P; Agogué, H; Courties, C; Joux, F

2001-04-01

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.

Does the High Nucleic Acid Content of Individual Bacterial Cells Allow Us To Discriminate between Active Cells and Inactive Cells in Aquatic Systems?

PubMed Central

Lebaron, Philippe; Servais, Pierre; Agogué, Helene; Courties, Claude; Joux, Fabien

2001-01-01

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems. PMID:11282632

Live-cell Imaging with Genetically Encoded Protein Kinase Activity Reporters.

PubMed

Maryu, Gembu; Miura, Haruko; Uda, Youichi; Komatsubara, Akira T; Matsuda, Michiyuki; Aoki, Kazuhiro

2018-04-25

Protein kinases play pivotal roles in intracellular signal transduction, and dysregulation of kinases leads to pathological results such as malignant tumors. Kinase activity has hitherto been measured by biochemical methods such as in vitro phosphorylation assay and western blotting. However, these methods are less useful to explore spatial and temporal changes in kinase activity and its cell-to-cell variation. Recent advances in fluorescent proteins and live-cell imaging techniques enable us to visualize kinase activity in living cells with high spatial and temporal resolutions. Several genetically encoded kinase activity reporters, which are based on the modes of action of kinase activation and phosphorylation, are currently available. These reporters are classified into single-fluorophore kinase activity reporters and Förster (or fluorescence) resonance energy transfer (FRET)-based kinase activity reporters. Here, we introduce the principles of genetically encoded kinase activity reporters, and discuss the advantages and disadvantages of these reporters.Key words: kinase, FRET, phosphorylation, KTR.

Hyperspectral Imaging and Spectroscopy of Fluorescently Coupled Acyl-CoA: Cholesterol Acyltransferase in Insect Cells

NASA Technical Reports Server (NTRS)

Malak, H.; Mahtani, H.; Herman, P.; Vecer, J.; Lu, X.; Chang, T. Y.; Richmond, Robert C.; Whitaker, Ann F. (Technical Monitor)

2001-01-01

A high-performance hyperspectral imaging module with high throughput of light suitable for low-intensity fluorescence microscopic imaging and subsequent analysis, including single-pixel-defined emission spectroscopy, was tested on Sf21 insect cells expressing green fluorescence associated with recombinant green fluorescent protein linked or not with the membrane protein acyl-CoA:cholesterol acyltransferase. The imager utilized the phenomenon of optical activity as a new technique providing information over a spectral range of 220-1400 nm, and was inserted between the microscope and an 8-bit CCD video-rate camera. The resulting fluorescence image did not introduce observable image aberrations. The images provided parallel acquisition of well resolved concurrent spatial and spectral information such that fluorescence associated with green fluorescent protein alone was demonstrated to be diffuse within the Sf21 insect cell, and that green fluorescence associated with the membrane protein was shown to be specifically concentrated within regions of the cell cytoplasm. Emission spectra analyzed from different regions of the fluorescence image showed blue shift specific for the regions of concentration associated with the membrane protein.

Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging

PubMed Central

Hsieh, Ya-Ju; Ke, Chien-Chih; Yeh, Skye Hsin-Hsien; Lin, Chien-Feng; Chen, Fu-Du; Lin, Kang-Ping; Chen, Ran-Chou; Liu, Ren-Shyan

2014-01-01

Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase (fl), monomeric red fluorescence protein (mrfp), and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant (ttksr39) were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro and in vivo were determined by luciferase reporter assay, H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET). The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels of H-FEAU accumulation and significant cell death from ganciclovir (GCV) prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak for in vivo imaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter construct DsRedm-fl-ttksr39 for more effective and sensitive in vivo animal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications. PMID:24809057

Photo-triggered fluorescent theranostic prodrugs as DNA alkylating agents for mechlorethamine release and spatiotemporal monitoring.

PubMed

Cao, Yanting; Pan, Rong; Xuan, Weimin; Wei, Yongyi; Liu, Kejian; Zhou, Jiahong; Wang, Wei

2015-06-28

We describe a new theranostic strategy for selective delivery and spatiotemporal monitoring of mechlorethamine, a DNA alkylating agent. A photo-responsive prodrug is designed and composed of a photolabile o-nitrophenylethyl group, a DNA alkylating mechlorethamine drug and a coumarin fluorophore. Masking of the "N" in mechlorethamine in a positively charged state in the prodrug renders it inactive, non-toxic, selective and non-fluorescent. Indeed, the stable prodrug shows negligible cytotoxicity towards normal cells with and without UV activation and is completely non-fluorescent. However, upon photo-irradiation, the active mechlorethamine is released and induces efficient DNA cross-links, accompanied by a strong fluorescence enhancement (152 fold). Furthermore, DNA cross-linking activity from the release can be transformed into anticancer activity observed in in vitro studies of tumor cells. Importantly, the drug release progress and the movement can be conveniently monitored by fluorescence spectroscopy. The mechanistic study proves that the DNA cross-linking activity is mainly due to the release of DNA alkylating mechlorethamine. Altogether, the studies show the power of the theranostic strategy for efficient therapy in cancer treatment.

Ethidium bromide as a marker of mtDNA replication in living cells

NASA Astrophysics Data System (ADS)

Villa, Anna Maria; Fusi, Paola; Pastori, Valentina; Amicarelli, Giulia; Pozzi, Chiara; Adlerstein, Daniel; Doglia, Silvia Maria

2012-04-01

Mitochondrial DNA (mtDNA) in tumor cells was found to play an important role in maintaining the malignant phenotype. Using laser scanning confocal fluorescence microscopy (LSCFM) in a recent work, we reported a variable fluorescence intensity of ethidium bromide (EB) in mitochondria nucleoids of living carcinoma cells. Since when EB is bound to nucleic acids its fluorescence is intensified; a higher EB fluorescence intensity could reflect a higher DNA accessibility to EB, suggesting a higher mtDNA replication activity. To prove this hypothesis, in the present work we studied, by LSCFM, the EB fluorescence in mitochondria nucleoids of living neuroblastoma cells, a model system in which differentiation affects the level of mtDNA replication. A drastic decrease of fluorescence was observed after differentiation. To correlate EB fluorescence intensity to the mtDNA replication state, we evaluated the mtDNA nascent strands content by ligation-mediated real-time PCR, and we found a halved amount of replicating mtDNA molecules in differentiating cells. A similar result was obtained by BrdU incorporation. These results indicate that the low EB fluorescence of nucleoids in differentiated cells is correlated to a low content of replicating mtDNA, suggesting that EB may be used as a marker of mtDNA replication in living cells.

[The characters and specific features of new human embryonic stem cells lines].

PubMed

Krylova, T A; Kol'tsova, A M; Zenin, V V; Gordeeva, O F; Musorina, A S; Goriachaia, T S; Shlykova, S A; Kamenetskaia, Iu K; Pinaev, G P; Polianskaia, G G

2009-01-01

Four continuous human embryonic stem cell lines (SC1, SC2, SC3 and SC4), derived from the blastocysts has been described. The cell lines were cultivated on mitotically inactivated human feeder cells. The cell lines SC1 and SC2 have passed through 150 population doublings and the cell lines SC3 and SC4 -- near 120 populations doublings, which exceeds Hayflick limit sufficiently. These cell lines maintain high activity of alkaline phosphatase, expression of transcription factor OCT-4 and cell surface antigens (SSEA-4, TRA-1-60 and TRA-1-81), confirming their ESC status and human specificity. Immunofluorescent detection of antigens, characteristic of ectoderm, endoderm and mesoderm confirms the ability of these cells to retain their pluripotency under in vitro condition. PCR analysis revealed expression of six genes specific for pluripotent cells (OCT-4, NANOG, DPPA3/STELLA, TDGF/CRIPTO and LEFTYA). Correlation between the level of proliferative activity and the character of DNA-bound fluorescent staining was found. Fluorescent dyes, Hoechst 33342 and PI, produced diffuse staining of the nuclei in slowly proliferating cells of the SC1 and SC2 lines. In contrast, in actively proliferating cells of the SC3 and SC4 lines, the clear staining of the nuclei was observed. Upon changing the cultivation condition, proliferative activity of SC3 and SC4 lines decreased and became similar to that of SC1 and SC2 lines. The character of the fluorescent staining of all these lines was also shown to be similar. These results show that quality of the fluorescent staining with Hoechst 33342 and PI reflects the level of proliferation. Possible causes and mechanisms of this feature of human ESC are discussed.

Dielectrophoretic focusing integrated pulsed laser activated cell sorting

NASA Astrophysics Data System (ADS)

Zhu, Xiongfeng; Kung, Yu-Chun; Wu, Ting-Hsiang; Teitell, Michael A.; Chiou, Pei-Yu

2017-08-01

We present a pulsed laser activated cell sorter (PLACS) integrated with novel sheathless size-independent dielectrophoretic (DEP) focusing. Microfluidic fluorescence activated cell sorting (μFACS) systems aim to provide a fully enclosed environment for sterile cell sorting and integration with upstream and downstream microfluidic modules. Among them, PLACS has shown a great potential in achieving comparable performance to commercial aerosol-based FACS (>90% purity at 25,000 cells sec-1). However conventional sheath flow focusing method suffers a severe sample dilution issue. Here we demonstrate a novel dielectrophoresis-integrated pulsed laser activated cell sorter (DEP-PLACS). It consists of a microfluidic channel with 3D electrodes laid out to provide a tunnel-shaped electric field profile along a 4cmlong channel for sheathlessly focusing microparticles/cells into a single stream in high-speed microfluidic flows. All focused particles pass through the fluorescence detection zone along the same streamline regardless of their sizes and types. Upon detection of target fluorescent particles, a nanosecond laser pulse is triggered and focused in a neighboring channel to generate a rapidly expanding cavitation bubble for precise sorting. DEP-PLACS has achieved a sorting purity of 91% for polystyrene beads at a throughput of 1,500 particle/sec.

3D pulsed laser-triggered high-speed microfluidic fluorescence-activated cell sorter

PubMed Central

Chen, Yue; Wu, Ting-Hsiang; Kung, Yu-Chun; Teitell, Michael A.; Chiou, Pei-Yu

2014-01-01

We report a 3D microfluidic pulsed laser-triggered fluorescence-activated cell sorter capable of sorting at a throughput of 23,000 cells sec−1 with 90% purity in high-purity mode and at a throughput of 45,000 cells sec−1 with 45% purity in enrichment mode in one stage and in a single channel. This performance is realized by exciting laser-induced cavitation bubbles in a 3D PDMS microfluidic channel to generate high-speed liquid jets that deflect detected fluorescent cells and particles focused by 3D sheath flows. The ultrafast switching mechanism (20 μsec complete on-off cycle), small liquid jet perturbation volume, and three-dimensional sheath flow focusing for accurate timing control of fast (1.5 m sec−1) passing cells and particles are three critical factors enabling high-purity sorting at high-throughput in this sorter. PMID:23844418

A Micro Fluorescent Activated Cell Sorter for Astrobiology Applications

NASA Technical Reports Server (NTRS)

Platt, Donald W.; Hoover, Richard B.

2009-01-01

A micro-scale Fluorescent Activated Cell Sorter (microFACS) for astrobiology applications is under development. This device is designed to have a footprint of 7 cm x 7 cm x 4 cm and allow live-dead counts and sorting of cells that have fluorescent characteristics from staining. The FACS system takes advantage of microfluidics to create a cell sorter that can fit in the palm of the hand. A micron-scale channel allows cells to pass by a blue diode which causes emission of marker-expressed cells which are detected by a filtered photodetector. A small microcontroller then counts cells and operates high speed valves to select which chamber the cell is collected in (a collection chamber or a waste chamber). Cells with the expressed characteristic will be collected in the collection chamber. This system has been built and is currently being tested. We are also designing a system with integrated MEMS-based pumps and valves for a small and compact unit to fly on small satellite-based biology experiments.

Steady-State Acceptor Fluorescence Anisotropy Imaging under Evanescent Excitation for Visualisation of FRET at the Plasma Membrane

PubMed Central

Devauges, Viviane; Matthews, Daniel R.; Aluko, Justin; Nedbal, Jakub; Levitt, James A.; Poland, Simon P.; Coban, Oana; Weitsman, Gregory; Monypenny, James; Ng, Tony; Ameer-Beg, Simon M.

2014-01-01

We present a novel imaging system combining total internal reflection fluorescence (TIRF) microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET) imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor. PMID:25360776

Fluorescent sensors reporting the activity of ammonium transceptors in live cells

DOE PAGES

De Michele, Roberto; Ast, Cindy; Loqué, Dominique; ...

2013-07-02

Ammonium serves as key nitrogen source and metabolic intermediate, yet excess causes toxicity. Ammonium uptake is mediated by ammonium transporters, whose regulation is poorly understood. While transport can easily be characterized in heterologous systems, measuring transporter activity in vivo remains challenging. Here we developed a simple assay for monitoring activity in vivo by inserting circularly-permutated GFP into conformation-sensitive positions of two plant and one yeast ammonium transceptors (‘AmTrac’ and ‘MepTrac’). Addition of ammonium to yeast cells expressing the sensors triggered concentration-dependent fluorescence intensity (FI) changes that strictly correlated with the activity of the transporter. Fluorescence-based activity sensors present a novelmore » technology for monitoring the interaction of the transporters with their substrates, the activity of transporters and their regulation in vivo, which is particularly valuable in the context of analytes for which no radiotracers exist, as well as for cell-specific and subcellular transport processes that are otherwise difficult to track.« less

Pulsed laser triggered high speed microfluidic fluorescence activated cell sorter†‡

PubMed Central

Wu, Ting-Hsiang; Chen, Yue; Park, Sung-Yong; Hong, Jason; Teslaa, Tara; Zhong, Jiang F.; Di Carlo, Dino; Teitell, Michael A.

2014-01-01

We report a high speed and high purity pulsed laser triggered fluorescence activated cell sorter (PLACS) with a sorting throughput up to 20 000 mammalian cells s−1 with 37% sorting purity, 90% cell viability in enrichment mode, and >90% purity in high purity mode at 1500 cells s−1 or 3000 beads s−1. Fast switching (30 μs) and a small perturbation volume (~90 pL) is achieved by a unique sorting mechanism in which explosive vapor bubbles are generated using focused laser pulses in a single layer microfluidic PDMS channel. PMID:22361780

HAI-178 antibody-conjugated fluorescent magnetic nanoparticles for targeted imaging and simultaneous therapy of gastric cancer

NASA Astrophysics Data System (ADS)

Wang, Can; Bao, Chenchen; Liang, Shujing; Zhang, Lingxia; Fu, Hualin; Wang, Yutian; Wang, Kan; Li, Chao; Deng, Min; Liao, Qiande; Ni, Jian; Cui, Daxiang

2014-05-01

The successful development of safe and highly effective nanoprobes for targeted imaging and simultaneous therapy of in vivo gastric cancer is a great challenge. Herein we reported for the first time that anti-α-subunit of ATP synthase antibody, HAI-178 monoclonal antibody-conjugated fluorescent magnetic nanoparticles, was successfully used for targeted imaging and simultaneous therapy of in vivo gastric cancer. A total of 172 specimens of gastric cancer tissues were collected, and the expression of α-subunit of ATP synthase in gastric cancer tissues was investigated by immunohistochemistry method. Fluorescent magnetic nanoparticles were prepared and conjugated with HAI-178 monoclonal antibody, and the resultant HAI-178 antibody-conjugated fluorescent magnetic nanoparticles (HAI-178-FMNPs) were co-incubated with gastric cancer MGC803 cells and gastric mucous GES-1 cells. Gastric cancer-bearing nude mice models were established, were injected with prepared HAI-178-FMNPs via tail vein, and were imaged by magnetic resonance imaging and small animal fluorescent imaging system. The results showed that the α-subunit of ATP synthase exhibited high expression in 94.7% of the gastric cancer tissues. The prepared HAI-178-FMNPs could target actively MGC803 cells, realized fluorescent imaging and magnetic resonance imaging of in vivo gastric cancer, and actively inhibited growth of gastric cancer cells. In conclusion, HAI-178 antibody-conjugated fluorescent magnetic nanoparticles have a great potential in applications such as targeted imaging and simultaneous therapy of in vivo early gastric cancer cells in the near future.

Encapsulation of single cells into monodisperse droplets by fluorescence-activated droplet formation on a microfluidic chip.

PubMed

Hu, Rui; Liu, Pian; Chen, Pu; Wu, Liang; Wang, Yao; Feng, Xiaojun; Liu, Bi-Feng

2016-06-01

Random compartmentalization of cells by common droplet formation methods, i.e., T-junction and flow-focusing, results in low occupancy of droplets by single cells. To resolve this issue, a fluorescence-activated droplet formation method was developed for the on-command generation of droplets and encapsulation of single cells. In this method, droplets containing one cell were generated by switching on/off a two-phase hydrodynamic gating valve upon optical detection of single cells. To evaluate the developed method, flow visualization experiments were conducted with fluorescein. Results indicated that picoliter droplets of uniform sizes (RSD<4.9%) could be generated. Encapsulation of single fluorescent polystyrene beads demonstrated an average of 94.3% droplets contained one bead. Further application of the developed methods to the compartmentalization of individual HeLa cells indicated 82.5% occupancy of droplets by single cells, representing a 3 fold increase in comparison to random compartmentalization. Copyright © 2016 Elsevier B.V. All rights reserved.

Application of magnetic carriers to two examples of quantitative cell analysis

NASA Astrophysics Data System (ADS)

Zhou, Chen; Qian, Zhixi; Choi, Young Suk; David, Allan E.; Todd, Paul; Hanley, Thomas R.

2017-04-01

The use of magnetophoretic mobility as a surrogate for fluorescence intensity in quantitative cell analysis was investigated. The objectives of quantitative fluorescence flow cytometry include establishing a level of labeling for the setting of parameters in fluorescence activated cell sorters (FACS) and the determination of levels of uptake of fluorescently labeled substrates by living cells. Likewise, the objectives of quantitative magnetic cytometry include establishing a level of labeling for the setting of parameters in flowing magnetic cell sorters and the determination of levels of uptake of magnetically labeled substrates by living cells. The magnetic counterpart to fluorescence intensity is magnetophoretic mobility, defined as the velocity imparted to a suspended cell per unit of magnetic ponderomotive force. A commercial velocimeter available for making this measurement was used to demonstrate both applications. Cultured Gallus lymphoma cells were immunolabeled with commercial magnetic beads and shown to have adequate magnetophoretic mobility to be separated by a novel flowing magnetic separator. Phagocytosis of starch nanoparticles having magnetic cores by cultured Chinese hamster ovary cells, a CHO line, was quantified on the basis of magnetophoretic mobility.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bertoncello, I.; Hodgson, G.S.; Bradley, T.R.

A multiparameter cell separative procedure is described that enables normal transplantable hemopoietic stem cells that preferentially home to the marrow of lethally irradiated mice to be enriched and separated from the majority of spleen colony-forming cells that are assayed 13 days after transplantation (CFU-S13). First, bone marrow cells are centrifuged in a discontinuous bovine serum albumin gradient. Low-density cells are harvested and labeled with the supravital cationic fluorochrome rhodamine 123 (Rh123). Labeled cells are analyzed using a fluorescence-activated cell sorter, and cells are sorted on the basis of relative Rh123 fluorescence within a predetermined forward versus 90 degrees red lightmore » scatter window that has been optimized for the recovery and enrichment of cells with marrow repopulating ability (MRA). Cells with MRA were characterized by relatively low Rh123 fluorescence and could be separated from a fraction that fluoresced more intensely and contained the majority of CFU-S13 but low MRA. Cells with platelet repopulating ability cofractionate with MRA whereas cells with erythroid repopulating ability remain associated with CFU-S13.« less

Time-lapse monitoring of TLR2 ligand internalization with newly developed fluorescent probes.

PubMed

Arai, Yohei; Yokoyama, Kouhei; Kawahara, Yuki; Feng, Qi; Ohta, Ippei; Shimoyama, Atsushi; Inuki, Shinsuke; Fukase, Koichi; Kabayama, Kazuya; Fujimoto, Yukari

2018-05-23

As a mammalian toll-like receptor family member protein, TLR2 recognizes lipoproteins from bacteria and modulates the immune response by inducing the expression of various cytokines. We have developed fluorescence-labeled TLR2 ligands with either hydrophilic or hydrophobic fluorescence groups. The labeled ligands maintained the inflammatory IL-6 induction activity and enabled us to observe the internalization and colocalization of the TLR2 ligands using live-cell imaging. The time-lapse monitoring in the live-cell imaging of the fluorescence-labeled TLR2 ligand showed that TLR2/CD14 expression in the host cells enhanced the internalization of TLR2 ligand molecules.

Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level

PubMed Central

Baubet, Valérie; Le Mouellic, Hervé; Campbell, Anthony K.; Lucas-Meunier, Estelle; Fossier, Philippe; Brûlet, Philippe

2000-01-01

Monitoring calcium fluxes in real time could help to understand the development, the plasticity, and the functioning of the central nervous system. In jellyfish, the chemiluminescent calcium binding aequorin protein is associated with the green fluorescent protein and a green bioluminescent signal is emitted upon Ca2+ stimulation. We decided to use this chemiluminescence resonance energy transfer between the two molecules. Calcium-sensitive bioluminescent reporter genes have been constructed by fusing green fluorescent protein and aequorin, resulting in much more light being emitted. Chemiluminescent and fluorescent activities of these fusion proteins have been assessed in mammalian cells. Cytosolic Ca2+ increases were imaged at the single-cell level with a cooled intensified charge-coupled device camera. This bifunctional reporter gene should allow the investigation of calcium activities in neuronal networks and in specific subcellular compartments in transgenic animals. PMID:10860991

Light-induced rapid Ca2+ response and MAPK phosphorylation in the cells heterologously expressing human OPN5

PubMed Central

Sugiyama, Takashi; Suzuki, Hirobumi; Takahashi, Takeo

2014-01-01

Molecular imaging is a powerful tool for investigating intracellular signalling, but it is difficult to acquire conventional fluorescence imaging from photoreceptive cells. Here we demonstrated that human opsin5 (OPN5) photoreceptor mediates light-induced Ca2+ response in human embryonic kidney (HEK293) and mouse neuroblastoma (Neuro2a) cell lines using a luminescence imaging system with a fluorescent indicator and a newly synthesized bioluminescent indicator. Weak light fluorescence and bioluminescence imaging revealed rapid and transient light-stimulated Ca2+ release from thapsigargin-sensitive Ca2+ stores, whereas long-lasting Ca2+ elevation was observed using a conventional fluorescence imaging system. Bioluminescence imaging also demonstrated that OPN5 activation in HEK293 cells induced a decrease in pertussis toxin–sensitive cAMP, confirming previous reports. In addition, ultraviolet radiation induced the phosphorylation of mitogen-activated protein kinases when OPN5 was stimulated in Neuro2a cells. These findings suggest that the combination of these imaging approaches may provide a new means to investigate the physiological characteristics of photoreceptors. PMID:24941910

A novel bicistronic sensor vector for detecting caspase-3 activation.

PubMed

Vagner, Tatyana; Mouravlev, Alexandre; Young, Deborah

2015-01-01

Apoptosis is involved in pathological cell death of a wide range of human diseases. One of the most important biochemical markers of apoptosis is activation of caspase-3. Ability to detect caspase-3 activation early in the pathological process is important for determining the timing for interfering with apoptosis initiation and prevention of cell damage. Techniques allowing detection of caspase-3 activity at a single cell level show increased sensitivity, compared to biochemical assays; therefore, we developed a novel bicistronic caspase-3 sensor vector enabling detection of caspase-3 activity in individual cells. We employed green fluorescent protein (GFP) as a reporter for caspase-3 activation in our constructs and assessed the functionality of the generated constructs in transiently transfected Neuro2A and HEK293 cells under basal conditions and following application of okadaic acid (OA) or staurosporine (STS) to induce apoptosis. To ensure responsiveness of the new sensor vector to active caspase-3, we co-transfected the sensor with plasmid(s) overexpressing active caspase-3 and quantified GFP fluorescence using a plate reader. We observed an increase in GFP expression in cells transfected with the new bicistronic caspase-3 sensor in response to both OA and STS. We also showed a significant increase in GFP fluorescence intensity in cells co-expressing the sensor with the plasmid(s) encoding active caspase-3. We generated a novel bicistronic caspase-3 sensor vector which relies on a transcription factor/response element system. The obtained sensor combines high sensitivity of the single cell level detection with the possibility of automated quantification. Copyright © 2015 Elsevier Inc. All rights reserved.

Detection of Mitochondrial Caspase Activity in Real Time In Situ in Live Cells

NASA Astrophysics Data System (ADS)

Zhang, Yingpei; Haskins, Catherine; Lopez-Cruzan, Marisa; Zhang, Jianhua; Centonze, Victoria E.; Herman, Brian

2004-08-01

Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP caspase 3 substrate YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.

Efficient globally optimal segmentation of cells in fluorescence microscopy images using level sets and convex energy functionals.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2012-10-01

In high-throughput applications, accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression and the understanding of cell function. We propose an approach for segmenting cell nuclei which is based on active contours using level sets and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We consider three different well-known energy functionals for active contour-based segmentation and introduce convex formulations of these functionals. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images from different experiments comprising different cell types. We have also performed a quantitative comparison with previous segmentation approaches. Copyright © 2012 Elsevier B.V. All rights reserved.

Live morphological analysis of taxol-induced cytoplasmic vacuolization [corrected] in human lung adenocarcinoma cells.

PubMed

Wang, Xiao-Ping; Chen, Tong-Sheng; Sun, Lei; Cai, Ji-Ye; Wu, Ming-Qian; Mok, Martin

2008-12-01

Taxol (paclitaxel), one of the most active cancer chemotherapeutic agents, can cause programmed cell death (PCD) and cytoplasmic vacuolization. The objective of this study was to analyze the morphological characteristics induced by taxol. Human lung adenocarcinoma (ASTC-a-1) cells were exposed to various concentration of taxol. CCK-8 was used to assay the cell viability. Atomic force microscopy (AFM), plasmid transfection and confocal fluorescence microscopy were performed to image the cells morphological change induced by taxol. Fluorescence resonance energy transfer (FRET) was used to monitor the caspase-3 activation in living cells during taxol-induced cell death. Cells treated with taxol exhibited significant swelling and cytoplasmic vacuolization which may be due to endoplasmic reticulum (ER) vacuolization. Caspase-3 was not activated during taxol-induced cytoplasmic vacuolization and cell death. These findings suggest that taxol induces caspase-3-independent cytoplasmic vacuolization, cell swelling and cell death through ER vacuolization.

Single-cell analysis of radiotracers' uptake by fluorescence microscopy: direct and droplet approach

NASA Astrophysics Data System (ADS)

Gallina, M. E.; Kim, T. J.; Vasquez, J.; Tuerkcan, S.; Abbyad, P.; Pratx, G.

2017-02-01

Radionuclides are used for sensitive and specific detection of small molecules in vivo and in vitro. Recently, radioluminescence microscopy extended their use to single-cell studies. Here we propose a new single-cell radioisotopic assay that improves throughput while adding sorting capabilities. The new method uses fluorescence-based sensor for revealing single-cell interactions with radioactive molecular markers. This study focuses on comparing two different experimental approaches. Several probes were tested and Dihydrorhodamine 123 was selected as the best compromise between sensitivity, brightness and stability. The sensor was incorporated either directly within the cell cytoplasm (direct approach), or it was coencapsulated with radiolabeled single-cells in oil-dispersed water droplets (droplet approach). Both approaches successfully activated the fluorescence signal following cellular uptake of 18F-fluorodeoxyglucose (FDG) and external Xrays exposure. The direct approach offered single-cell resolution and longtime stability ( > 20 hours), moreover it could discriminate FDG uptake at labelling concentration as low as 300 μCi/ml. In cells incubated with Dihydrorhodamine 123 after exposure to high radiation doses (8-16 Gy), the fluorescence signal was found to increase with the depletion of ROS quenchers. On the other side, the droplet approach required higher labelling concentrations (1.00 mCi/ml), and, at the current state of art, three cells per droplet are necessary to produce a fluorescent signal. This approach, however, is independent on cellular oxidative stress and, with further improvements, will be more suitable for studying heterogeneous populations. We anticipate this technology to pave the way for the analysis of single-cell interactions with radiomarkers by radiofluorogenic-activated single-cell sorting.

Investigating the Functional Role of Prostate-Specific Membrane Antigen and its Enzymatic Activity in Prostate Cancer Metastasis

DTIC Science & Technology

2008-02-01

fluorescent probes for live cell imaging . PSMA distribution of cells grown on different extracellular matrices will be characterized to provide guidance...PCa migration, using in vitro cell model systems and live - cell imaging methods, we characterized the role of PSMA in cell motility and adhesion. Using...Generated fluorescently conjugated anti-PSMA antibodies for live cell imaging . 2. Optimized the siRNA-PSMA transfection and achieved an approximately

Intracarotid injection of fluorescence activated cell-sorted CD49d-positive neural stem cells improves targeted cell delivery and behavior after stroke in a mouse stroke model.

PubMed

Guzman, Raphael; De Los Angeles, Alejandro; Cheshier, Samuel; Choi, Raymond; Hoang, Stanley; Liauw, Jason; Schaar, Bruce; Steinberg, Gary

2008-04-01

Intravascular delivery of neural stem cells (NSCs) after stroke has been limited by the low efficiency of transendothelial migration. Vascular cell adhesion molecule-1 is an endothelial adhesion molecule known to be upregulated early after stroke and is responsible for the firm adhesion of inflammatory cells expressing the surface integrin, CD49d. We hypothesize that enriching for NSCs that express CD49d and injecting them into the carotid artery would improve targeted cell delivery to the injured brain. Mouse NSCs were analyzed for the expression of CD49d by fluorescence activated cell sorting. A CD49d-enriched (CD49d(+)) (>95%) and -depleted (CD49d(-); <5%) NSC population was obtained by cell sorting. C57/Bl6 mice underwent left-sided hypoxia-ischemia surgery and were assigned to receive 3 x 10(5) CD49d(+), CD49d(-) NSCs, or vehicle injection into the left common carotid artery 48 hours after stroke. Behavioral recovery was measured using a rotarod for 2 weeks after cell injection. Fluorescence activated cell sorting analysis revealed 25% CD49d(+) NSCs. In a static adhesion assay, NSCs adhered to vascular cell adhesion molecule-1 in a dose-dependent manner. Significantly more NSCs were found in the cortex, the hippocampus, and the subventricular zone in the ischemic hemisphere in animals receiving CD49d(+) NSCs as compared with CD49d(-) NSCs (P<0.05). Animals treated with CD49d(+) cells showed a significantly better behavioral recovery as compared with CD49d(-) and vehicle-treated animals. We show that enrichment of NSCs by fluorescence activated cell sorting for the surface integrin, CD49d, and intracarotid delivery promotes cell homing to the area of stroke in mice and improves behavioral recovery.

Fluorescent-Antibody Measurement Of Cancer-Cell Urokinase

NASA Technical Reports Server (NTRS)

Morrison, Dennis R.

1993-01-01

Combination of laboratory techniques provides measurements of amounts of urokinase in and between normal and cancer cells. Includes use of fluorescent antibodies specific against different forms of urokinase-type plasminogen activator, (uPA), fluorescence microscopy, quantitative analysis of images of sections of tumor tissue, and flow cytometry of different uPA's and deoxyribonucleic acid (DNA) found in suspended-tumor-cell preparations. Measurements provide statistical method for indicating or predicting metastatic potentials of some invasive tumors. Assessments of metastatic potentials based on such measurements used in determining appropriate follow-up procedures after surgical removal of tumors.

ABCG2 transporter inhibitor restores the sensitivity of triple negative breast cancer cells to aminolevulinic acid-mediated photodynamic therapy.

PubMed

Palasuberniam, Pratheeba; Yang, Xue; Kraus, Daniel; Jones, Patrick; Myers, Kenneth A; Chen, Bin

2015-08-18

Photosensitizer protoporphyrin IX (PpIX) fluorescence, intracellular localization and cell response to photodynamic therapy (PDT) were analyzed in MCF10A normal breast epithelial cells and a panel of human breast cancer cells including estrogen receptor (ER) positive, human epidermal growth factor receptor 2 (HER2) positive and triple negative breast cancer (TNBC) cells after treatment with PpIX precursor aminolevulinic acid (ALA). Although PpIX fluorescence was heterogeneous in different cells, TNBC cells showed significantly lower PpIX level than MCF10A and ER- or HER2-positive cells. PpIX fluorescence in TNBC cells also had much less mitochondrial localization than other cells. There was an inverse correlation between PpIX fluorescence and cell viability after PDT. Breast cancer cells with the highest PpIX fluorescence were the most sensitive to ALA-PDT and TNBC cells with the lowest PpIX level were resistant to PDT. Treatment of TNBC cells with ABCG2 transporter inhibitor Ko143 significantly increased ALA-PpIX fluorescence, enhanced PpIX mitochondrial accumulation and sensitized cancer cells to ALA-PDT. Ko143 treatment had little effect on PpIX production and ALA-PDT in normal and ER- or HER2-positive cells. These results demonstrate that enhanced ABCG2 activity renders TNBC cell resistance to ALA-PDT and inhibiting ABCG2 transporter is a promising approach for targeting TNBC with ALA-based modality.

A dark green fluorescent protein as an acceptor for measurement of Förster resonance energy transfer.

PubMed

Murakoshi, Hideji; Shibata, Akihiro C E; Nakahata, Yoshihisa; Nabekura, Junichi

2015-10-15

Measurement of Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) is a powerful method for visualization of intracellular signaling activities such as protein-protein interactions and conformational changes of proteins. Here, we developed a dark green fluorescent protein (ShadowG) that can serve as an acceptor for FLIM-FRET. ShadowG is spectrally similar to monomeric enhanced green fluorescent protein (mEGFP) and has a 120-fold smaller quantum yield. When FRET from mEGFP to ShadowG was measured using an mEGFP-ShadowG tandem construct with 2-photon FLIM-FRET, we observed a strong FRET signal with low cell-to-cell variability. Furthermore, ShadowG was applied to a single-molecule FRET sensor to monitor a conformational change of CaMKII and of the light oxygen voltage (LOV) domain in HeLa cells. These sensors showed reduced cell-to-cell variability of both the basal fluorescence lifetime and response signal. In contrast to mCherry- or dark-YFP-based sensors, our sensor allowed for precise measurement of individual cell responses. When ShadowG was applied to a separate-type Ras FRET sensor, it showed a greater response signal than did the mCherry-based sensor. Furthermore, Ras activation and translocation of its effector ERK2 into the nucleus could be observed simultaneously. Thus, ShadowG is a promising FLIM-FRET acceptor.

Long Term Non-Invasive Imaging of Embryonic Stem Cells Using Reporter Genes

PubMed Central

Sun, Ning; Lee, Andrew; Wu, Joseph C.

2013-01-01

Development of non-invasive and accurate methods to track cell fate following delivery will greatly expedite transition of embryonic stem (ES) cell therapy to the clinic. Here we describe a protocol for the in vivo monitoring of stem cell survival, proliferation, and migration using reporter genes. We established stable ES cell lines constitutively expressing double fusion (DF; enhanced green fluorescent protein and firefly luciferase) or triple fusion (TF; monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase) reporter genes using lentiviral transduction. We used fluorescence activated cell sorting to purify these populations in vitro, bioluminescence imaging and positron emission tomography imaging to track them in vivo, and fluorescence immunostaining to confirm the results ex vivo. Unlike other methods of cell tracking such as iron particle and radionuclide labeling, reporter genes are inherited genetically and can be used to monitor cell proliferation and survival for the lifetime of transplanted cells and their progeny. PMID:19617890

Live visualization of genomic loci with BiFC-TALE

PubMed Central

Hu, Huan; Zhang, Hongmin; Wang, Sheng; Ding, Miao; An, Hui; Hou, Yingping; Yang, Xiaojing; Wei, Wensheng; Sun, Yujie; Tang, Chao

2017-01-01

Tracking the dynamics of genomic loci is important for understanding the mechanisms of fundamental intracellular processes. However, fluorescent labeling and imaging of such loci in live cells have been challenging. One of the major reasons is the low signal-to-background ratio (SBR) of images mainly caused by the background fluorescence from diffuse full-length fluorescent proteins (FPs) in the living nucleus, hampering the application of live cell genomic labeling methods. Here, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies, we developed a novel method for labeling genomic loci (BiFC-TALE), which largely reduces the background fluorescence level. Using BiFC-TALE, we demonstrated a significantly improved SBR by imaging telomeres and centromeres in living cells in comparison with the methods using full-length FP. PMID:28074901

Live visualization of genomic loci with BiFC-TALE.

PubMed

Hu, Huan; Zhang, Hongmin; Wang, Sheng; Ding, Miao; An, Hui; Hou, Yingping; Yang, Xiaojing; Wei, Wensheng; Sun, Yujie; Tang, Chao

2017-01-11

Tracking the dynamics of genomic loci is important for understanding the mechanisms of fundamental intracellular processes. However, fluorescent labeling and imaging of such loci in live cells have been challenging. One of the major reasons is the low signal-to-background ratio (SBR) of images mainly caused by the background fluorescence from diffuse full-length fluorescent proteins (FPs) in the living nucleus, hampering the application of live cell genomic labeling methods. Here, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies, we developed a novel method for labeling genomic loci (BiFC-TALE), which largely reduces the background fluorescence level. Using BiFC-TALE, we demonstrated a significantly improved SBR by imaging telomeres and centromeres in living cells in comparison with the methods using full-length FP.

Cell segmentation in time-lapse fluorescence microscopy with temporally varying sub-cellular fusion protein patterns.

PubMed

Bunyak, Filiz; Palaniappan, Kannappan; Chagin, Vadim; Cardoso, M

2009-01-01

Fluorescently tagged proteins such as GFP-PCNA produce rich dynamically varying textural patterns of foci distributed in the nucleus. This enables the behavioral study of sub-cellular structures during different phases of the cell cycle. The varying punctuate patterns of fluorescence, drastic changes in SNR, shape and position during mitosis and abundance of touching cells, however, require more sophisticated algorithms for reliable automatic cell segmentation and lineage analysis. Since the cell nuclei are non-uniform in appearance, a distribution-based modeling of foreground classes is essential. The recently proposed graph partitioning active contours (GPAC) algorithm supports region descriptors and flexible distance metrics. We extend GPAC for fluorescence-based cell segmentation using regional density functions and dramatically improve its efficiency for segmentation from O(N(4)) to O(N(2)), for an image with N(2) pixels, making it practical and scalable for high throughput microscopy imaging studies.

Near-Membrane Refractometry Using Supercritical Angle Fluorescence.

PubMed

Brunstein, Maia; Roy, Lopamudra; Oheim, Martin

2017-05-09

Total internal reflection fluorescence (TIRF) microscopy and its variants are key technologies for visualizing the dynamics of single molecules or organelles in live cells. Yet truly quantitative TIRF remains problematic. One unknown hampering the interpretation of evanescent-wave excited fluorescence intensities is the undetermined cell refractive index (RI). Here, we use a combination of TIRF excitation and supercritical angle fluorescence emission detection to directly measure the average RI in the "footprint" region of the cell during image acquisition. Our RI measurement is based on the determination on a back-focal plane image of the critical angle separating evanescent and far-field fluorescence emission components. We validate our method by imaging mouse embryonic fibroblasts and BON cells. By targeting various dyes and fluorescent-protein chimeras to vesicles, the plasma membrane, as well as mitochondria and the endoplasmic reticulum, we demonstrate local RI measurements with subcellular resolution on a standard TIRF microscope, with a removable Bertrand lens as the only modification. Our technique has important applications for imaging axial vesicle dynamics and the mitochondrial energy state or detecting metabolically more active cancer cells. Copyright © 2017. Published by Elsevier Inc.

False HDAC Inhibition by Aurone Compound.

PubMed

Itoh, Yukihiro; Suzuki, Miki; Matsui, Taiji; Ota, Yosuke; Hui, Zi; Tsubaki, Kazunori; Suzuki, Takayoshi

2016-01-01

Fluorescence assays are useful tools for estimating enzymatic activity. Their simplicity and manageability make them suitable for screening enzyme inhibitors in drug discovery studies. However, researchers need to pay attention to compounds that show auto-fluorescence and quench fluorescence, because such compounds lower the accuracy of the fluorescence assay systems by producing false-positive or negative results. In this study, we found that aurone compound 7, which has been reported as a histone deacetylase (HDAC) inhibitor, gave false-positive results. Although compound 7 was identified by an in vitro HDAC fluorescence assay, it did not show HDAC inhibitory activity in a cell-based assay, leading us to suspect its in vitro HDAC inhibitory activity. As a result of verification experiments, we found that compound 7 interferes with the HDAC fluorescence assay by quenching the HDAC fluorescence signal. Our findings underscore the faults of fluorescence assays and call attention to careless interpretation.

A Label-Free Microfluidic Biosensor for Activity Detection of Single Microalgae Cells Based on Chlorophyll Fluorescence

PubMed Central

Wang, Junsheng; Sun, Jinyang; Song, Yongxin; Xu, Yongyi; Pan, Xinxiang; Sun, Yeqing; Li, Dongqing

2013-01-01

Detection of living microalgae cells is very important for ballast water treatment and analysis. Chlorophyll fluorescence is an indicator of photosynthetic activity and hence the living status of plant cells. In this paper, we developed a novel microfluidic biosensor system that can quickly and accurately detect the viability of single microalgae cells based on chlorophyll fluorescence. The system is composed of a laser diode as an excitation light source, a photodiode detector, a signal analysis circuit, and a microfluidic chip as a microalgae cell transportation platform. To demonstrate the utility of this system, six different living and dead algae samples (Karenia mikimotoi Hansen, Chlorella vulgaris, Nitzschia closterium, Platymonas subcordiformis, Pyramidomonas delicatula and Dunaliella salina) were tested. The developed biosensor can distinguish clearly between the living microalgae cells and the dead microalgae cells. The smallest microalgae cells that can be detected by using this biosensor are 3 μm ones. Even smaller microalgae cells could be detected by increasing the excitation light power. The developed microfluidic biosensor has great potential for in situ ballast water analysis. PMID:24287532

Dual-Color Fluorescence Imaging to Monitor CYP3A4 and CYP3A7 Expression in Human Hepatic Carcinoma HepG2 and HepaRG Cells

PubMed Central

Kubiura, Musashi; Hayashi, Ayaka; Ohbayashi, Tetsuya; Kazuki, Yasuhiro; Chesné, Christophe; Oshimura, Mitsuo; Tada, Masako

2014-01-01

Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP) and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps. PMID:25101946

Dual-color fluorescence imaging to monitor CYP3A4 and CYP3A7 expression in human hepatic carcinoma HepG2 and HepaRG cells.

PubMed

Tsuji, Saori; Kawamura, Fumihiko; Kubiura, Musashi; Hayashi, Ayaka; Ohbayashi, Tetsuya; Kazuki, Yasuhiro; Chesné, Christophe; Oshimura, Mitsuo; Tada, Masako

2014-01-01

Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP) and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps.

Measuring mRNA copy-number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization (smFISH)

PubMed Central

Skinner, Samuel O.; Sepúlveda, Leonardo A.; Xu, Heng; Golding, Ido

2014-01-01

We present a method for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed Escherichia coli cells. A set of fluorescently-labeled oligonucleotide probes are hybridized to the target mRNA, so that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The number of target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete “spots” as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy-number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 days, with another 2-3 days typically required for image and data analysis. PMID:23680982

The human prothrombin kringle-2 derived peptide, NSA9, is internalized into bovine capillary endothelial cells through endocytosis and energy-dependent pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang, Hyun Sook; Kim, Soung Soo

Human prothrombin kringle-2 and its partial peptide, NSA9 (NSAVQLVEN), have been reported to have potent anti-angiogenic activities. Here, the internalization mechanism of NSA9 into bovine capillary endothelial (BCE) cells was examined using lactate dehydrogenase (LDH) release assay, fluorescence microscopy, and flow cytometry. LDH release assay results suggested that the integrity of the BCE cell membrane was unaffected by NSA9. Fluorescence microscopy indicated that internalized NSA9 was localized in the cytoplasm around the nucleus, and showed a punctuated fluorescence pattern, which is indicative of endocytic vesicles. Also, the cellular internalization of NSA9 is significantly inhibited by depletion of the cellular ATPmore » pool, endocytosis inhibitors such as chloroquine and nocodazole, and incubation at low temperature (4 deg C). In addition, the anti-proliferative activity of NSA9 against BCE cells was diminished in the presence of endocytosis or metabolic inhibitors. In conclusion, these results strongly suggest that NSA9 might exert its anti-proliferative activity through internalization into BCE cells by endocytosis and energy-dependent pathways.« less

Elucidation of the new generation fluorescent protein tdTomato for space related radiobiological research

NASA Astrophysics Data System (ADS)

Chishti, Arif Ali; Baumstark-Khan, Christa; Hellweg, Christine; Reitz, Guenther

Astronauts in space are exposed to a potentially harmful radiation field, which does not exist in its quality and quantity on earth. Radiation exposure in space can lead to delayed or acute health effects. A successful long-term space mission requires better risk estimation and development of appropriate countermeasures, therefore study of the cellular radiation response is necessary. Ionizing radiation can provoke active cellular responses (cell cycle arrest, DNA repair, apoptosis or other forms of cell type). Exposure to ionizing radiation also activates various signaling pathways in human cells. In the cellular radiation-response, two pivotal signal transduction pathways have to be comprehensively studied i.e. the p53-pathway and NF-κB-pathway. Discovery of fluorescent proteins has revolutionized biological research by making it possible to carry out functional studies in living cells and understanding complex signaling pathways. Previously the green fluorescent proteins EGFP and d2EGFP were used for signaling pathway studies. In this work the new red fluorescent protein tdTomato will be used for comprehensive investigation of NF-κB and other transcription factor activation after exposure of human cells to ionizing radiation (X-rays, heavy ions; space conditions). tdTomato has many advantages over EGFP because of its high fluorescence signals and a better signal/noise ratio in human cells. The previously constructed reporter system with d2EGFP was used to evaluate NF-kB activation after exposure to heavy ion particles of different biological effectiveness. The sensitivity threshold of this system was determined to be 2 particle traversals per cell nucleus. In the current study a more sensitive reporter assay will be constructed using a GAL4-VP16 turbo system that comprises a receptor plasmid and a reporter plasmid. This reporter assay will be designed and constructed with tdTomato and evaluation will be done with different molecular techniques.

Penetration of short fluorescence-labeled peptides into the nucleus in HeLa cells and in vitro specific interaction of the peptides with deoxyribooligonucleotides and DNA.

PubMed

Fedoreyeva, L I; Kireev, I I; Khavinson, V Kh; Vanyushin, B F

2011-11-01

Marked fluorescence in cytoplasm, nucleus, and nucleolus was observed in HeLa cells after incubation with each of several fluorescein isothiocyanate-labeled peptides (epithalon, Ala-Glu-Asp-Gly; pinealon, Glu-Asp-Arg; testagen, Lys-Glu-Asp-Gly). This means that short biologically active peptides are able to penetrate into an animal cell and its nucleus and, in principle they may interact with various components of cytoplasm and nucleus including DNA and RNA. It was established that various initial (intact) peptides differently affect the fluorescence of the 5,6-carboxyfluorescein-labeled deoxyribooligonucleotides and DNA-ethidium bromide complexes. The Stern-Volmer constants characterizing the degree of fluorescence quenching of various single- and double-stranded fluorescence-labeled deoxyribooligonucleotides with short peptides used were different depending on the peptide primary structures. This indicates the specific interaction between short biologically active peptides and nucleic acid structures. On binding to them, the peptides discriminate between different nucleotide sequences and recognize even their cytosine methylation status. Judging from corresponding constants of the fluorescence quenching, the epithalon, pinealon, and bronchogen (Ala-Glu-Asp-Leu) bind preferentially with deoxyribooligonucleotides containing CNG sequence (CNG sites are targets for cytosine DNA methylation in eukaryotes). Epithalon, testagen, and pinealon seem to preferentially bind with CAG- but bronchogen with CTG-containing sequences. The site-specific interactions of peptides with DNA can control epigenetically the cell genetic functions, and they seem to play an important role in regulation of gene activity even at the earliest stages of life origin and in evolution.

Permeabilization of the plasma membrane of L1210 mouse leukemia cells using lithotripter shock waves.

PubMed

Gambihler, S; Delius, M; Ellwart, J W

1994-09-01

Permeabilization of L1210 cells by lithotripter shock waves in vitro was monitored by evaluating the accumulation of fluorescein-labeled dextrans with a relative molecular mass ranging from 3,900-2,000,000. Incubation with labeled dextran alone caused a dose- and time-dependent increase in cellular fluorescence as determined by flow cytometry, with a vesicular distribution pattern in the cells consistent with endocytotic uptake. Shock wave exposure prior to incubation with labeled dextran revealed similar fluorescence intensities compared to incubation with labeled dextran alone. When cells were exposed to shock waves in the presence of labeled dextran, mean cellular fluorescence was further increased, indicating additional internalization of the probe. Confocal laser scanning microscopy confirmed intracellular fluorescence of labeled dextran with a diffuse distribution pattern. Fluorescence-activated cell sorting with subsequent determination of proliferation revealed that permeabilized cells were viable and able to proliferate. Permeabilization of the membrane of L1210 cells by shock waves in vitro allowed loading of dextrans with a relative molecular mass up to 2,000,000. Permeabilization of tumor cells by shock waves provides a useful tool for introducing molecules into cells which might be of interest for drug targeting in tumor therapy in vivo.

Analysis of caspase-3 in ASTC-a-1 cells treated with mitomycin C using acceptor photobleaching techniques

NASA Astrophysics Data System (ADS)

Wang, Huiying; Chen, Tongsheng; Sun, Lei

2008-02-01

Caspase-3 is a key activated death protease, which catalyzes the specific cleavage of many cellular proteins and induces DNA cleavage eventually. In this report, cells were treated with mitomycin C (MMC) at different concentration and its activity was detected by cell counting kit (CCK-8). Based on results of CCK-8, cells were treated with 10μg/mL MMC and Hoechst 33258 has been used to observe cell apoptosis. Fluorescence resonance energy transfer (FRET) and confocal microscopy have been used to the effect of MMC on the caspase3 activation in living cells. Human lung adenocarcinoma cells (ASTC-a-1) was transfected with plasmid SCAT3 (pSCAT3)/CKAR FRET receptor. Acceptor photobleaching techniques of FRET plasmid has been used to destruct fluorophore of cells stably expressing SCAT3 reporter on a fluorescence confocal microscope. The activity of caspase3 can be analyzed by FRET dynamics of SCAT3 in living cells. Our results show that MM C can induce ASTC-a-1 cell apoptosis through activation of caspase3.

An imidazole functionalized pentameric thiophene displays different staining patterns in normal and malignant cells

NASA Astrophysics Data System (ADS)

Nilsson, Peter; Magnusson, Karin; Appelqvist, Hanna; Cieslar-Pobuda, Artur; Bäck, Marcus; Kågedal, Bertil; Jonasson, Jon; Los, Marek

2015-10-01

Molecular tools for fluorescent imaging of cells and their components are vital for understanding the function and activity of cells. Here, we report an imidazole functionalized pentameric oligothiophene, p-HTIm, that can be utilized for fluorescent imaging of cells. p-HTIm fluorescence in normal cells appeared in a peripheral punctate pattern partially co-localized with lysosomes, whereas a one-sided perinuclear Golgi associated localization of the dye was observed in malignant cells. The uptake of p-HTIm was temperature dependent and the intracellular target was reached within 1 h after staining. The ability of p-HTIm to stain cells was reduced when the imidazole side chain was chemically altered, verifying that specific imidazole side-chain functionalities are necessary for achieving the observed cellular staining. Our findings confirm that properly functionalized oligothiophenes can be utilized as fluorescent tools for vital staining of cells and that the selectivity towards distinct intracellular targets are highly dependent on the side-chain functionalities along the conjugated thiophene backbone.

Dark proteins: effect of inclusion body formation on quantification of protein expression.

PubMed

Iafolla, Marco A J; Mazumder, Mostafizur; Sardana, Vandit; Velauthapillai, Tharsan; Pannu, Karanbir; McMillen, David R

2008-09-01

Plasmid-borne gene expression systems have found wide application in the emerging fields of systems biology and synthetic biology, where plasmids are used to implement simple network architectures, either to test systems biology hypotheses about issues such as gene expression noise or as a means of exerting artificial control over a cell's dynamics. In both these cases, fluorescent proteins are commonly applied as a means of monitoring the expression of genes in the living cell, and efforts have been made to quantify protein expression levels through fluorescence intensity calibration and by monitoring the partitioning of proteins among the two daughter cells after division; such quantification is important in formulating the predictive models desired in systems and synthetic biology research. A potential pitfall of using plasmid-based gene expression systems is that the high protein levels associated with expression from plasmids can lead to the formation of inclusion bodies, insoluble aggregates of misfolded, nonfunctional proteins that will not generate fluorescence output; proteins caught in these inclusion bodies are thus "dark" to fluorescence-based detection methods. If significant numbers of proteins are incorporated into inclusion bodies rather than becoming biologically active, quantitative results obtained by fluorescent measurements will be skewed; we investigate this phenomenon here. We have created two plasmid constructs with differing average copy numbers, both incorporating an unregulated promoter (P(LtetO-1) in the absence of TetR) expressing the GFP derivative enhanced green fluorescent protein (EGFP), and inserted them into Escherichia coli bacterial cells (a common model organism for work on the dynamics of prokaryotic gene expression). We extracted the inclusion bodies, denatured them, and refolded them to render them active, obtaining a measurement of the average number of EGFP per cell locked into these aggregates; at the same time, we used calibrated fluorescent intensity measurements to determine the average number of active EGFP present per cell. Both measurements were carried out as a function of cellular doubling time, over a range of 45-75 min. We found that the ratio of inclusion body EGFP to active EGFP varied strongly as a function of the cellular growth rate, and that the number of "dark" proteins in the aggregates could in fact be substantial, reaching ratios as high as approximately five proteins locked into inclusion bodies for every active protein (at the fastest growth rate), and dropping to ratios well below 1 (for the slowest growth rate). Our results suggest that efforts to compare computational models to protein numbers derived from fluorescence measurements should take inclusion body loss into account, especially when working with rapidly growing cells. 2008 Wiley-Liss, Inc.

Rapid in situ assessment of physiological activities in bacterial biofilms using fluorescent probes

NASA Technical Reports Server (NTRS)

Yu, F. P.; McFeters, G. A.

1994-01-01

Two rapid in situ enumeration methods using fluorescent probes were used to assess the physiological activities of Klebsiella pneumoniae biofilms on stainless steel. Fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and rhodamine 123 (Rh 123), were chosen to perform this study. CTC is a soluble redox indicator which can be reduced by respiring bacteria to fluorescent CTC-formazan crystals. Rh 123 is incorporated into bacteria with respect to cellular proton motive force. The intracellular accumulation of these fluorescent dyes can be determined using epifluorescence microscopy. The results obtained with these two fluorescent probes in situ were compared to the plate count (PC) and in situ direct viable count (DVC) methods. Viable cell densities within biofilms determined by the three in situ methods were comparable and always showed approximately 2-fold higher values than those obtained with the PC method. As an additional advantage, the results were observed after 2 h, which was shorter than the 4 h incubation time required for the DVC method and 24 h for colony formation. The results indicate that staining with CTC and Rh 123 provides rapid information regarding cell numbers and physiological activities of bacteria within biofilms.

Complex Networks, Fractals and Topology Trends for Oxidative Activity of DNA in Cells for Populations of Fluorescing Neutrophils in Medical Diagnostics

NASA Astrophysics Data System (ADS)

Galich, N. E.

A novel nonlinear statistical method of immunofluorescence data analysis is presented. The data of DNA fluorescence due to oxidative activity in neutrophils nuclei of peripheral blood is analyzed. Histograms of photon counts statistics are generated using flow cytometry method. The histograms represent the distributions of fluorescence flash frequency as functions of intensity for large populations∼104-105 of fluorescing cells. We have shown that these experiments present 3D-correlations of oxidative activity of DNA for full chromosomes set in cells with spatial resolution of measurements is about few nanometers in the flow direction the jet of blood. Detailed analysis showed that large-scale correlations in oxidative activity of DNA in cells are described as networks of small- worlds (complex systems with logarithmic scaling) with self own small-world networks for given donor at given time for all states of health. We observed changes in fractal networks of oxidative activity of DNA in neutrophils in vivo and during medical treatments for classification and diagnostics of pathologies for wide spectra of diseases. Our approach based on analysis of changes topology of networks (fractal dimension) at variation the scales of networks. We produce the general estimation of health status of a given donor in a form of yes/no of answers (healthy/sick) in the dependence on the sign of plus/minus in the trends change of fractal dimensions due to decreasing the scale of nets. We had noted the increasing biodiversity of neutrophils and stochastic (Brownian) character of intercellular correlations of different neutrophils in the blood of healthy donor. In the blood of sick people we observed the deterministic cell-cell correlations of neutrophils and decreasing their biodiversity.

Development of novel small molecules for imaging and drug release

NASA Astrophysics Data System (ADS)

Cao, Yanting

Small organic molecules, including small molecule based fluorescent probes, small molecule based drugs or prodrugs, and smart multifunctional fluorescent drug delivery systems play important roles in biological research, drug discovery, and clinical practices. Despite the significant progress made in these fields, the development of novel and diverse small molecules is needed to meet various demands for research and clinical applications. My Ph.D study focuses on the development of novel functional molecules for recognition, imaging and drug release. In the first part, a turn-on fluorescent probe is developed for the detection of intracellular adenosine-5'-triphosphate (ATP) levels based on multiplexing recognitions. Considering the unique and complicated structure of ATP molecules, a fluorescent probe has been implemented with improved sensitivity and selectivity due to two synergistic binding recognitions by incorporating of 2, 2'-dipicolylamine (Dpa)-Zn(II) for targeting of phospho anions and phenylboronic acid group for cis-diol moiety. The novel probe is able to detect intracellular ATP levels in SH-SY5Y cells. Meanwhile, the advantages of multiplexing recognition design concept have been demonstrated using two control molecules. In the second part, a prodrug system is developed to deliver multiple drugs within one small molecule entity. The prodrug is designed by using 1-(2-nitrophenyl)ethyl (NPE) as phototrigger, and biphenol biquaternary ammonium as the prodrug. With controlled photo activation, both DNA cross-linking agents mechlorethamine and o-quinone methide are delivered and released at the preferred site, leading to efficient DNA cross-links formation and cell death. The prodrug shows negligible cytotoxicity towards normal skin cells (Hekn cells) with and without UV activation, but displays potent activity towards cancer cells (HeLa cells) upon UV activation. The multiple drug release system may hold a great potential for practical application. In the last part, a new photo-initiated fluorescent anticancer prodrug for DNA alkylating agent mechlorethamine releasing and monitoring has been developed. The theranostic prodrug consists a photolabile NPE group, an inactive form of mechlorethamine and a nonfluorescent coumarin in one small molecule. It is demonstrated that the prodrug shows negligible cytotoxicity towards normal skin cells (Hekn cells) with and without UV activation, while the original parent drug mechlorethamine can be photocontrol-released and induces effective DNA cross-linking activity. Importantly, the drug release progress can be conveniently monitored by the 'off-on' fluorescence enhancement in cells. Moreover, the selective prodrug is not only cell permeable but also nuclear permeable. Therefore, the prodrug serves as a promising drug delivery system for spatiotemporal control release and monitoring of an anticancer drug to obtain the optimal treatment efficacy.

In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry

PubMed Central

Hatzenpichler, Roland; Scheller, Silvan; Tavormina, Patricia L; Babin, Brett M; Tirrell, David A; Orphan, Victoria J

2014-01-01

Here we describe the application of a new click chemistry method for fluorescent tracking of protein synthesis in individual microorganisms within environmental samples. This technique, termed bioorthogonal non-canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of the non-canonical amino acid L-azidohomoalanine (AHA), a surrogate for l-methionine, followed by fluorescent labelling of AHA-containing cellular proteins by azide-alkyne click chemistry. BONCAT was evaluated with a range of phylogenetically and physiologically diverse archaeal and bacterial pure cultures and enrichments, and used to visualize translationally active cells within complex environmental samples including an oral biofilm, freshwater and anoxic sediment. We also developed combined assays that couple BONCAT with ribosomal RNA (rRNA)-targeted fluorescence in situ hybridization (FISH), enabling a direct link between taxonomic identity and translational activity. Using a methanotrophic enrichment culture incubated under different conditions, we demonstrate the potential of BONCAT-FISH to study microbial physiology in situ. A direct comparison of anabolic activity using BONCAT and stable isotope labelling by nano-scale secondary ion mass spectrometry (15NH3 assimilation) for individual cells within a sediment-sourced enrichment culture showed concordance between AHA-positive cells and 15N enrichment. BONCAT-FISH offers a fast, inexpensive and straightforward fluorescence microscopy method for studying the in situ activity of environmental microbes on a single-cell level. PMID:24571640

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

PubMed Central

Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

2013-01-01

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0. PMID:23685703

Direct imaging of ER calcium with targeted-esterase induced dye loading (TED).

PubMed

Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

2013-05-07

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca(2+) indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca(2+) indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca(2+) indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca(2+) indicator and a hydrophilic fluorescent dye/Ca(2+) complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.

Microfluidics platform for single-shot dose-response analysis of chloride channel-modulating compounds.

PubMed

Jin, Byung-Ju; Ko, Eun-A; Namkung, Wan; Verkman, A S

2013-10-07

We previously developed cell-based kinetics assays of chloride channel modulators utilizing genetically encoded yellow fluorescent proteins. Fluorescence platereader-based high-throughput screens yielded small-molecule activators and inhibitors of the cAMP-activated chloride channel CFTR and calcium-activated chloride channels, including TMEM16A. Here, we report a microfluidics platform for single-shot determination of concentration-activity relations in which a 1.5 × 1.5 mm square area of adherent cultured cells is exposed for 5-10 min to a pseudo-logarithmic gradient of test compound generated by iterative, two-component channel mixing. Cell fluorescence is imaged following perfusion with an iodide-containing solution to give iodide influx rate at each location in the image field, thus quantifying modulator effects over a wide range of concentrations in a single measurement. IC50 determined for CFTR and TMEM16A activators and inhibitors by single-shot microfluidics were in agreement with conventional plate reader measurements. The microfluidics approach developed here may accelerate the discovery and characterization of chloride channel-targeted drugs.

Reaction-Based Off-On Near-infrared Fluorescent Probe for Imaging Alkaline Phosphatase Activity in Living Cells and Mice.

PubMed

Tan, Yi; Zhang, Ling; Man, Ka Ho; Peltier, Raoul; Chen, Ganchao; Zhang, Huatang; Zhou, Liyi; Wang, Feng; Ho, Derek; Yao, Shao Q; Hu, Yi; Sun, Hongyan

2017-03-01

Alkaline phosphatases are a group of enzymes that play important roles in regulating diverse cellular functions and disease pathogenesis. Hence, developing fluorescent probes for in vivo detection of alkaline phosphatase activity is highly desirable for studying the dynamic phosphorylation in living organisms. Here, we developed the very first reaction-based near-infrared (NIR) probe (DHXP) for sensitive detection of alkaline phosphatase activity both in vitro and in vivo. Our studies demonstrated that the probe displayed an up to 66-fold fluorescence increment upon incubation with alkaline phosphatases, and the detection limit of our probe was determined to be 0.07 U/L, which is lower than that of most of alkaline phosphatase probes reported in literature. Furthermore, we demonstrated that the probe can be applied to detecting alkaline phosphatase activity in cells and mice. In addition, our probe possesses excellent biocompatibility and rapid cell-internalization ability. In light of these prominent properties, we envision that DHXP will add useful tools for investigating alkaline phosphatase activity in biomedical research.

A biosensor generated via high throughput screening quantifies cell edge Src dynamics

PubMed Central

Gulyani, Akash; Vitriol, Eric; Allen, Richard; Wu, Jianrong; Gremyachinskiy, Dmitriy; Lewis, Steven; Dewar, Brian; Graves, Lee M.; Kay, Brian K.; Kuhlman, Brian; Elston, Tim; Hahn, Klaus M.

2011-01-01

Fluorescent biosensors for living cells currently require laborious optimization and a unique design for each target. They are limited by the availability of naturally occurring ligands with appropriate target specificity. Here we describe a biosensor based on an engineered fibronectin monobody scaffold that can be tailored to bind different targets via high throughput screening. This Src family kinase (SFK) biosensor was made by derivatizing a monobody specific for activated SFK with a bright dye whose fluorescence increases upon target binding. We identified sites for dye attachment and alterations to eliminate vesiculation in living cells, providing a generalizable scaffold for biosensor production. This approach minimizes cell perturbation because it senses endogenous, unmodified target, and because sensitivity is enhanced by direct dye excitation. Automated correlation of cell velocities and SFK activity revealed that SFK are activated specifically during protrusion. Activity correlates with velocity, and peaks 1–2 microns from the leading edge. PMID:21666688

Real-time intravital imaging of pH variation associated with osteoclast activity.

PubMed

Maeda, Hiroki; Kowada, Toshiyuki; Kikuta, Junichi; Furuya, Masayuki; Shirazaki, Mai; Mizukami, Shin; Ishii, Masaru; Kikuchi, Kazuya

2016-08-01

Intravital imaging by two-photon excitation microscopy (TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, as well as their undesirable physicochemical properties for TPEM imaging. Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and a fluorescence switch and then imaged target cell activity by injecting the SMP into living mice. The combination of the rationally designed SMP with a fluorescent protein as a reporter of cell localization enabled quantitation of osteoclast activity and time-lapse imaging of its in vivo function associated with changes in cell deformation and membrane fluctuations. Real-time imaging revealed heterogenic behaviors of osteoclasts in vivo and provided insights into the mechanism of bone resorption.

Determination of Cancer Cell-Based pH-Sensitive Fluorescent Carbon Nanoparticles of Cross-Linked Polydopamine by Fluorescence Sensing of Alkaline Phosphatase Activity on Coated Surfaces and Aqueous Solution.

PubMed

Kang, Eun Bi; Choi, Cheong A; Mazrad, Zihnil Adha Islamy; Kim, Sung Han; In, Insik; Park, Sung Young

2017-12-19

The tumor-specific sensitive fluorescence sensing of cellular alkaline phosphatase (ALP) activity on the basis of host-guest specific and pH sensitivity was conducted on coated surfaces and aqueous states. Cross-linked fluorescent nanoparticles (C-FNP) consisting of β-cyclodextrin (β-CD)/boronic acid (BA) and fluorescent hyaluronic acid [FNP(HA)] were conjugated to fluorescent polydopamine [FNP(pDA)]. To determine the quenching effect of this system, hydrolysis of 4-nitrophenyl phosphate (NPP) to 4-nitrophenol (NP) was performed in the cavity of β-CD in the presence of ALP activated photoinduced electron transfer (PET) between NP and C-FNP. At an ALP level of 30-1000 U/L, NP caused off-emission of C-FNP because of their specific host-guest recognition. Fluorescence can be recovered under pH shock due to cleavage of the diol bond between β-CD and BA, resulting in release of NP from the fluorescent system. Sensitivity of the assays was assessed by confocal imaging not only in aqueous states, but also for the first time on coated surfaces in MDAMB-231 and MDCK cells. This novel system demonstrated high sensitivity to ALP through generation of good electron donor/acceptor pair during the PET process. Therefore, this fluorescence sensor system can be used to enhance ALP monitoring and cancer diagnosis on both coated surfaces and in aqueous states in clinical settings.

Imaging and Detection of Carboxylesterase in Living Cells and Zebrafish Pretreated with Pesticides by a New Near-Infrared Fluorescence Off-On Probe.

PubMed

Li, Dongyu; Li, Zhao; Chen, Weihua; Yang, Xingbin

2017-05-24

A new near-infrared fluorescence off-on probe was developed and applied to fluorescence imaging of carboxylesterase in living HepG-2 cells and zebrafish pretreated with pesticides (carbamate, organophosphorus, and pyrethroid). The probe was readily prepared by connecting (4-acetoxybenzyl)oxy as a quenching and recognizing moiety to a stable hemicyanine skeleton that can be formed via the decomposition of IR-780. The fluorescence off-on response of the probe to carboxylesterase is based on the enzyme-catalyzed spontaneous hydrolysis of the carboxylic ester bond, followed by a further fragmentation of the phenylmethyl unit and thereby the fluorophore release. Compared with the only existing near-infrared carboxylesterase probe, the proposed probe exhibits superior analytical performance, such as near-infrared fluorescence emission over 700 nm as well as high selectivity and sensitivity, with a detection limit of 4.5 × 10 -3 U/mL. More importantly, the probe is cell membrane permeable, and its applicability has been successfully demonstrated for monitoring carboxylesterase activity in living HepG-2 cells and zebrafish pretreated with pesticides, revealing that pesticides can effectively inhibit the activity of carboxylesterase. The superior properties of the probe make it of great potential use in indicating pesticide exposure.

Partial alanine scan of mast cell degranulating peptide (MCD): importance of the histidine- and arginine residues.

PubMed

Buku, Angeliki; Mendlowitz, Milton; Condie, Barry A; Price, Joseph A

2004-06-01

The influence of the two histidine and two arginine residues of mast cell degranulating peptide (MCD) in activity and binding was studied by replacing these amino acids in the MCD sequence with L-alanine. Their histamine releasing activity was determined on rat peritoneal mast cells. Their binding affinity to the FcepsilonRIalpha binding subunit of the human mast cell receptor protein, was carried out using fluorescence polarization. The histamine assay showed that replacement of His13 by Ala o ccurred without loss of activity compared with the activity of MCD. Alanine substitutions for Arg7 and His8 resulted in an approximately 40 fold increase, and for Arg16 in a 14-fold increase in histamine-releasing activity of MCD. The binding affinities of the analogs were tested by competitive displacement of bound fluorescent MCD peptide from the FcepsilonRIalpha binding protein of the mast cell receptor by the Ala analogs using fluorescence polarization. The analogs Ala8 (for His) and Ala16 (for Arg) showed the same binding affinities as MCD, whereas analog Ala7 (for Arg) and analog Ala13 (for His) showed slightly better binding affinity than the parent compound. This study showed that the introduction of alanine residues in these positions resulted in MCD agonists of diverse potency. These findings will be useful in further MCD structure-activity studies.

Assessment of FUN-1 vital dye staining: Yeast with a block in the vacuolar sorting pathway have impaired ability to form CIVS when stained with FUN-1 fluorescent dye.

PubMed

Essary, Brandin D; Marshall, Pamela A

2009-08-01

FUN-1 [2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-phenylquinolinium iodide] is a fluorescent dye used in studies of yeast and other fungi to monitor cell viability in the research lab and to assay for active fungal infection in the clinical setting. When the plasma membrane is intact, fungal cells internalize FUN-1 and the dye is seen as diffuse green cytosolic fluorescence. FUN-1 is then transported to the vacuole in metabolically active wild type cells and subsequently is compacted into fluorescent red cylindrical intravacuolar structures (CIVS) by an unknown transport pathway. This dye is used to determine yeast viability, as only live cells form CIVS. However, in live Saccharomyces cerevisiae with impaired protein sorting to the yeast vacuole, we report decreased to no CIVS formation, depending on the cellular location of the block in the sorting pathway. Cells with a block in vesicle-mediated transport from the Golgi to prevacuolar compartment (PVC) or with a block in recycling from the PVC to the Golgi demonstrate a substantial impairment in CIVS formation. Instead, the FUN-1 dye is seen either in small punctate structures under fluorescence or as diffuse red cytosol under white light. Thus, researchers using FUN-1 should be cognizant of the limitations of this procedure in determining cell viability as there are viable yeast mutants with impaired CIVS formation.

Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bennett, Kristen; Sadler, Natalie C.; Wright, Aaron T.

Nitrosomonas europaeais an aerobic nitrifying bacterium that oxidizes ammonia (NH 3) to nitrite (NO 2 ₋) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH 4 +-dependent O 2uptake byN. europaeaby 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide usingmore » a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization–tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA.« less

Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

PubMed Central

Bennett, Kristen; Sadler, Natalie C.; Wright, Aaron T.; Yeager, Chris

2016-01-01

Nitrosomonas europaea is an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2−) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4+-dependent O2 uptake by N. europaea by 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, and de novo protein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization–tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA. PMID:26826234

Measurement of caspase-2 activation during different anti-tumor drugs induced apoptosis by FRET technique

NASA Astrophysics Data System (ADS)

Lin, Juqiang; Zeng, Shaoqun; Luo, Qingming; Rong, Chen; Zhang, Zhihong

2007-11-01

Caspase-2 is important for the engagement of the mitochondrial apoptotic pathway, in the presence of DNA-damaging agents, such as cisplatin; however, the mechanism by which caspase-2 executes apoptosis remains obscure. In this study, we carried out the measurements of the dynamics of caspase-2 activation in a single living cell by a FRET (fluorescence resonance energy transfer) probe. A FRET probe was constructed that encoded a CRS (caspase-2 recognition site) fused with a cyan fluorescent protein (CFP) and a red fluorescent protein (DsRed) (CFP-CRS-DsRed). Using this probe, we found that during TRAIL-induced apoptosis, caspase-2 was not activated, and caspase-2 activation occurred in etoposide and cisplatin treated cells. However, during cisplatin-induced apoptosis caspase-2 activation was initiated much earlier than that of etoposide. Cisplatin and etoposide is one of the most broadly used drugs in the Clinical applications of cancer chemotherapy, and TRAIL, which belongs to the TNF family proteins, can selectively induce apoptosis in many transformed cells but not in normal cells. Most of anticancer drugs can induce apoptosis mediated by the activation of caspase pathway. Thus, the perfect synergistic effect group of multi-drug can be selected by using our FRET probe.

[Phloretin induces apoptosis of BEL-7402 cells in vitro].

PubMed

Luo, Hui; Wang, Ya-jun; Chen, Jie; Liu, Jiang-qin; Zhang, Hai-tao

2008-07-01

To examine the effect of phloretin on apoptosis of BEL-7402 cells. The viability changes of BEL- 7402 cells as a result of phloretin-induced toxicity were analyzed using MTT assay, and the cell morphology changes were observed with fluorescence microscope. Flow cytometry was used to analyze the cell cycle and mitochondrial membrane potential changes, and chromogenic substrate assay performed to detect caspase activity. Phloretin induced obvious cytotoxicity against BEL-7402 cells with IC50 of 89.23 microg/mL. The growth curve demonstrated decreased growth of the cells as phloretin concentration increased. Cell apoptosis occurred 24 h after treatment with 40-160 microg/mL phloretin. Morphological, the cells exposed to phloretin exhibited nuclear chromatin condensation and increased fluorescence intensity. The activity of caspase-9 reached the peak level 12 h after phloretin exposure, and leak levels of caspase-6 and caspase-3 activities occurred 18 and 24 h after the exposure, respectively. Phloretin can induce BEL-7402 cell apoptosis though the mitochondrial pathway.

A ratiometric fluorescent molecular probe for visualization of mitochondrial temperature in living cells.

PubMed

Homma, Mitsumasa; Takei, Yoshiaki; Murata, Atsushi; Inoue, Takafumi; Takeoka, Shinji

2015-04-11

Mitochondrial thermodynamics is the key to understand cellular activities related to homeostasis and energy balance. Here, we report the first ratiometric fluorescent molecular probe (Mito-RTP) that is selectively localized in the mitochondria and visualize the temperature. We confirmed that Mito-RTP could work as a ratiometric thermometer in a cuvette and living cells.

Feedback-mediated cancer therapy: a FRET-based nanoreporter approach

NASA Astrophysics Data System (ADS)

Sarkar, Suproteem K.; Khater, Yashika; Kulkarni, Ashish; Sengupta, Shiladitya

2014-08-01

A theranostic nanoparticle system was developed by integrating a chemotherapeutic agent with an "activatable" fluorescent tracer. The system signals tumor death by monitoring the activity of caspase-3, a product of apoptosis, and can therefore screen the treatment sensitivity of a particular tumor. The polymer nanoparticles (Poly [isobutylene-alt-maleic anhydride]) were formed through reprecipitation and contained paclitaxel, a chemotherapy drug, and fluorescein isothiocyanate, a fluorescent dye. The dye's fluorescence was quenched through Förster resonance energy transfer (FRET) by a quencher that was connected to the dye by a peptide chain. With sizes ranging from 200-250 nm, the nanoparticles were stable for two weeks. The nanoparticles were tested in vitro with responsive Lewis Lung Carcinoma (LLC) cells and taxane-resistant cells. Upon cell death by paclitaxel exposure, caspase-3 cleaved the peptide chain connecting the dye and the quencher, causing the system to fluoresce. When LLC cells were treated with the system, the nanoreporters fluoresced, but when resistant cells were tested, and when the drug was removed from the system, the nanoreporters did not fluoresce. Since the system screens if a drug can successfully kill a particular tumor, it offers a novel and promising approach to personalized medicine.

Visualization of nucleic acids with synthetic exciton-controlled fluorescent oligonucleotide probes.

PubMed

Wang, Dan Ohtan; Okamoto, Akimitsu

2015-01-01

Engineered probes to adapt new photochemical properties upon recognition of target nucleic acids offer powerful tools to DNA and RNA visualization technologies. Herein, we describe a rapid and effective visualization method of nucleic acids in both fixed and living cells with hybridization-sensitive fluorescent oligonucleotide probes. These probes are efficiently quenched in an aqueous environment due to the homodimeric, excitonic interactions between fluorophores but become highly fluorescent upon hybridization to DNA or RNA with complementary sequences. The fast hybridization kinetics and quick fluorescence activation of the new probes allow applications to simplify the conventional fluorescent in situ hybridization protocols and reduce the amount of time to process the samples. Furthermore, hybridization-sensitive fluorescence emission of the probes allows monitoring dynamic behaviors of RNA in living cells.

Fast globally optimal segmentation of cells in fluorescence microscopy images.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2011-01-01

Accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression in high-throughput screening applications. We propose a new approach for segmenting cell nuclei which is based on active contours and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images of different cell types. We have also performed a quantitative comparison with previous segmentation approaches.

Smart imaging of acute lung injury: exploration of myeloperoxidase activity using in vivo endoscopic confocal fluorescence microscopy.

PubMed

Chagnon, Frédéric; Bourgouin, Alexandra; Lebel, Réjean; Bonin, Marc-André; Marsault, Eric; Lepage, Martin; Lesur, Olivier

2015-09-15

The pathophysiology of acute lung injury (ALI) is well characterized, but its real-time assessment at bedside remains a challenge. When patients do not improve after 1 wk despite supportive therapies, physicians have to consider open lung biopsy (OLB) to identify the process(es) at play. Sustained inflammation and inadequate repair are often observed in this context. OLB is neither easy to perform in a critical setting nor exempt from complications. Herein, we explore intravital endoscopic confocal fluorescence microscopy (ECFM) of the lung in vivo combined with the use of fluorescent smart probe(s) activated by myeloperoxidase (MPO). MPO is a granular enzyme expressed by polymorphonuclear neutrophils (PMNs) and alveolar macrophages (AMs), catalyzing the synthesis of hypoclorous acid, a by-product of hydrogen peroxide. Activation of these probes was first validated in vitro in relevant cells (i.e., AMs and PMNs) and on MPO-non-expressing cells (as negative controls) and then tested in vivo using three rat models of ALI and real-time intravital imaging with ECFM. Semiquantitative image analyses revealed that in vivo probe-related cellular/background fluorescence was associated with corresponding enhanced lung enzymatic activity and was partly prevented by specific MPO inhibition. Additional ex vivo phenotyping was performed, confirming that fluorescent cells were neutrophil elastase(+) (PMNs) or CD68(+) (AMs). This work is a first step toward "virtual biopsy" of ALI without OLB. Copyright © 2015 the American Physiological Society.

Measurement of intracellular nitric oxide (NO) production in shrimp haemocytes by flow cytometry.

PubMed

Xian, Jian-An; Guo, Hui; Li, Bin; Miao, Yu-Tao; Ye, Jian-Min; Zhang, Sheng-Peng; Pan, Xun-Bin; Ye, Chao-Xia; Wang, An-Li; Hao, Xuan-Ming

2013-12-01

A flow cytometric method to measure the production of intracellular nitric oxide (NO) was adapted for use with shrimp haemocytes. We applied fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) for NO detection in haemocytes from the tiger shrimp Penaeus monodon, and used flow cytometry to quantify fluorescence intensity in individual haemocyte. The optimized protocol for intracellular NO analysis consists to incubate haemocytes with DAF-FM DA at 10 μM for 60 min to determine the mean fluorescence intensity. Result showed that NO was also produced in the untreated shrimp haemocytes. NO level in granular cells and semigranular cells were much higher than that in hyaline cells. Defined by different characteristic of NO content, three subsets of haemocytes were observed. Zymosan A at dose of 10 or 100 particles per haemocyte triggered higher DAF-FM fluorescence intensity in granular and semigranular cells, than PMA that had no significant impact on all three cell types. These results indicate that granular and semigranular cells are the primary cells for NO generation. Cytochalasin B significantly inhibited the NO level induced by zymosan A. NG-Monomethyl-L-arginine (L-NMMA) and diphenylene iodonium chloride (DPI) significantly suppressed the DAF-FM fluorescence in haemocytes, but apocynin could not modulate it, indicating that the DAF-FM fluorescence was closely related to the activity of NO-synthase pathway. The NO donor sodium nitroprusside (SNP) improved the DAF-FM fluorescence in haemocytes, while the NO scavenger C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) significantly decreased the fluorescence, demonstrating that the fluorescence intensity of DAF-FM is mainly dependent on the intracellular NO level.

Deep brain optical measurements of cell type-specific neural activity in behaving mice.

PubMed

Cui, Guohong; Jun, Sang Beom; Jin, Xin; Luo, Guoxiang; Pham, Michael D; Lovinger, David M; Vogel, Steven S; Costa, Rui M

2014-01-01

Recent advances in genetically encoded fluorescent sensors enable the monitoring of cellular events from genetically defined groups of neurons in vivo. In this protocol, we describe how to use a time-correlated single-photon counting (TCSPC)-based fiber optics system to measure the intensity, emission spectra and lifetime of fluorescent biosensors expressed in deep brain structures in freely moving mice. When combined with Cre-dependent selective expression of genetically encoded Ca(2+) indicators (GECIs), this system can be used to measure the average neural activity from a specific population of cells in mice performing complex behavioral tasks. As an example, we used viral expression of GCaMPs in striatal projection neurons (SPNs) and recorded the fluorescence changes associated with calcium spikes from mice performing a lever-pressing operant task. The whole procedure, consisting of virus injection, behavior training and optical recording, takes 3-4 weeks to complete. With minor adaptations, this protocol can also be applied to recording cellular events from other cell types in deep brain regions, such as dopaminergic neurons in the ventral tegmental area. The simultaneously recorded fluorescence signals and behavior events can be used to explore the relationship between the neural activity of specific brain circuits and behavior.

Integrated light and scanning electron microscopy of GFP-expressing cells.

PubMed

Peddie, Christopher J; Liv, Nalan; Hoogenboom, Jacob P; Collinson, Lucy M

2014-01-01

Integration of light and electron microscopes provides imaging tools in which fluorescent proteins can be localized to cellular structures with a high level of precision. However, until recently, there were few methods that could deliver specimens with sufficient fluorescent signal and electron contrast for dual imaging without intermediate staining steps. Here, we report protocols that preserve green fluorescent protein (GFP) in whole cells and in ultrathin sections of resin-embedded cells, with membrane contrast for integrated imaging. Critically, GFP is maintained in a stable and active state within the vacuum of an integrated light and scanning electron microscope. For light microscopists, additional structural information gives context to fluorescent protein expression in whole cells, illustrated here by analysis of filopodia and focal adhesions in Madin Darby canine kidney cells expressing GFP-Paxillin. For electron microscopists, GFP highlights the proteins of interest within the architectural space of the cell, illustrated here by localization of the conical lipid diacylglycerol to cellular membranes. © 2014 Elsevier Inc. All rights reserved.

Real Time Measures of Prestin Charge and Fluorescence during Plasma Membrane Trafficking Reveal Sub-Tetrameric Activity

PubMed Central

Bian, Shumin; Navaratnam, Dhasakumar; Santos-Sacchi, Joseph

2013-01-01

Prestin (SLC26a5) is the outer hair cell integral membrane motor protein that drives cochlear amplification, and has been described as an obligate tetramer. We studied in real time the delivery of YFP-prestin to the plasma membrane of cells from a tetracycline-inducible cell line. Following the release of temperature block to reinstate trans Golgi network delivery of the integral membrane protein, we measured nonlinear capacitance (NLC) and membrane fluorescence during voltage clamp. Prestin was delivered exponentially to the plasma membrane with a time constant of less than 10 minutes, with both electrical and fluorescence methods showing high temporal correlation. However, based on disparity between estimates of prestin density derived from either fluorescence or NLC, we conclude that sub-tetrameric forms of prestin contribute to our electrical and fluorescence measures. Thus, in agreement with previous observations we find that functional prestin is not an obligate tetramer. PMID:23762468

NMDA Receptor Activity in Circulating Red Blood Cells: Methods of Detection.

PubMed

Makhro, Asya; Kaestner, Lars; Bogdanova, Anna

2017-01-01

Abundance and activity of N-methyl-D-aspartate (NMDA) in circulating red blood cells contributes to the maintenance of intracellular Ca 2+ in these cells and, by doing that, controls red cell volume, membrane stability, and O 2 carrying capacity. Detection of the NMDA receptor activity in red blood cells is challenging as the number of its copies is low and shows substantial cell-to-cell heterogeneity. Receptor abundance is reliably assessed using the radiolabeled antagonist ([ 3 H]MK-801) binding technique. Uptake of Ca 2+ following the NMDA receptor activation is detected in cells loaded with Ca 2+ -sensitive fluorescent dye Fluo-4 AM. Both microfluorescence live-cell imaging and flow cytometry may be used for fluorescence intensity detection. Automated patch clamp is currently used for recording of electric currents triggered by the stimulation of the NMDA receptor. These currents are mediated by the Ca 2+ -sensitive K + (Gardos) channels that open upon Ca 2+ uptake via the active NMDA receptor. Furthermore, K + flux through the Gardos channels induced by the NMDA receptor stimulation in red blood cells may be detected using unidirectional K + ( 86 Rb + ) influx.

Microspectrofluorometry for metabolic control analysis and the study of organelle morphogenesis in cell differentiation and transformation

NASA Astrophysics Data System (ADS)

Hirschberg, Joseph G.; Kohen, Elli; Kohen, Cahide; Pinon, Raul

1994-02-01

Microspectrofluorometry has been used in conjunction with fluorescence micrography for metabolic control analysis in normal and genetically deficient human fibroblasts, as well as human melanoma cells. These studies point to the role of mitochondria as the `cell's policeman' with regard to metabolic control. Cytotoxic agents active on mitochondrial structure and function (i.e. anthralin, azelaic acid) produce an unleashing of extramitochondrial pathways characterized by large and out-of-control NAD(P)H transients elicited by microinjected substrates. An interesting aspect has been the demonstration of an active nuclear energy metabolism, by NAD(P)H fluorescence excited at 365 nm, which may help to link cell bioenergetics to gene expression in the eukaryotes by the use of DNA probes. The metabolic control analysis of cell bioenergetics has been extended to the pathways involved in the cell's handling of cytotoxic agents. Non invasive fluorescence equipment offers possibilities for diagnostics and therapeutics in dermatology. Structure and function studies can be carried out at considerably enhanced resolution and with on-line interpretation by introducing scanning nearfield optics microscopy (SNOM) and real-time interactive parameter experimentation control (RIPEC).

Portable Immune-Assessment System

NASA Technical Reports Server (NTRS)

Pierson, Duane L.; Stowe, Raymond P.; Mishra, Saroj K.

1995-01-01

Portable immune-assessment system developed for use in rapidly identifying infections or contaminated environment. System combines few specific fluorescent reagents for identifying immune-cell dysfunction, toxic substances, buildup of microbial antigens or microbial growth, and potential identification of pathogenic microorganisms using fluorescent microplate reader linked to laptop computer. By using few specific dyes for cell metabolism, DNA/RNA conjugation, specific enzyme activity, or cell constituents, one makes immediate, onsite determination of person's health or of contamination of environment.

Single-Cell Resolution Imaging of Retinal Ganglion Cell Apoptosis In Vivo Using a Cell-Penetrating Caspase-Activatable Peptide Probe

PubMed Central

Qiu, Xudong; Johnson, James R.; Wilson, Bradley S.; Gammon, Seth T.; Piwnica-Worms, David; Barnett, Edward M.

2014-01-01

Peptide probes for imaging retinal ganglion cell (RGC) apoptosis consist of a cell-penetrating peptide targeting moiety and a fluorophore-quencher pair flanking an effector caspase consensus sequence. Using ex vivo fluorescence imaging, we previously validated the capacity of these probes to identify apoptotic RGCs in cell culture and in an in vivo rat model of N-methyl- D-aspartate (NMDA)-induced neurotoxicity. Herein, using TcapQ488, a new probe designed and synthesized for compatibility with clinically-relevant imaging instruments, and real time imaging of a live rat RGC degeneration model, we fully characterized time- and dose-dependent probe activation, signal-to-noise ratios, and probe safety profiles in vivo. Adult rats received intravitreal injections of four NMDA concentrations followed by varying TcapQ488 doses. Fluorescence fundus imaging was performed sequentially in vivo using a confocal scanning laser ophthalmoscope and individual RGCs displaying activated probe were counted and analyzed. Rats also underwent electroretinography following intravitreal injection of probe. In vivo fluorescence fundus imaging revealed distinct single-cell probe activation as an indicator of RGC apoptosis induced by intravitreal NMDA injection that corresponded to the identical cells observed in retinal flat mounts of the same eye. Peak activation of probe in vivo was detected 12 hours post probe injection. Detectable fluorescent RGCs increased with increasing NMDA concentration; sensitivity of detection generally increased with increasing TcapQ488 dose until saturating at 0.387 nmol. Electroretinography following intravitreal injections of TcapQ488 showed no significant difference compared with control injections. We optimized the signal-to-noise ratio of a caspase-activatable cell penetrating peptide probe for quantitative non-invasive detection of RGC apoptosis in vivo. Full characterization of probe performance in this setting creates an important in vivo imaging standard for functional evaluation of future probe analogues and provides a basis for extending this strategy into glaucoma-specific animal models. PMID:24586415

Labeling proteins inside living cells using external fluorophores for microscopy.

PubMed

Teng, Kai Wen; Ishitsuka, Yuji; Ren, Pin; Youn, Yeoan; Deng, Xiang; Ge, Pinghua; Lee, Sang Hak; Belmont, Andrew S; Selvin, Paul R

2016-12-09

Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.

Discovery of potent cytotoxic ortho-aryl chalcones as new scaffold targeting tubulin and mitosis with affinity-based fluorescence.

PubMed

Zhu, Cuige; Zuo, Yinglin; Wang, Ruimin; Liang, Baoxia; Yue, Xin; Wen, Gesi; Shang, Nana; Huang, Lei; Chen, Yu; Du, Jun; Bu, Xianzhang

2014-08-14

A series of new ortho-aryl chalcones have been designed and synthesized. Many of these compounds were found to exhibit significant antiproliferation activity toward a panel of cancer cell lines. Selected compounds show potent cytotoxicity against several drug resistant cell lines including paclitaxel (Taxol) resistant human ovarian carcinoma cells, vincristine resistant human ileocecum carcinoma cells, and doxorubicin resistant human breast carcinoma cells. Further investigation revealed that active analogues could inhibit the microtubule polymerization by binding to colchicine site and thus induce multipolar mitosis, G2/M phase arrest, and apoptosis of cancer cells. Furthermore, affinity-based fluorescence enhancement was observed during the binding of active compounds with tubulin, which greatly facilitated the determination of tubulin binding site of the compounds. Finally, selected compound 26 was found to exhibit obvious in vivo antitumor activity in A549 tumor xenografts model. Our systematic studies implied a new scaffold targeting tubulin and mitosis for novel antitumor drug discovery.

Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

NASA Astrophysics Data System (ADS)

Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

2016-09-01

Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction.

Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B

PubMed Central

Mouchacca, Pierre; Schmitt-Verhulst, Anne-Marie; Boyer, Claude

2013-01-01

To evaluate acquisition and activation of cytolytic functions during immune responses we generated knock in (KI) mice expressing Granzyme B (GZMB) as a fusion protein with red fluorescent tdTomato (GZMB-Tom). As for GZMB in wild type (WT) lymphocytes, GZMB-Tom was absent from naïve CD8 and CD4 T cells in GZMB-Tom-KI mice. It was rapidly induced in most CD8 T cells and in a subpopulation of CD4 T cells in response to stimulation with antibodies to CD3/CD28. A fraction of splenic NK cells expressed GZMB-Tom ex vivo with most becoming positive upon culture in IL-2. GZMB-Tom was present in CTL granules and active as a protease when these degranulated into cognate target cells, as shown with target cells expressing a specific FRET reporter construct. Using T cells from mice expressing GZMB-Tom but lacking perforin, we show that the transfer of fluorescent GZMB-Tom into target cells was dependent on perforin, favoring a role for perforin in delivery of GZMB at the target cells’ plasma membranes. Time-lapse video microscopy showed Ca++ signaling in CTL upon interaction with cognate targets, followed by relocalization of GZMB-Tom-containing granules to the synaptic contact zone. A perforin-dependent step was next visualized by the fluorescence signal from the non-permeant dye TO-PRO-3 at the synaptic cleft, minutes before the labeling of the target cell nucleus, characterizing a previously undescribed synaptic event in CTL cytolysis. Transferred OVA-specific GZMB-Tom-expressing CD8 T cells acquired GZMB-Tom expression in Listeria monocytogenes-OVA infected mice as soon as 48h after infection. These GZMB-Tom positive CD8 T cells localized in the splenic T-zone where they interacted with CD11c positive dendritic cells (DC), as shown by GZMB-Tom granule redistribution to the T/DC contact zone. GZMB-Tom-KI mice thus also provide tools to visualize acquisition and activation of cytolytic function in vivo. PMID:23840635

Fluorescent indicators for Akt/protein kinase B and dynamics of Akt activity visualized in living cells.

PubMed

Sasaki, Kazuki; Sato, Moritoshi; Umezawa, Yoshio

2003-08-15

Akt/protein kinase B (PKB) is a serine/threonine kinase that regulates a variety of cellular responses. To provide information on the spatial and temporal dynamics of Akt/PKB activity, we have developed genetically encoded fluorescent indicators for Akt/PKB. The indicators contain two green fluorescent protein mutants, an Akt/PKB substrate domain, flexible linker sequence, and phosphorylation recognition domain. A phosphorylation of the substrate domain in the indicators caused change in the emission ratio based on fluorescent resonance energy transfer between the two green fluorescent protein mutants. To let the fluorescent indicators behave as endothelial nitric-oxide synthase and Bad, which are endogenous Akt/PKB substrates, they were fused with the Golgi target domain and mitochondria target domain, respectively. The indicators thus colocalized with the endogenous substrates conferred their susceptibilities to phosphorylation by Akt/PKB. We showed that the Golgi-localized indicator responded to the stimulation with 17beta-estradiol (E2) and insulin in endothelial cells. In addition, E2 elicited the phosphorylation of the mitochondria-localized indicator in the endothelial cells, but no phosphorylation was observed by E2 or by insulin of the diffusible indicator that has no targeting domain. The difference in the results with the three indicators suggests that the activated Akt/PKB is localized to subcellular compartments, including the Golgi apparatus and/or mitochondria, rather than diffusing in the cytosol, thereby efficiently phosphorylating its substrate proteins. E2 triggered the phosphorylation of the mitochondria-localized indicator, whereas insulin did not induce this phosphorylation, which suggests that the localization of the activated Akt/PKB to the mitochondria is directed differently between insulin and E2 via distinct mechanisms.

Cell-Permeable, MMP-2 Activatable, Nickel Ferrite and His-Tagged Fusion Protein Self-Assembled Fluorescent Nanoprobe for Tumor Magnetic-Targeting and Imaging.

PubMed

Sun, Lu; Xie, Shuping; Qi, Jing; Liu, Ergang; Liu, Di; Liu, Quan; Chen, Sunhui; He, Huining; Yang, Victor C

2017-11-15

Matrix metalloproteinases (MMPs) activatable imaging probe has been explored for tumor detection. However, activation of the probe is mainly done in the extracellular space without intracellular uptake of the probe for more sensitivity. Although cell-penetrating peptides (CPPs) have been demonstrated to enable intracellular delivery of the imaging probe, they nevertheless encounter off-target delivery of the cargos to normal tissues. Herein, we have developed a dual MMP-2-activatable and tumor cell-permeable magnetic nanoprobe to simultaneously achieve selective and intracellular tumor imaging. This novel imaging probe was constructed by self-assembling a hexahistidine-tagged (His-tagged) fluorescent fusion protein chimera and nickel ferrite nanoparticles via a chelation mechanism. The His-tagged fluorescent protein chimera consisted of a red fluorescent protein mCherry that acted as the fluorophore, the low-molecular-weight protamine peptide as the CPP, and the MMP-2 cleavage sequence fused with the hexahistidine tag, whereas the nickel ferrite nanoparticles functioned as the His-tagged protein binder and also the fluorescent quencher. Both in vitro and in vivo results revealed that this imaging probe would not only remain nonpermeable to normal tissues, thereby offsetting the nonselective cellular uptake, but was also suppressed of fluorescent signals during magnetic tumor-targeting in the circulation, primarily because of the masking of the CPP activity and quenching of the fluorophore by the associated NiFe 2 O 4 nanoparticles. However, these properties were recovered or "turned on" by the action of tumor-associated MMP-2 stimuli, leading to cell penetration of the nanoprobes as well as fluorescence restoration and visualization within the tumor cells. In this regard, the presented tumor-activatable and cell-permeable system deems to be an appealing platform to achieve selective tumor imaging and intracellular protein delivery. Its impact is therefore significant, far-reaching, and wide-spread.

Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells.

PubMed

Ganini, Douglas; Leinisch, Fabian; Kumar, Ashutosh; Jiang, JinJie; Tokar, Erik J; Malone, Christine C; Petrovich, Robert M; Mason, Ronald P

2017-08-01

Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells [1,2]. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems [3]. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as the red fluorescent protein TagRFP [4]. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H 2 O 2 ) per molecule of chromophore [5,6]. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H 2 O 2 [6], this study investigates the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Immature eGFP and TagRFP catalytically generate the free radical superoxide anion (O 2 •- ) and H 2 O 2 in the presence of NADH. Generation of the free radical O 2 •- and H 2 O 2 by eGFP in the presence of NADH affects the gene expression of cells. Many biological pathways are altered, such as a decrease in HIF1α stabilization and activity. The biological pathways altered by eGFP are known to be implicated in the pathophysiology of many diseases associated with oxidative stress; therefore, it is critical that such experiments using fluorescent proteins are validated with alternative methodologies and the results are carefully interpreted. Since cells inevitably experience oxidative stress when fluorescent proteins are expressed, the use of this tool for cell labeling and in vivo cell tracing also requires validation using alternative methodologies. Published by Elsevier B.V.

Red-Emitting Fluorescent Probe for Detection of γ-Glutamyltranspeptidase and Its Application of Real-Time Imaging under Oxidative Stress in Cells and in Vivo.

PubMed

Liu, Feiyan; Wang, Zhen; Wang, Wenli; Luo, Jian-Guang; Kong, Lingyi

2018-06-19

γ-Glutamyltranspeptidase (GGT) plays critical roles in regulating various physiological/pathophysiological processes including the intracellular redox homeostasis. However, an effective fluorescent probe for dissecting the relationships between GGT and oxidative stress in vivo remains largely unexplored. Herein, we present a light-up fluorescent probe (DCDHF-Glu) with long wavelength emission (613 nm) for the highly sensitive and selective detection of GGT using dicyanomethylenedihydrofuran derivative as the fluorescent reporter and γ-glutamyl group as the enzyme-active trigger. DCDHF-Glu is competent to real-time image endogenous GGT in live cells and mice. In particular, DCDHF-Glu enables the direct real-time visualization of the upregulation of GGT under drug-induced oxidative stress in the HepG2 cells and the LO2 cells, as well as in vivo, vividly implying its excellent capacity in elucidation of GGT function in GGT-related biological events.

Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.

PubMed

Liu, Ling; Cheung, Tom H; Charville, Gregory W; Rando, Thomas A

2015-10-01

The prospective isolation of purified stem cell populations has dramatically altered the field of stem cell biology, and it has been a major focus of research across tissues in different organisms. Muscle stem cells (MuSCs) are now among the most intensely studied stem cell populations in mammalian systems, and the prospective isolation of these cells has allowed cellular and molecular characterizations that were not dreamed of a decade ago. In this protocol, we describe how to isolate MuSCs from limb muscles of adult mice by fluorescence-activated cell sorting (FACS). We provide a detailed description of the physical and enzymatic dissociation of mononucleated cells from limb muscles, a procedure that is essential in order to maximize cell yield. We also describe a FACS-based method that is used subsequently to obtain highly pure populations of either quiescent or activated MuSCs (VCAM(+)CD31(-)CD45(-)Sca1(-)). The isolation process takes ∼5-6 h to complete. The protocol also allows for the isolation of endothelial cells, hematopoietic cells and mesenchymal stem cells from muscle tissue.

Biochemical characterization of prostate-specific membrane antigen from canine prostate carcinoma cells.

PubMed

Wu, Lisa Y; Johnson, Jacqueline M; Simmons, Jessica K; Mendes, Desiree E; Geruntho, Jonathan J; Liu, Tiancheng; Dirksen, Wessel P; Rosol, Thomas J; Davis, William C; Berkman, Clifford E

2014-05-01

Prostate-specific membrane antigen (PSMA) remains an important target for diagnostic and therapeutic application for human prostate cancer. Model cell lines have been recently developed to study canine prostate cancer but their PSMA expression and enzymatic activity have not been elucidated. The present study was focused on determining PSMA expression in these model canine cell lines and the use of fluorescent small-molecule enzyme inhibitors to detect canine PSMA expression by flow cytometry. Western blot and RT-PCR were used to determine the transcriptional and translational expression of PSMA on the canine cell lines Leo and Ace-1. An endpoint HPLC-based assay was used to monitor the enzymatic activity of canine PSMA and the potency of enzyme inhibitors. Flow cytometry was used to detect the PSMA expressed on Leo and Ace-1 cells using a fluorescently tagged PSMA enzyme inhibitor. Canine PSMA expression on the Leo cell line was confirmed by Western blot and RT-PCR, the enzyme activity, and flow cytometry. Kinetic parameters Km and Vmax of PSMA enzymatic activity for the synthetic substrate (PABGγG) were determined to be 393 nM and 220 pmol min(-1)  mg protein(-1) , respectively. The inhibitor core 1 and fluorescent inhibitor 2 were found to be potent reversible inhibitors (IC50  = 13.2 and 1.6 nM, respectively) of PSMA expressed on the Leo cell line. Fluorescent labeling of Leo cells demonstrated that the fluorescent PSMA inhibitor 2 can be used for the detection of PSMA-positive canine prostate tumor cells. Expression of PSMA on Ace-1 was low and not detectable by flow cytometry. The results described herein have demonstrated that PSMA is expressed on canine prostate tumor cells and exhibits similar enzymatic characteristics as human PSMA. The findings show that the small molecule enzyme inhibitors currently being studied for use in diagnosis and therapy of human prostate cancer can also be extended to include canine prostate cancer. Importantly, the findings demonstrate that the potential of the inhibitors for use in diagnosis and therapy can be evaluated in an immunocompetent animal model that naturally develops prostate cancer before use in humans. © 2014 Wiley Periodicals, Inc.

Facile synthesis of carbon dot and residual carbon nanobeads: Implications for ion sensing, medicinal and biological applications.

PubMed

Gaddam, Rohit Ranganathan; Mukherjee, Sudip; Punugupati, Neelambaram; Vasudevan, D; Patra, Chitta Ranjan; Narayan, Ramanuj; Vsn Kothapalli, Raju

2017-04-01

Synthesis of carbon dots (Cdots) via chemical route involves disintegration of carbon materials into nano-domains, wherein, after extraction of Cdots, the remaining carbon material is discarded. The present work focuses on studying even the leftover carbon residue namely, carbon nanobeads (CNBs) as an equally important material for applications on par with that of carbon dot. It employs oxidative treatment of carbonised gum olibanum resin (GOR) to produce the carbons namely Cdots and CNBs (as the residue). The Cdots (~5-10nm) exhibit blue-green fluorescence with an optical absorption at ~300nm unlike the CNBs (40-50nm) which fail to exhibit fluorescence. The fluorescence behaviour exhibited by Cdots were utilized for heavy metal ion sensing of Pb 2+ , Hg 2+ and Cd 2+ ions in aqueous media. Interestingly, both Cdots and CNBs are biocompatible to normal cell lines but cytotoxic to cancer cell lines, observed during several in vitro experiments (cell viability assay, cell cycle assay, apoptosis assay, ROS determination assay, caspase-9 activity assay). Additionally, Cdots exhibit bright green fluorescence in B16F10 cells. The Cdots and CNB's demonstrate multifunctional activities (sensor, cellular imaging and cancer therapy) in biomedical applications. Copyright © 2016 Elsevier B.V. All rights reserved.

UV fluorescence excitation spectroscopy as a non-invasive predictor of epidermal proliferation and clinical performance of cosmetic formulations

NASA Astrophysics Data System (ADS)

Maidhof, Robert; Liebel, Frank; Hwang, Cheng; Ruvolo, Eduardo; Lyga, John

2017-02-01

The epidermis is the outermost layer of skin and is composed of cells primarily containing keratin. It consists of about ten layers of living cells (keratinocytes) and ten layers of dead cells (corneocytes). These cells are continually shed from the outside and replaced from the inside in a process called desquamation which is controlled by two biological events - proliferation and differentiation. One method to non-invasively study biological changes in the skin is using fluorescence excitation spectroscopy. Several characteristic excitation-emission peaks occur in skin that have been related to the epidermal and dermal composition. The magnitude of the peak that occurs at 295nm excitation (F295) has been linked to changes in skin proliferation, cell turnover, epidermal thickening, and skin aging. We hypothesize that changes in this fluorescent signal could be used to assess the potential activity of cosmetic anti-aging compounds to deliver a benefit to skin. Previous work with retinol and glycolic acid, two commonly used actives that effect epidermal proliferation and exfoliation, has demonstrated an increase in F295 (attributed to tryptophan excitation fluorescence). In this study we present the results of a placebo controlled study that aims to correlate changes in F295 with biological performance (epidermal thickening and Ki67 expression).

Target-cancer cell specific activatable fluorescence imaging Probes: Rational Design and in vivo Applications

PubMed Central

Kobayashi, Hisataka; Choyke, Peter L.

2010-01-01

CONSPECTUS Conventional imaging methods, such as angiography, computed tomography, magnetic resonance imaging and radionuclide imaging, rely on contrast agents (iodine, gadolinium, radioisotopes) that are “always on”. While these agents have proven clinically useful, they are not sufficiently sensitive because of the inadequate target to background ratio. A unique aspect of optical imaging is that fluorescence probes can be designed to be activatable, i.e. only “turned on” under certain conditions. These probes can be designed to emit signal only after binding a target tissue, greatly increasing sensitivity and specificity in the detection of disease. There are two basic types of activatable fluorescence probes; 1) conventional enzymatically activatable probes, which exist in the quenched state until activated by enzymatic cleavage mostly outside of the cells, and 2) newly designed target-cell specific activatable probes, which are quenched until activated in targeted cells by endolysosomal processing that results when the probe binds specific cell-surface receptors and is subsequently internalized. Herein, we present a review of the rational design and in vivo applications of target-cell specific activatable probes. Designing these probes based on their photo-chemical (e.g. activation strategy), pharmacological (e.g. biodistribution), and biological (e.g. target specificity) properties has recently allowed the rational design and synthesis of target-cell specific activatable fluorescence imaging probes, which can be conjugated to a wide variety of targeting molecules. Several different photo-chemical mechanisms have been utilized, each of which offers a unique capability for probe design. These include: self-quenching, homo- and hetero-fluorescence resonance energy transfer (FRET), H-dimer formation and photon-induced electron transfer (PeT). In addition, the repertoire is further expanded by the option for reversibility or irreversibility of the signal emitted using the aforementioned mechanisms. Given the wide range of photochemical mechanisms and properties, target-cell specific activatable probes possess considerable flexibility and can be adapted to specific diagnostic needs. Herein, we summarize the chemical, pharmacological, and biological basis of target-cell specific activatable imaging probes and discuss methods to successfully design such target-cell specific activatable probes for in vivo cancer imaging. PMID:21062101

[Effects of algicidal bacterium BS03 (Microbulbifer sp.) on the growth and antioxidant systems of Alexandrium tamarense].

PubMed

Fu, Lijun; Li, Dong; Wu, Chengji; Zheng, Tianling

2012-06-04

We studied the algicidal mechanism of extracellular substances of algicidal bacteria strain BS03 (Microbulbifer sp.) on photosynthetic characteristics, antioxident enzyme system and cysteine-dependent aspartate specific protease-3 (Caspase-3) of Alexandrium tamarense. We tested photosynthetic pigments, chlorophyll fluorescence efficiency, antioxidant systems and caspase-3 activity in the algae cells treated with 0.5%, 1.0% , 1.5% and 2.0% BS03 cell-free filtrate after 12, 24, 36 and 48 h. (1) The chlorophyll-a and chlorophyll fluorescence efficiency Fv/Fm decreased with the increase of BS03 cell-free filtrate and treatment time. Carotenoids contents of A. tamarense cells treated with low BS03 (0.5% and 1.0%) cell-free filtrate were higher than the control. (2) Antioxident enzyme activities varied as treatment time and concentration. Malodialdehyde (MDA) contents increased significantly with BS03 cell-free filtrate treatment. (3) Caspase-3 protease activities of algal cells increased by BS03 cell-free filtrate. BS03 inhibited the photosynthesis whereas enhanced the lipid peroxidation of the cellular membrane of Alexandrium tamarense, indicating its algicidal activity.

Imaging of Ras/Raf activity induced by low energy laser irradiation in living cell using FRET

NASA Astrophysics Data System (ADS)

Wang, Fang; Chen, Tong-Sheng; Xing, Da

2005-01-01

Ras/Raf signaling pathway is an important signaling pathway that governs cell proliferation, differential and apoptosis. Low-energy laser irradiation (LELI) was found to modulate various processes. Generally, cell proliferation is induced by low doses LELI and apoptosis is induced by high doses LELI. Mechanism of biological effect of LELI has not been clear. Recently, activation of MEK (mitogen-activated protein kinase) and ERK (extracellular-signal-regulated kinase), which are downstream protein kinases of Ras/Raf, are observed during LELI-induced cell proliferation by immunoprecipitation and western blot analysis. RaichuRas reporter consisting of fusions of H-ras, the Ras-binding domain of Raf (RafRBD), a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP). Therefore, intramolecular binding of GTP-Ras to RafRBD brings CFP close to YFP and increases FRET between CFP and YFP. Human lung adenocarcinoma cell line (ASTC-a-1) was transfected with the plasmid (pRaichuRas) and then treated with LELI at dose of 60J/cm2. Effect of LELI on Ras/Raf in physiological condition of living cells was observed by fluorescence resonance energy transfer (FRET) technique during lung adenocarcinoma cell apoptosis induced by high dose (60J/cm2) LELI. Experimental results showed that after high dose LELI treatment, the binding of Ras and Raf decreases obviously, Ras/Raf signaling pathway deregulates and cell apoptosis occurs.

Efficiency of fluorescence in situ hybridization for bacterial cell identification in temporary river sediments with contrasting water content.

PubMed

Fazi, Stefano; Amalfitano, Stefano; Pizzetti, Ilaria; Pernthaler, Jakob

2007-09-01

We studied the efficiency of two hybridization techniques for the analysis of benthic bacterial community composition under varying sediment water content. Microcosms were set up with sediments from four European temporary rivers. Wet sediments were dried, and dry sediments were artificially rewetted. The percentage of bacterial cells detected by fluorescence in situ hybridization with fluorescently monolabeled probes (FISH) significantly increased from dry to wet sediments, showing a positive correlation with the community activity measured via incorporation of (3)H leucine. FISH and signal amplification by catalyzed reporter deposition (CARD-FISH) could significantly better detect cells with low activity in dried sediments. Through the application of an optimized cell permeabilization protocol, the percentage of hybridized cells by CARD-FISH showed comparable values in dry and wet conditions. This approach was unrelated to (3)H leucine incorporation rates. Moreover, the optimized protocol allowed a significantly better visualization of Gram-positive Actinobacteria in the studied samples. CARD-FISH is, therefore, proposed as an effective technique to compare bacterial communities residing in sediments with contrasting water content, irrespective of differences in the activity state of target cells. Considering the increasing frequencies of flood and drought cycles in European temporary rivers, our approach may help to better understand the dynamics of microbial communities in such systems.

Binding effect of fluorescence labeled glycyrrhetinic acid with GA receptors in hepatocellular carcinoma cells.

PubMed

Sun, Yu-Qi; Dai, Chun-Mei; Zheng, Yan; Shi, Shu-Dan; Hu, Hai-Yang; Chen, Da-Wei

2017-11-01

Glycyrrhetinic acid (GA) is a natural active component from licorice, which is broadly used in traditional Chinese medicine. Lots of glycyrrhetinic acid receptors (GA-R) are proved to locate on the surface of liver cells. Many reports about the hepatocellular carcinoma (HCC) treatment were dependent on GA modified carriers. However, the reality of GA-R in HCC cells was not clear. In this paper, 18β-glycyrrhetinic acid (18β-GA) was labeled with fluorescence (FITC) by chemical synthesis. Together with the binding effect of fluorescence labeled glycyrrhetinic acid (FITC-GA), the competitive action of 18β-GA with GA-R was investigated in HCC cells. The results showed that in HepG2 cells, 18β-GA and FITC-GA presented similar cytotoxicity. The specific binding saturation of GA showed the dissociation constant (K d ) was 7.457±2.122pmol/L and the maximum binding counts (B max ) was 2.385±0.175pmol/2.5×10 6 cells, respectively. FITC-GA bound to cytomembrane specifically and 18β-GA competed to bind the sites significantly in HepG2 cells. Therefore, there is binding effect between fluorescence labeled GA and GA-R. The GA-R on HCC cells is confirmed as expected, which provides a useful reference of active target modified by GA and a novel approach for receptors and ligands study. Copyright © 2017 Elsevier Inc. All rights reserved.

Microplate-compatible total internal reflection fluorescence microscopy for receptor pharmacology

NASA Astrophysics Data System (ADS)

Chen, Minghan; Zaytseva, Natalya V.; Wu, Qi; Li, Min; Fang, Ye

2013-05-01

We report the use of total internal reflection fluorescence (TIRF) microscopy for analyzing receptor pharmacology and the development of a microplate-compatible TIRF imaging system. Using stably expressed green fluorescence protein tagged β2-adrenergic receptor as the reporter, we found that the activation of different receptors results in distinct kinetic signatures of the TIRF intensity of cells. These TIRF signatures closely resemble the characteristics of their respective label-free dynamic mass redistribution signals in the same cells. This suggests that TIRF in microplate can be used for profiling and screening drugs.

Poly β-cyclodextrin/TPdye nanomicelle-based two-photon nanoprobe for caspase-3 activation imaging in live cells and tissues.

PubMed

Yan, Huijuan; He, Leiliang; Zhao, Wenjie; Li, Jishan; Xiao, Yue; Yang, Ronghua; Tan, Weihong

2014-11-18

Two-photon excitation (TPE) with near-infrared (NIR) photons as the excitation source has important advantages over conventional one-photon excitation (OPE) in the field of biomedical imaging. β-cyclodextrin polymer (βCDP)-based two-photon absorption (TPA) fluorescent nanomicelle exhibits desirable two-photon-sensitized fluorescence properties, high photostability, high cell-permeability and excellent biocompatibility. By combination of the nanostructured two-photon dye (TPdye)/βCDP nanomicelle with the TPE technique, herein we have designed a TPdye/βCDP nanomicelle-based TPA fluorescent nanoconjugate for enzymatic activity assay in biological fluids, live cells and tissues. This sensing system is composed of a trans-4-[p-(N,N-diethylamino)styryl]-N-methylpyridinium iodide (DEASPI)/βCDP nanomicelle as TPA fluorophore and carrier vehicle for delivery of a specific peptide sequence to live cell through fast endocytosis, and an adamantine (Ad)-GRRRDEVDK-BHQ2 (black hole quencher 2) peptide (denoted as Ad-DEVD-BHQ2) anchored on the DEASPI/βCDP nanomicelle's surface to form TPA DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate by the βCD/Ad host-guest inclusion strategy. Successful in vitro and in vivo enzymatic activities assay of caspase-3 was demonstrated with this sensing strategy. Our results reveal that this DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate not only is a robust, sensitive and selective sensor for quantitative assay of caspase-3 in the complex biological environment but also can be efficiently delivered into live cells as well as tissues and act as a "signal-on" fluorescent biosensor for specific, high-contrast imaging of enzymatic activities. This DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate provides a new opportunity to screen enzyme inhibitors and evaluate the apoptosis-associated disease progression. Moreover, our design also provides a methodology model scheme for development of future TPdye/βCDP nanomicelle-based two-photon fluorescent probes for in vitro or in vivo determination of biological or biologically relevant species.

Differentiating quiescent cancer cell populations in heterogeneous samples with fluorescence lifetime imaging

NASA Astrophysics Data System (ADS)

Heaster, Tiffany M.; Walsh, Alex J.; Skala, Melissa C.

2016-03-01

Measurement of relative fluorescence intensities of NAD(P)H and FAD with fluorescence lifetime imaging (FLIM) allows metabolic characterization of cancerous populations and correlation to treatment response. However, quiescent populations of cancer cells introduce heterogeneity to the tumor and exhibit resistance to standard therapies, requiring a better understanding of this influence on treatment outcome. Significant differences were observed between proliferating and quiescent cell populations upon comparison of respective redox ratios (p<0.05) and FAD lifetimes (p<0.05) across monolayers and in mixed samples. These results demonstrate that metabolic activity may function as a marker for separation and characterization of proliferating and quiescent cancer cells within mixed samples, contributing to comprehensive investigation of heterogeneity-dependent drug resistance.

Fluorescence-based assay as a new screening tool for toxic chemicals

PubMed Central

Moczko, Ewa; Mirkes, Evgeny M.; Cáceres, César; Gorban, Alexander N.; Piletsky, Sergey

2016-01-01

Our study involves development of fluorescent cell-based diagnostic assay as a new approach in high-throughput screening method. This highly sensitive optical assay operates similarly to e-noses and e-tongues which combine semi-specific sensors and multivariate data analysis for monitoring biochemical processes. The optical assay consists of a mixture of environmental-sensitive fluorescent dyes and human skin cells that generate fluorescence spectra patterns distinctive for particular physico-chemical and physiological conditions. Using chemometric techniques the optical signal is processed providing qualitative information about analytical characteristics of the samples. This integrated approach has been successfully applied (with sensitivity of 93% and specificity of 97%) in assessing whether particular chemical agents are irritating or not for human skin. It has several advantages compared with traditional biochemical or biological assays and can impact the new way of high-throughput screening and understanding cell activity. It also can provide reliable and reproducible method for assessing a risk of exposing people to different harmful substances, identification active compounds in toxicity screening and safety assessment of drugs, cosmetic or their specific ingredients. PMID:27653274

Fluorescence-based assay as a new screening tool for toxic chemicals.

PubMed

Moczko, Ewa; Mirkes, Evgeny M; Cáceres, César; Gorban, Alexander N; Piletsky, Sergey

2016-09-22

Our study involves development of fluorescent cell-based diagnostic assay as a new approach in high-throughput screening method. This highly sensitive optical assay operates similarly to e-noses and e-tongues which combine semi-specific sensors and multivariate data analysis for monitoring biochemical processes. The optical assay consists of a mixture of environmental-sensitive fluorescent dyes and human skin cells that generate fluorescence spectra patterns distinctive for particular physico-chemical and physiological conditions. Using chemometric techniques the optical signal is processed providing qualitative information about analytical characteristics of the samples. This integrated approach has been successfully applied (with sensitivity of 93% and specificity of 97%) in assessing whether particular chemical agents are irritating or not for human skin. It has several advantages compared with traditional biochemical or biological assays and can impact the new way of high-throughput screening and understanding cell activity. It also can provide reliable and reproducible method for assessing a risk of exposing people to different harmful substances, identification active compounds in toxicity screening and safety assessment of drugs, cosmetic or their specific ingredients.

Fluorescence-based assay as a new screening tool for toxic chemicals

NASA Astrophysics Data System (ADS)

Moczko, Ewa; Mirkes, Evgeny M.; Cáceres, César; Gorban, Alexander N.; Piletsky, Sergey

2016-09-01

Our study involves development of fluorescent cell-based diagnostic assay as a new approach in high-throughput screening method. This highly sensitive optical assay operates similarly to e-noses and e-tongues which combine semi-specific sensors and multivariate data analysis for monitoring biochemical processes. The optical assay consists of a mixture of environmental-sensitive fluorescent dyes and human skin cells that generate fluorescence spectra patterns distinctive for particular physico-chemical and physiological conditions. Using chemometric techniques the optical signal is processed providing qualitative information about analytical characteristics of the samples. This integrated approach has been successfully applied (with sensitivity of 93% and specificity of 97%) in assessing whether particular chemical agents are irritating or not for human skin. It has several advantages compared with traditional biochemical or biological assays and can impact the new way of high-throughput screening and understanding cell activity. It also can provide reliable and reproducible method for assessing a risk of exposing people to different harmful substances, identification active compounds in toxicity screening and safety assessment of drugs, cosmetic or their specific ingredients.

Imaging of activated caspase-3 in living cell by fluorescence resonance energy transfer during photosensitization-induced apoptosis

NASA Astrophysics Data System (ADS)

Wu, Yunxia; Xing, Da; Chen, Qun; Tang, Yonghong

2005-01-01

Photodynamic therapy (PDT) is a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in cell, and activation of caspase-3 is considered to be the final step in many apoptosis pathways. The changes of caspase-3 activation in cell during TNFα- and photodynamic therapy-induced apoptosis was measured by fluorescence resonance energy transfer (FRET) analysis. FRET probe consisting of fusions of an enhanced cyan fluorescent protein (ECFP), Venus and a linker peptide containing the caspase-3 cleavage sequence DEVD was utilized. Therefore, activated caspase-3 cleaved the linker peptide of FRET probe and disrupted the FRET signal. Human lung adenocarcinoma cell line (ASTC-a-1) were stably transfected with the plasmid (ECFP-DEVD-Venus) and then were treated by TNF-α and PDT, respectively. Experimental results indicated that caspase-3 activation resulted in cleavage of linker peptide and subsequent disruption of the FRET signal during TNFα- and photodynamic therapy-induced apoptosis, and that the activation of caspase-3 induced by photodynamic therapy was faster than that induce by TNF-α. The study supports that using FRET technique and different recombinant substrates as FRET probes could be used to detect the process of PDT-induced apoptosis and provide a new means to investigate apoptotic mechanism of PDT.

'Green mice' display limitations in enhanced green fluorescent protein expression in retina and optic nerve cells.

PubMed

Caminos, Elena; Vaquero, Cecilia F; García-Olmo, Dolores C

2014-12-01

Characterization of retinal cells, cell transplants and gene therapies may be helped by pre-labeled retinal cells, such as those transfected with vectors for green fluorescent protein expression. The aim of this study was to analyze retinal cells and optic nerve components from transgenic green mice (GM) with the 'enhanced' green fluorescent protein (EGFP) gene under the control of the CAG promoter (a chicken β-actin promoter and a cytomegalovirus enhancer). The structural analysis and electroretinography recordings showed a normal, healthy retina. Surprisingly, EGFP expression was not ubiquitously located in the retina and optic nerve. Epithelial cells, photoreceptors and bipolar cells presented high green fluorescence levels. In contrast, horizontal cells, specific amacrine cells and ganglion cells exhibited a null EGFP expression level. The synaptic terminals of rod bipolar cells displayed a high green fluorescence level when animals were kept in the dark. Immature retinas exhibited different EGFP expression patterns to those noted in adults. Axons and glial cells in the optic nerve revealed a specific regional EGFP expression pattern, which correlated with the presence of myelin. These results suggest that EGFP expression might be related to the activity of both the CAG promoter and β-actin in mature retinal neurons and oligodendrocytes. Moreover, EGFP expression might be regulated by light in both immature and adult animals. Since GM are used in numerous retina bioassays, it is essential to know the differential EGFP expression in order to select cells of interest for each study.

Microspectroscopic Study of Liposome-to-cell Interaction Revealed by Förster Resonance Energy Transfer.

PubMed

Yefimova, Svetlana L; Kurilchenko, Irina Yu; Tkacheva, Tatyana N; Kavok, Nataliya S; Todor, Igor N; Lukianova, Nataliya Yu; Chekhun, Vasyl F; Malyukin, Yuriy V

2014-03-01

We report the Förster resonance energy transfer (FRET)-labeling of liposomal vesicles as an effective approach to study in dynamics the interaction of liposomes with living cells of different types (rat hepatocytes, rat bone marrow, mouse fibroblast-like cells and human breast cancer cells) and cell organelles (hepatocyte nuclei). The in vitro experiments were performed using fluorescent microspectroscopic technique. Two fluorescent dyes (DiO as the energy donor and DiI as an acceptor) were preloaded in lipid bilayers of phosphatidylcholine liposomes that ensures the necessary distance between the dyes for effective FRET. The change in time of the donor and acceptor relative fluorescence intensities was used to visualize and trace the liposome-to-cell interaction. We show that FRET-labeling of liposome vesicles allows one to reveal the differences in efficiency and dynamics of these interactions, which are associated with composition, fluidity, and metabolic activity of cell plasma membranes.

Early T-cell activation biophysics

PubMed Central

Henry, Nelly; Hivroz, Claire

2009-01-01

The T-cell is one of the main players in the mammalian immune response. It ensures antigen recognition at the surface of antigen-presenting cells in a complex and highly sensitive and specific process, in which the encounter of the T-cell receptor with the agonist peptide associated with the major histocompatibility complex triggers T-cell activation. While signaling pathways have been elucidated in increasing detail, the mechanism of TCR triggering remains highly controversial despite active research published in the past 10 years. In this paper, we present a short overview of pending questions on critical initial events associated with T-cell triggering. In particular, we examine biophysical approaches already in use, as well as future directions. We suggest that the most recent advances in fluorescence super-resolution imaging, coupled with the new classes of genetic fluorescent probes, will play an important role in elucidation of the T-cell triggering mechanism. Beyond this aspect, we predict that exploration of mechanical cues in the triggering process will provide new clues leading to clarification of the entire mechanism. PMID:20514131

Nano-fEM: protein localization using photo-activated localization microscopy and electron microscopy.

PubMed

Watanabe, Shigeki; Richards, Jackson; Hollopeter, Gunther; Hobson, Robert J; Davis, Wayne M; Jorgensen, Erik M

2012-12-03

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated (1-3). However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated (4-7). However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot (8-10). We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

Impact of metal binding on the antitumor activity and cellular imaging of a metal chelator cationic imidazopyridine derivative.

PubMed

Roy, Mithun; Chakravarthi, Balabhadrapatruni V S K; Jayabaskaran, Chelliah; Karande, Anjali A; Chakravarty, Akhil R

2011-05-14

A new water soluble cationic imidazopyridine species, viz. (1E)-1-((pyridin-2-yl)methyleneamino)-3-(3-(pyridin-2-yl)imidazo[1,5-a]pyridin-2(3H)-yl)propan-2-ol (1), as a metal chelator is prepared as its PF(6) salt and characterized. Compound 1 shows fluorescence at 438 nm on excitation at 342 nm in Tris-HCl buffer giving a fluorescence quantum yield (φ) of 0.105 and a life-time of 5.4 ns. Compound 1, as an avid DNA minor groove binder, shows pUC19 DNA cleavage activity in UV-A light of 365 nm forming singlet oxygen species in a type-II pathway. The photonuclease potential of 1 gets enhanced in the presence of Fe(2+), Cu(2+) or Zn(2+). Compound 1 itself displays anticancer activity in HeLa, HepG2 and Jurkat cells with an enhancement on addition of the metal ions. Photodynamic effect of 1 at 365 nm also gets enhanced in the presence of Fe(2+) and Zn(2+). Fluorescence-based cell cycle analysis shows a significant dead cell population in the sub-G1 phase of the cell cycle suggesting apoptosis via ROS generation. A significant change in the nuclear morphology is observed from Hoechst 33258 and an acridine orange/ethidium bromide (AO/EB) dual nuclear staining suggesting apoptosis in cells when treated with 1 alone or in the presence of the metal ions. Apoptosis is found to be caspase-dependent. Fluorescence imaging to monitor the distribution of 1 in cells shows that 1 in the presence of metal ions accumulates predominantly in the cytoplasm. Enhanced uptake of 1 into the cells within 12 h is observed in the presence of Fe(2+) and Zn(2+).

Evaluation of nanoparticles as endocytic tracers in cellular microbiology

NASA Astrophysics Data System (ADS)

Zhang, Yuying; Hensel, Michael

2013-09-01

The study of pathogen interactions with eukaryotic host cells requires the introduction of fluorescent probes to visualize processes such as endocytosis, intracellular transport or host cell manipulation by the pathogen. Here, three types of fluorescent nanoparticles (NPs), i.e. Rhodamine-labeled polymethacrylate (PMA) NPs, silica NPs and gold NPs, were employed to label the host cellular endolysosomal system and monitor manipulations by the pathogen Salmonella enterica. Using live cell imaging, we investigated the performance of NPs in cellular uptake, labeling of endocytic vesicles and lysosomes, as well as interaction with the pathogen. We show that fluorescent gold and silica, but not PMA NPs appropriately label host cell structures and efficiently track rearrangements of the host endosomal system by the activities of intracellular Salmonella. Silica NPs slightly aggregated and located in Salmonella-induced compartments as isolated dots, while gold NPs distributed uniformly inside such structures. Both silica and gold NPs exhibited no adverse impact on either host cells or pathogens, and are versatile tools for infection biology.The study of pathogen interactions with eukaryotic host cells requires the introduction of fluorescent probes to visualize processes such as endocytosis, intracellular transport or host cell manipulation by the pathogen. Here, three types of fluorescent nanoparticles (NPs), i.e. Rhodamine-labeled polymethacrylate (PMA) NPs, silica NPs and gold NPs, were employed to label the host cellular endolysosomal system and monitor manipulations by the pathogen Salmonella enterica. Using live cell imaging, we investigated the performance of NPs in cellular uptake, labeling of endocytic vesicles and lysosomes, as well as interaction with the pathogen. We show that fluorescent gold and silica, but not PMA NPs appropriately label host cell structures and efficiently track rearrangements of the host endosomal system by the activities of intracellular Salmonella. Silica NPs slightly aggregated and located in Salmonella-induced compartments as isolated dots, while gold NPs distributed uniformly inside such structures. Both silica and gold NPs exhibited no adverse impact on either host cells or pathogens, and are versatile tools for infection biology. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr01550e

Selective Activation of N,N'-Diacyl Rhodamine Pro-fluorophores Paired with Releasing Enzyme, Porcine Liver Esterase (PLE).

PubMed

Abney, Kristopher K; Ramos-Hunter, Susan J; Romaine, Ian M; Godwin, J Shawn; Sulikowski, Gary A; Weaver, Charles David

2018-04-21

This study reports the synthesis and testing of a family of rhodamine pro-fluorophores and an enzyme capable of converting pro-fluorophores to Rhodamine 110. We prepared a library of simple N,N'-diacyl rhodamines and investigated Porcine Liver Esterase (PLE) as an enzyme to activate rhodamine-based pro-fluorophores. A PLE-expressing cell line generated an increase in fluorescence rapidly upon pro-fluorophore addition demonstrating the rhodamine pro-fluorophores are readily taken up and fluorescent upon PLE-mediated release. Rhodamine pro-fluorophore amides trifluoroacetamide (TFAm) and proponamide (PAm) appeared to be the best substrates using a cell-based assay using PLE expressing HEK293. Our pro-fluorophore series showed diffusion into live cells and resisted endogenous hydrolysis. The use of our engineered cell line containing the exogenous enzyme PLE demonstrated the rigorousness of amide masking when compared to cells not containing PLE. This simple and selective pro-fluorophore rhodamine pair with PLE offers the potential to be used in vitro and in vivo fluorescence based assays. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression of GFP under the control of the RNA helicase VASA permits fluorescence-activated cell sorting isolation of human primordial germ cells.

PubMed

Tilgner, Katarzyna; Atkinson, Stuart P; Yung, Sun; Golebiewska, Anna; Stojkovic, Miodrag; Moreno, Ruben; Lako, Majlinda; Armstrong, Lyle

2010-01-01

The isolation of significant numbers of human primordial germ cells at several developmental stages is important for investigations of the mechanisms by which they are able to undergo epigenetic reprogramming. Only small numbers of these cells can be obtained from embryos of appropriate developmental stages, so the differentiation of human embryonic stem cells is essential to obtain sufficient numbers of primordial germ cells to permit epigenetic examination. Despite progress in the enrichment of human primordial germ cells using fluorescence-activated cell sorting (FACS), there is still no definitive marker of the germ cell phenotype. Expression of the widely conserved RNA helicase VASA is restricted to germline cells, but in contrast to species such as Mus musculus in which reporter constructs expressing green fluorescent protein (GFP) under the control of a Vasa promoter have been developed, such reporter systems are lacking in human in vitro models. We report here the generation and characterization of human embryonic stem cell lines stably carrying a VASA-pEGFP-1 reporter construct that expresses GFP in a population of differentiating human embryonic stem cells that show expression of characteristic markers of primordial germ cells. This population shows a different pattern of chromatin modifications to those obtained by FACS enrichment of Stage Specific Antigen one expressing cells in our previous publication.

Mini G protein probes for active G protein-coupled receptors (GPCRs) in live cells.

PubMed

Wan, Qingwen; Okashah, Najeah; Inoue, Asuka; Nehmé, Rony; Carpenter, Byron; Tate, Christopher G; Lambert, Nevin A

2018-05-11

G protein-coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase-mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus, and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells and should be useful for discovering and characterizing G protein subtype-biased ligands. © 2018 Wan et al.

Cytotoxicity and DNA cleavage with core-shell nanocomposites functionalized by a KH domain DNA binding peptide

NASA Astrophysics Data System (ADS)

Bazak, Remon; Ressl, Jan; Raha, Sumita; Doty, Caroline; Liu, William; Wanzer, Beau; Salam, Seddik Abdel; Elwany, Samy; Paunesku, Tatjana; Woloschak, Gayle E.

2013-11-01

A nanoconjugate was composed of metal oxide nanoparticles decorated with peptides and fluorescent dye and tested for DNA cleavage following UV light activation. The peptide design was based on a DNA binding domain, the so called KH domain of the hnRNPK protein. This ``KH peptide'' enabled cellular uptake of nanoconjugates and their entry into cell nuclei. The control nanoconjugate carried no peptide; it consisted only of the metal oxide nanoparticle prepared as Fe3O4@TiO2 nanocomposite and the fluorescent dye alizarin red S. These components of either construct are responsible for nanoconjugate activation by UV light and the resultant production of reactive oxygen species (ROS). Production of ROS at different subcellular locations causes damage to different components of cells: only nanoconjugates inside cell nuclei can be expected to cause DNA cleavage. Degradation of cellular DNA with KH peptide decorated nanoconjugates exceeded the DNA damage obtained from control, no-peptide nanoconjugate counterparts. Moreover, caspase activation and cell death were more extensive in the same cells.A nanoconjugate was composed of metal oxide nanoparticles decorated with peptides and fluorescent dye and tested for DNA cleavage following UV light activation. The peptide design was based on a DNA binding domain, the so called KH domain of the hnRNPK protein. This ``KH peptide'' enabled cellular uptake of nanoconjugates and their entry into cell nuclei. The control nanoconjugate carried no peptide; it consisted only of the metal oxide nanoparticle prepared as Fe3O4@TiO2 nanocomposite and the fluorescent dye alizarin red S. These components of either construct are responsible for nanoconjugate activation by UV light and the resultant production of reactive oxygen species (ROS). Production of ROS at different subcellular locations causes damage to different components of cells: only nanoconjugates inside cell nuclei can be expected to cause DNA cleavage. Degradation of cellular DNA with KH peptide decorated nanoconjugates exceeded the DNA damage obtained from control, no-peptide nanoconjugate counterparts. Moreover, caspase activation and cell death were more extensive in the same cells. Electronic supplementary information (ESI) available: http://janus.northwestern.edu/wololab/auxiliary/supplementary_data_2013.docx. See DOI: 10.1039/c3nr02203j

P2X antagonists inhibit styryl dye entry into hair cells.

PubMed

Crumling, M A; Tong, M; Aschenbach, K L; Liu, L Qian; Pipitone, C M; Duncan, R K

2009-07-21

The styryl pyridinium dyes, FM1-43 and AM1-43, are fluorescent molecules that can permeate the mechanotransduction channels of hair cells, the sensory receptors of the inner ear. When these dyes are applied to hair cells, they enter the cytoplasm rapidly, resulting in a readily detectable intracellular fluorescence that is often used as a molecular indication of mechanotransduction channel activity. However, such dyes can also permeate the ATP receptor, P2X(2). Therefore, we explored the contribution of P2X receptors to the loading of hair cells with AM1-43. The chick inner ear was found to express P2X receptors and to release ATP, similar to the inner ear of mammals, allowing for the endogenous stimulation of P2X receptors. The involvement of these receptors was evaluated pharmacologically, by exposing the sensory epithelium of the chick inner ear to 5 microM AM1-43 under different experimental conditions and measuring the fluorescence in hair cells after fixation of the tissue. Pre-exposure of the tissue to 5 mM EGTA for 15 min, which should eliminate most of the gating "tip links" of the mechanotransduction channels, deceased fluorescence by only 44%. In contrast, P2X receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid [PPADS], suramin, 2',3'-O-(2,4,6-trinitrophenyl) ATP [TNP-ATP], and d-tubocurarine) had greater effects on dye loading. PPADS, suramin, and TNP-ATP all decreased intracellular AM1-43 fluorescence in hair cells by at least 69% when applied at a concentration of 100 microM. The difference between d-tubocurarine-treated and control fluorescence was statistically insignificant when d-tubocurarine was applied at a concentration that blocks the mechanotransduction channel (200 microM). At a concentration that also blocks P2X(2) receptors (2 mM), d-tubocurarine decreased dye loading by 72%. From these experiments, it appears that AM1-43 can enter hair cells through endogenously activated P2X receptors. Thus, the contribution of P2X receptors to dye entry should be considered when using styryl pyridinium dyes to detect hair cell mechanotransduction channel activity, especially in the absence of explicit mechanical stimulation of stereocilia.

Coupling Binding to Catalysis: Using Yeast Cell Surface Display to Select Enzymatic Activities.

PubMed

Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

2015-01-01

We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence-activated cell sorting.

Investigation of selective induction of breast cancer cells to death with treatment of plasma-activated medium

NASA Astrophysics Data System (ADS)

Hashizume, Hiroshi; Tanaka, Hiromasa; Nakamura, Kae; Kano, Hiroyuki; Ishikawa, Kenji; Kikkawa, Fumitaka; Mizuno, Masaaki; Hori, Masaru

2015-09-01

The applications of plasma in medicine have much attention. We previously showed that plasma-activated medium (PAM) induced glioblastoma cells to apoptosis. However, it has not been elucidated the selectivity of PAM in detail. In this study, we investigated the selective effect of PAM on the death of human breast normal and cancer cells, MCF10A and MCF7, respectively, and observed the selective death with fluorescent microscopy. For the investigation of cell viability with PAM treatment, we prepared various PAMs according to the strengths, and treated each of cells with PAMs. Week PAM treatment only decreased the viability of MCF7 cells, while strong PAM treatment significantly affected both viabilities of MCF7 and MCF10A cells. For the fluorescent observation, we prepared the mixture of MCF7 and fluorescent-probed MCF10A cells, and seeded them. After the treatment of PAMs, the images showed that only MCF7 cells damaged in the mixture with week PAM treatment. These results suggested that a specific range existed with the selective effect in the strength of PAM. This work was partly supported by a Grant-in-Aid for Scientific Research on Innovative Areas ``Plasma Medical Innovation'' Grant No. 24108002 and 24108008 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Rapid Isolation of Antibody from a Synthetic Human Antibody Library by Repeated Fluorescence-Activated Cell Sorting (FACS)

PubMed Central

Yim, Sung Sun; Bang, Hyun Bae; Kim, Young Hwan; Lee, Yong Jae; Jeong, Gu Min; Jeong, Ki Jun

2014-01-01

Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS). First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv) was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show KD values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼106). These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required. PMID:25303314

Isolation of circulating tumor cells by immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS) for molecular profiling.

PubMed

Magbanua, Mark Jesus M; Park, John W

2013-12-01

Circulating tumor cells (CTCs) are cells shed by the primary tumor into the blood stream capable of initiating distant metastasis. In the past decade, numerous assays have been developed to reliably detect these extremely rare cells. However, methods for purification of CTCs with little or no contamination of normal blood cells for molecular profiling are limited. We have developed a novel protocol to isolate CTCs by combining immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS). The two-part assay includes (1) immunomagnetic capture using magnetic beads conjugated to monoclonal antibody against an epithelial cell adhesion marker (EpCAM) to enrich for tumor cells; and (2) FACS analysis using EpCAM to purify tumor cells away from mononuclear cells of hematopoietic lineage. Downstream molecular analyses of single and pooled cells confirmed the isolation of highly pure CTCs with characteristics typical that of malignant cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Active-site-matched fluorescent probes for rapid and direct detection of vicinal-sulfydryl-containing peptides/proteins in living cells.

PubMed

Pan, Xiaohong; Liang, Ziye; Li, Jing; Wang, Shanshan; Kong, Fanpeng; Xu, Kehua; Tang, Bo

2015-01-26

Vicinal-sulfydryl-containing peptides/proteins (VSPPs) play a crucial role in human pathologies. Fluorescent probes that are capable of detecting intracellular VSPPs in vivo would be useful tools to explore the mechanisms of some diseases. In this study, by regulating the spatial separation of two maleimide groups in a fluorescent dye to match that of two active cysteine residues contained in the conserved amino acid sequence (-CGPC-) of human thioredoxin, two active-site-matched fluorescent probes, o-Dm-Ac and m-Dm-Ac, were developed for real-time imaging of VSPPs in living cells. As a result, the two probes can rapidly respond to small peptide models and reduced proteins, such as WCGPCK (W-6), WCGGPCK (W-7), and WCGGGPCK (W-8), reduced bovine serum albumin (rBSA), and reduced thioredoxin (rTrx). Moreover, o-Dm-Ac displays a higher binding sensitivity with the above-mentioned peptides and proteins, especially with W-7 and rTrx. Furthermore, o-Dm-Ac was successfully used to rapidly and directly detect VSPPs both in vitro and in living cells. Thus, a novel probe-design strategy was proposed and the synthesized probe applied successfully in imaging of target proteins in situ. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

CD81 Controls Sustained T Cell Activation Signaling and Defines the Maturation Stages of Cognate Immunological Synapses

PubMed Central

Rocha-Perugini, V.; Zamai, M.; González-Granado, J. M.; Barreiro, O.; Tejera, E.; Yañez-Mó, M.; Caiolfa, V. R.

2013-01-01

In this study, we investigated the dynamics of the molecular interactions of tetraspanin CD81 in T lymphocytes, and we show that CD81 controls the organization of the immune synapse (IS) and T cell activation. Using quantitative microscopy, including fluorescence recovery after photobleaching (FRAP), phasor fluorescence lifetime imaging microscopy-Föster resonance energy transfer (phasorFLIM-FRET), and total internal reflection fluorescence microscopy (TIRFM), we demonstrate that CD81 interacts with ICAM-1 and CD3 during conjugation between T cells and antigen-presenting cells (APCs). CD81 and ICAM-1 exhibit distinct mobilities in central and peripheral areas of early and late T cell-APC contacts. Moreover, CD81–ICAM-1 and CD81-CD3 dynamic interactions increase over the time course of IS formation, as these molecules redistribute throughout the contact area. Therefore, CD81 associations unexpectedly define novel sequential steps of IS maturation. Our results indicate that CD81 controls the temporal progression of the IS and the permanence of CD3 in the membrane contact area, contributing to sustained T cell receptor (TCR)-CD3-mediated signaling. Accordingly, we find that CD81 is required for proper T cell activation, regulating CD3ζ, ZAP-70, LAT, and extracellular signal-regulated kinase (ERK) phosphorylation; CD69 surface expression; and interleukin-2 (IL-2) secretion. Our data demonstrate the important role of CD81 in the molecular organization and dynamics of the IS architecture that sets the signaling threshold in T cell activation. PMID:23858057

Fluid shear stress enhances the cell volume decrease of osteoblast cells by increasing the expression of the ClC-3 chloride channel

PubMed Central

LIU, LI; CAI, SIYI; QIU, GUIXING; LIN, JIN

2016-01-01

ClC-3 is a volume-sensitive chloride channel that is responsible for cell volume adjustment and regulatory cell volume decrease (RVD). In order to evaluate the effects of fluid shear stress (FSS) stimulation on the osteoblast ClC-3 chloride channel, MC3T3-E1 cells were stimulated by FSS in the experimental group. Fluorescence quantitative polymerase chain reaction was used to detect changes in ClC-3 mRNA expression, the chloride ion fluorescent probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) was used to detect the chloride channel activity, and whole-cell patch clamping was used to monitor the changes in the volume-sensitive chloride current activated by a hypotonic environment following mechanical stimulation. The results show that the expression of the osteoblast chloride channel ClC-3 was significantly higher in the FSS group compared with the control group. MQAE fluorescence intensity was significantly reduced in the FSS group compared to the control group, suggesting that mechanical stimulation increased chloride channel activity and increased the efflux of intracellular chloride ions. Image analysis of osteoblast volume changes showed that osteoblast RVD was enhanced by mechanical stimulation. Whole-cell patch clamping showed that the osteoblast volume-sensitive chloride current was larger in the stimulated group compared to the control group, suggesting that elevated ClC-3 chloride channel expression results in an increased volume-sensitive chloride current. In conclusion, FSS stimulation enhances the RVD of osteoblast cell by increasing the expression of the ClC-3 and enhancing the chloride channel activity. PMID:27073622

Immunomagnetic cell separation, imaging, and analysis using Captivate ferrofluids

NASA Astrophysics Data System (ADS)

Jones, Laurie; Beechem, Joseph M.

2002-05-01

We have developed applications of CaptivateTM ferrofluids, paramagnetic particles (approximately 200 nm diameter), for isolating and analyzing cell populations in combination with fluorescence-based techniques. Using a microscope-mounted magnetic yoke and sample insertion chamber, fluorescent images of magnetically captured cells were obtained in culture media, buffer, or whole blood, while non-magnetically labeled cells sedimented to the bottom of the chamber. We combined this immunomagnetic cell separation and imaging technique with fluorescent staining, spectroscopy, and analysis to evaluate cell surface receptor-containing subpopulations, live/dead cell ratios, apoptotic/dead cell ratios, etc. The acquired images were analyzed using multi-color parameters, as produced by nucleic acid staining, esterase activity, or antibody labeling. In addition, the immunomagnetically separated cell fractions were assessed through microplate analysis using the CyQUANT Cell Proliferation Assay. These methods should provide an inexpensive alternative to some flow cytometric measurements. The binding capacities of the streptavidin- labled Captivate ferrofluid (SA-FF) particles were determined to be 8.8 nmol biotin/mg SA-FF, using biotin-4- fluorescein, and > 106 cells/mg SA-FF, using several cell types labeled with biotinylated probes. For goat anti- mouse IgG-labeled ferrofluids (GAM-FF), binding capacities were established to be approximately 0.2 - 7.5 nmol protein/mg GAM-FF using fluorescent conjugates of antibodies, protein G, and protein A.

Low cost quantitative digital imaging as an alternative to qualitative in vivo bioassays for analysis of active aflatoxin B1.

PubMed

Rasooly, Reuven; Do, Paula M; Hernlem, Bradley J

2016-06-15

Aflatoxin B1 (AFB1) producing fungi contaminate food and feed and are a major health concern. To minimize the sources and incidence of AFB1 illness there is a need to develop affordable, sensitive mobile devices for detection of active AFB1. In the present study we used a low cost fluorescence detector and describe two quantitative assays for detection of detoxified and active AFB1 demonstrating that AFB1 concentration can be measured as intensity of fluorescence. When the assay plate containing increasing concentrations of AFB1 is illuminated with a 366 nm ultraviolet lamp, AFB1 molecules absorb photons and emit blue light with peak wavelength of 432 nm. The fluorescence intensity increased in dose dependent manner. However, this method cannot distinguish between active AFB1 which poses a threat to health, and the detoxified AFB1 which exhibits no toxicity. To measure the toxin activity, we used a cell based assay that makes quantification more robust and is capable of detecting multiple samples simultaneously. It is an alternative to the qualitative duckling bioassay which is the "gold-standard" assay currently being used for quantitative analysis of active AFB1. AFB1 was incubated with transduced Vero cells expressing the green fluorescence protein (GFP) gene. After excitation with blue light at 475 nm, cells emitted green light with emission peak at 509 nm. The result shows that AFB1 inhibits protein expression in a concentration dependent manner resulting in proportionately less GFP fluorescence in cells exposed to AFB1. The result also indicates strong positive linear relationship with R(2)=0.90 between the low cost CCD camera and a fluorometer, which costs 100 times more than a CCD camera. This new analytical method for measuring active AFB1 is low in cost and combined with in vitro assay, is quantitative. It also does not require the use of animals and may be useful especially for laboratories in regions with limited resources. Published by Elsevier B.V.

Droplet based microfluidics for highthroughput screening of antibody secreting cells

NASA Astrophysics Data System (ADS)

Cai, Liheng; Heyman, John; Mazutis, Linas; Ung, Lloyd; Guerra, Rodrigo; Aubrecht, Donald; Weitz, David

2014-03-01

We present a droplet based microfluidic platform that allows highthroughput screening of antibody secreting cells. We coencapsulate single cells, fluorescent probes, and detection beads into emulsion droplets with diameter of 40 micron. The beads capture antibodies secreted by cells, resulting in a pronounced fluorescent signal that activates dielectrophoresis sorting at rate about 500 droplets per second. Moreover, we demonstrate that Reverse Transcription Polymerase Chain Reaction (RT-PCR) can be successfully applied to the cell encapsulated in a single sorted droplet. Our work highlights the potential of droplet based microfluidics as a platform to generate recombinant antibodies.

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

PubMed Central

Pan, Zui; Zhao, Xiaoli; Brotto, Marco

2012-01-01

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7. The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12. PMID:22349010

Fluorescence-based measurement of store-operated calcium entry in live cells: from cultured cancer cell to skeletal muscle fiber.

PubMed

Pan, Zui; Zhao, Xiaoli; Brotto, Marco

2012-02-13

Store operated Ca(2+) entry (SOCE), earlier termed capacitative Ca(2+) entry, is a tightly regulated mechanism for influx of extracellular Ca(2+) into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca(2+) stores. Since Ca(2+) is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear. The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca(2+) measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca(2+) using the ratio of F(340nm;) and F(380nm;) (510 nm for emission wavelength) of the ratiometric Ca(2+) indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca(2+) release and Ca(2+) extrusion, a Mn(2+) quenching assay is frequently used. Mn(2+) is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca(2+) pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn(2+) into the cells represents a measurement of activity of SOCE. Ratiometric measurement and the Mn(+2) quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca(2+) indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules.

Preventive antitumor activity against hepatocellular carcinoma (HCC) induced by immunization with fusions of dendritic cells and HCC cells in mice.

PubMed

Homma, S; Toda, G; Gong, J; Kufe, D; Ohno, T

2001-11-01

The prevention of recurrence of hepatocellular carcinoma (HCC) after treatment is very important for improvement of the prognosis of HCC patients. Dendritic cells (DCs) are potent antigen-presenting cells that can prime naive T cells to induce a primary immune response. We attempted to induce preventive antitumor immunity against HCC by immunizing BALB/c mice with fusions of DCs and HCC cells. Murine bone marrow-derived DCs and a murine HCC cell line. BNL cells, were fused by treatment with 50% polyethyleneglvcol (PEG). Fusion efficacy was assessed by the analysis of fusions of BNL cells stained with red fluorescent dye and DCs stained with green fluorescent dye. Mice injected intravenously with DC/BNL fusions were challenged by BNL cell inoculation. About 30% of the PEG-treated non-adherent cells with both fluorescences were considered to be fusion cells. The cell fraction of DC/BNL fusions showed phenotypes of DCs, MHC class II, CD80, CD86, and intercellular adhesion molecule (ICAM)-1, which were not expressed on BNL cells. Mice immunized with the fusions were protected against the inoculation of BNL tumor cells, whereas injection with a mixture of DCs and BNL cells not treated with PEG did not provide significant resistance against BNL cell inoculation. Splenocytes from DC/BNL fusion-immunized mice showed lytic activity against BNL cells. These results demonstrate that immunization with fusions of DCs and HCC cells is capable of inducing preventive antitumor immunity against HCC.

Fluorescent ATP analog mant-ATP reports dynein activity in the isolated Chlamydomonas axoneme

NASA Astrophysics Data System (ADS)

Feofilova, Maria; Howard, Jonathon

Eukaryotic flagella are long rod-like extensions of cells, which play a fundamental role in single cell movement, as well as in fluid transport. Flagella contain a highly evolutionary conserved mechanical structure called the axoneme. The motion of the flagellum is generated by dynein motor proteins located all along the length of the axoneme. How the force production of motors is controlled spatially and temporally is still an open question. Therefore, monitoring dynein activity in the axonemal structure is expected to provide novel insights in regulation of the beat. We use high sensitivity fluorescence microscopy to monitor the binding and hydrolysis kinetics of the fluorescently labeled ATP analogue mant-ATP (2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate), which is known to support dynein activity. By studying the kinetics of mant-ATP fluorescence, we identified distinct mant-ATP binding sites in the axoneme. The application of this method to axonemes with reduced amounts of dynein, showed evidence that one of the sites is associated with binding to dynein. In the future, we would like to use this method to find the spatial distribution of dynein activity in the axoneme.

Modification of the cellular antioxidant activity (CAA) assay to study phenolic antioxidants in a Caco-2 cell line.

PubMed

Kellett, Mary E; Greenspan, Phillip; Pegg, Ronald B

2018-04-01

In vitro assays are widely used to analyze the antioxidant potential of compounds, but they cannot accurately predict antioxidant behavior in living systems. Cell-based assays, like the cellular antioxidant activity (CAA) assay, are gaining importance as they provide a biological perspective. When the CAA assay was employed to study phenolic antioxidants using hepatocarcinoma (HepG2) cells, quercetin showed antioxidant activity in HepG2 cells; 25 and 250μM quercetin reduced fluorescence by 17.1±0.9% and 58.6±2.4%, respectively. (+)-Catechin, a phenolic antioxidant present in many foods, bestowed virtually no CAA in HepG2 cells. When Caco-2 cells were employed, more robust antioxidant activity was observed; 50μM (+)-catechin and quercetin reduced fluorescence by 54.1±1.4% and 63.6±0.9%, respectively. Based on these results, likely due to differences in active membrane transport between the cell types, the Caco-2-based CAA assay appears to be a more appropriate method for the study of certain dietary phenolics. Copyright © 2017 Elsevier Ltd. All rights reserved.

CalQuo: automated, simultaneous single-cell and population-level quantification of global intracellular Ca2+ responses.

PubMed

Fritzsche, Marco; Fernandes, Ricardo A; Colin-York, Huw; Santos, Ana M; Lee, Steven F; Lagerholm, B Christoffer; Davis, Simon J; Eggeling, Christian

2015-11-13

Detecting intracellular calcium signaling with fluorescent calcium indicator dyes is often coupled with microscopy techniques to follow the activation state of non-excitable cells, including lymphocytes. However, the analysis of global intracellular calcium responses both at the single-cell level and in large ensembles simultaneously has yet to be automated. Here, we present a new software package, CalQuo (Calcium Quantification), which allows the automated analysis and simultaneous monitoring of global fluorescent calcium reporter-based signaling responses in up to 1000 single cells per experiment, at temporal resolutions of sub-seconds to seconds. CalQuo quantifies the number and fraction of responding cells, the temporal dependence of calcium signaling and provides global and individual calcium-reporter fluorescence intensity profiles. We demonstrate the utility of the new method by comparing the calcium-based signaling responses of genetically manipulated human lymphocytic cell lines.

PH-sensitive fluorescence detection by diffuse fluorescence tomography

NASA Astrophysics Data System (ADS)

Li, Jiao; Gao, Feng; Duan, Linjing; Wang, Xin; Zhang, Limin; Zhao, Huijuan

2012-03-01

The importance of cellular pH has been shown clearly in the study of cell activity, pathological feature, drug metabolism, etc. Monitoring pH changes of living cells and imaging the regions with abnormal pH values in vivo could provide the physiologic and pathologic information for the research of the cell biology, pharmacokinetics, diagnostics and therapeutics of certain diseases such as cancer. Thus, pH-sensitive fluorescence imaging of bulk tissues has been attracting great attention in the regime of near-infrared diffuse fluorescence tomography (DFT), an efficient small-animal imaging tool. In this paper, the feasibility of quantifying pH-sensitive fluorescence targets in turbid medium is investigated using both time-domain and steady-state DFT methods. By use of the specifically designed time-domain and continuous-wave systems and the previously proposed image reconstruction scheme, we validate the method through 2-dimensional imaging experiments on a small-animal-sized phantom with multiply targets of distinct pH values. The results show that the approach can localize the targets with reasonable accuracy and achieve quantitative reconstruction of the pH-sensitive fluorescent yield.

SRB-2: a promiscuous rainbow aptamer for live-cell RNA imaging.

PubMed

Sunbul, Murat; Jäschke, Andres

2018-06-21

The SRB-2 aptamer originally selected against sulforhodamine B is shown here to promiscuously bind to various dyes with different colors. Binding of SRB-2 to these dyes results in either fluorescence increase or decrease, making them attractive for fluorescence microscopy and biological assays. By systematically varying fluorophore structural elements and measuring dissociation constants, the principles of fluorophore recognition by SRB-2 were analyzed. The obtained structure-activity relationships allowed us to rationally design a novel, bright, orange fluorescent turn-on probe (TMR-DN) with low background fluorescence, enabling no-wash live-cell RNA imaging. This new probe improved the signal-to-background ratio of fluorescence images by one order of magnitude over best previously known probe for this aptamer. The utility of TMR-DN is demonstrated by imaging ribosomal and messenger RNAs, allowing the observation of distinct localization patterns in bacteria and mammalian cells. The SRB-2 / TMR-DN system is found to be orthogonal to the Spinach/DFHBI and MG/Malachite green aptamer/dye systems.

Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

PubMed Central

Peddie, Christopher J.; Blight, Ken; Wilson, Emma; Melia, Charlotte; Marrison, Jo; Carzaniga, Raffaella; Domart, Marie-Charlotte; O׳Toole, Peter; Larijani, Banafshe; Collinson, Lucy M.

2014-01-01

Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure. PMID:24637200

Towards pH-sensitive imaging of small animals with photon-counting difference diffuse fluorescence tomography

NASA Astrophysics Data System (ADS)

Li, Jiao; Wang, Xin; Yi, Xi; Zhang, Limin; Zhou, Zhongxing; Zhao, Huijuan; Gao, Feng

2012-09-01

The importance of cellular pH has been shown clearly in the study of cell activity, pathological feature, and drug metabolism. Monitoring pH changes of living cells and imaging the regions with abnormal pH-values, in vivo, could provide invaluable physiological and pathological information for the research of the cell biology, pharmacokinetics, diagnostics, and therapeutics of certain diseases such as cancer. Naturally, pH-sensitive fluorescence imaging of bulk tissues has been attracting great attentions from the realm of near infrared diffuse fluorescence tomography (DFT). Herein, the feasibility of quantifying pH-induced fluorescence changes in turbid medium is investigated using a continuous-wave difference-DFT technique that is based on the specifically designed computed tomography-analogous photon counting system and the Born normalized difference image reconstruction scheme. We have validated the methodology using two-dimensional imaging experiments on a small-animal-sized phantom, embedding an inclusion with varying pH-values. The results show that the proposed approach can accurately localize the target with a quantitative resolution to pH-sensitive variation of the fluorescent yield, and might provide a promising alternative method of pH-sensitive fluorescence imaging in addition to the fluorescence-lifetime imaging.

A monoclonal IgM directed against immunodominant catalase B of cell wall of Aspergillus fumigatus exerts anti-A. fumigatus activities.

PubMed

Chaturvedi, Ashok K; Kumar, Rohitashw; Kumar, Awanit; Shukla, Praveen K

2009-11-01

Aspergillus fumigatus, a ubiquitous fungus, has been reported to cause human diseases like allergic pulmonary aspergillosis, aspergilloma and invasive infection. Limited spectrum and emergence of resistance has become a serious problem with available antifungals. Therefore, an alternative approach is required for successful treatment of mycoses. In the present study, immunogenic protein profile of A. fumigatus cell wall was generated using two-dimensional-gel electrophoresis and three hybridomas producing monoclonal antibodies (MAbs; IgM) were selected after fusion experiments. Of these three MAbs, MAb-7 exhibited potent in vitro inhibitory activity, which was confirmed by MTT assay, fluorescence-activated cell sorter analysis and immuno-fluorescence studies, and the protein was identified as catalase B using MALDI-TOF-MS.

A genetically encoded fluorescent tRNA is active in live-cell protein synthesis

PubMed Central

Masuda, Isao; Igarashi, Takao; Sakaguchi, Reiko; Nitharwal, Ram G.; Takase, Ryuichi; Han, Kyu Young; Leslie, Benjamin J.; Liu, Cuiping; Gamper, Howard; Ha, Taekjip; Sanyal, Suparna

2017-01-01

Abstract Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering. PMID:27956502

Turn-on theranostic fluorescent nanoprobe by electrostatic self-assembly of carbon dots with doxorubicin for targeted cancer cell imaging, in vivo hyaluronidase analysis, and targeted drug delivery.

PubMed

Gao, Na; Yang, Wen; Nie, Hailiang; Gong, Yunqian; Jing, Jing; Gao, Loujun; Zhang, Xiaoling

2017-10-15

This paper reports a turn-on theranostic fluorescent nanoprobe P-CDs/HA-Dox obtained by electrostatic assembly of polyethylenimine (PEI)-modified carbon dots (P-CDs) and Hyaluronic acid (HA)-conjugated doxorubicin (Dox) for hyaluronidase (HAase) detection, self-targeted imaging and drug delivery. P-CDs/HA-Dox show weak emission in a physiological environment. By utilizing the high affinity of HA to CD44 receptors overexpressed on many cancer cells, P-CDs/HA-Dox are capable of targeting and penetrating into cancer cells, where they are activated by HAase. As a result, HA-Dox can be digested into small fragments, causing the release of Dox and thereby restoring the fluorescence of P-CDs. The theranostic fluorescent nanoprobe can effectively distinguish cancer cells from normal cells. The as-prepared nanoprobe achieves a sensitive assay of HAase with a detection limit of 0.65UmL -1 . Furthermore, upon Dox release, the Dox could efficiently induce apoptosis in HeLa cells, as confirmed by MTT assay. The design of such a turn-on theranostic fluorescent probe provides a new strategy for self-targeted and image-guided chemotherapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Product-induced gene expression, a product-responsive reporter assay used to screen metagenomic libraries for enzyme-encoding genes.

PubMed

Uchiyama, Taku; Miyazaki, Kentaro

2010-11-01

A reporter assay-based screening method for enzymes, which we named product-induced gene expression (PIGEX), was developed and used to screen a metagenomic library for amidases. A benzoate-responsive transcriptional activator, BenR, was placed upstream of the gene encoding green fluorescent protein and used as a sensor. Escherichia coli sensor cells carrying the benR-gfp gene cassette fluoresced in response to benzoate concentrations as low as 10 μM but were completely unresponsive to the substrate benzamide. An E. coli metagenomic library consisting of 96,000 clones was grown in 96-well format in LB medium containing benzamide. The library cells were then cocultivated with sensor cells. Eleven amidase genes were recovered from 143 fluorescent wells; eight of these genes were homologous to known bacterial amidase genes while three were novel genes. In addition to their activity toward benzamide, the enzymes were active toward various substrates, including d- and l-amino acid amides, and displayed enantioselectivity. Thus, we demonstrated that PIGEX is an effective approach for screening novel enzymes based on product detection.

Modifying Expression Modes of Human Neurotensin Receptor Type 1 Alters Sensing Capabilities for Agonists in Yeast Signaling Biosensor.

PubMed

Hashi, Hiroki; Nakamura, Yasuyuki; Ishii, Jun; Kondo, Akihiko

2018-04-01

Neurotensin receptor type 1 (NTSR1), a member of the G-protein-coupled receptor (GPCR) family, is naturally activated by binding of a neurotensin peptide, leading to a variety of physiological effects. The budding yeast Saccharomyces cerevisiae is a proven host organism for assaying the agonistic activation of human GPCRs. Previous studies showed that yeast cells can functionally express human NTSR1 receptor, permitting the detection of neurotensin-promoted signaling using a ZsGreen fluorescent reporter gene. However, the fluorescence intensity (sensitivity) of NTSR1-expressing yeast cells is low compared to that of yeast cells expressing other human GPCRs (e.g., human somatostatin receptors). The present study sought to increase the sensitivity of the NTSR1-expressing yeast for use as a fluorescent biosensor, including modification of the expression of human NTSR1 in yeast. Changes in the transcription, translation, and transport of the receptor are attempted by altering the promoter, consensus Kozak-like sequence, and secretion signal sequences of the NTSR1-encoding gene. The resulting yeast cells exhibited increased sensitivity to exogenously added peptide. The cells are further engineered by using cell-surface display technology to ensure that the agonistic peptides are secreted and tethered to the yeast cell wall, yielding cells with enhanced NTSR1 activation. This yeast biosensor holds promise for the identification of agonists to treat NTSR1-related diseases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A pH-sensitive fluor, CypHer 5, used to monitor agonist-induced G protein-coupled receptor internalization in live cells.

PubMed

Adie, E J; Kalinka, S; Smith, L; Francis, M J; Marenghi, A; Cooper, M E; Briggs, M; Michael, N P; Milligan, G; Game, S

2002-11-01

G protein-coupled receptors (GPCRs) are the largest family of proteins involved in transmembrane signal transduction and are actively studied because of their suitability as therapeutic small-molecule drug targets. Agonist activation of GPCRs almost invariably results in the receptor being desensitized. One of the key events in receptor desensitization is the sequestration of the receptor from the cell surface into acidic intracellular endosomes. Therefore, a convenient, generic, and noninvasive monitor of this process is desirable. A novel, pH-sensitive, red-excited fluorescent dye, CypHer 5, was synthesized. This dye is non-fluorescent at neutral pH and is fluorescent at acidic pH. Anti-epitope antibodies labeled with this dye were internalized in an agonist concentration- and time-dependent manner, following binding on live cells to a range of GPCRs that had been modified to incorporate the epitope tags in their extracellular N-terminal domain. This resulted in a large signal increase over background. When protonated, the red fluorescence of CypHer 5 provides a generic reagent suitable for monitoring the internalization of GPCRs into acidic vesicles. This approach should be amenable to the study of many other classes of cell surface receptors that also internalize following stimulation.

An innovative pre-targeting strategy for tumor cell specific imaging and therapy

NASA Astrophysics Data System (ADS)

Qin, Si-Yong; Peng, Meng-Yun; Rong, Lei; Jia, Hui-Zhen; Chen, Si; Cheng, Si-Xue; Feng, Jun; Zhang, Xian-Zheng

2015-08-01

A programmed pre-targeting system for tumor cell imaging and targeting therapy was established based on the ``biotin-avidin'' interaction. In this programmed functional system, transferrin-biotin can be actively captured by tumor cells with the overexpression of transferrin receptors, thus achieving the pre-targeting modality. Depending upon avidin-biotin recognition, the attachment of multivalent FITC-avidin to biotinylated tumor cells not only offered the rapid fluorescence labelling, but also endowed the pre-targeted cells with targeting sites for the specifically designed biotinylated peptide nano-drug. Owing to the successful pre-targeting, tumorous HepG2 and HeLa cells were effectively distinguished from the normal 3T3 cells via fluorescence imaging. In addition, the self-assembled peptide nano-drug resulted in enhanced cell apoptosis in the observed HepG2 cells. The tumor cell specific pre-targeting strategy is applicable for a variety of different imaging and therapeutic agents for tumor treatments.A programmed pre-targeting system for tumor cell imaging and targeting therapy was established based on the ``biotin-avidin'' interaction. In this programmed functional system, transferrin-biotin can be actively captured by tumor cells with the overexpression of transferrin receptors, thus achieving the pre-targeting modality. Depending upon avidin-biotin recognition, the attachment of multivalent FITC-avidin to biotinylated tumor cells not only offered the rapid fluorescence labelling, but also endowed the pre-targeted cells with targeting sites for the specifically designed biotinylated peptide nano-drug. Owing to the successful pre-targeting, tumorous HepG2 and HeLa cells were effectively distinguished from the normal 3T3 cells via fluorescence imaging. In addition, the self-assembled peptide nano-drug resulted in enhanced cell apoptosis in the observed HepG2 cells. The tumor cell specific pre-targeting strategy is applicable for a variety of different imaging and therapeutic agents for tumor treatments. Electronic supplementary information (ESI) available: Experimental details, peptide structures, molecular weights, and additional data. See DOI: 10.1039/c5nr03862f

Properties of a non-bioactive fluorescent derivative of differentiation-inducing factor-3, an anti-tumor agent found in Dictyostelium discoideum

PubMed Central

Kubohara, Yuzuru; Kikuchi, Haruhisa; Matsuo, Yusuke; Oshima, Yoshiteru; Homma, Yoshimi

2014-01-01

ABSTRACT Differentiation-inducing factor-3 (DIF-3), found in the cellular slime mold Dictyostelium discoideum, and its derivatives, such as butoxy-DIF-3 (Bu-DIF-3), are potent anti-tumor agents. To investigate the activity of DIF-like molecules in tumor cells, we recently synthesized a green fluorescent DIF-3 derivative, BODIPY-DIF-3G, and analyzed its bioactivity and cellular localization. In this study, we synthesized a red (orange) fluorescent DIF-3 derivative, BODIPY-DIF-3R, and compared the cellular localization and bioactivities of the two BODIPY-DIF-3s in HeLa human cervical cancer cells. Both fluorescent compounds penetrated the extracellular membrane within 0.5 h and localized mainly to the mitochondria. In formalin-fixed cells, the two BODIPY-DIF-3s also localized to the mitochondria, indicating that the BODIPY-DIF-3s were incorporated into mitochondria independently of the mitochondrial membrane potential. After treatment for 3 days, BODIPY-DIF-3G, but not BODIPY-DIF-3R, induced mitochondrial swelling and suppressed cell proliferation. Interestingly, the swollen mitochondria were stainable with BODIPY-DIF-3G but not with BODIPY-DIF-3R. When added to isolated mitochondria in vitro, BODIPY-DIF-3G increased dose-dependently the rate of O2 consumption, but BODIPY-DIF-3R did not. These results suggest that the bioactive BODIPY-DIF-3G suppresses cell proliferation, at least in part, by altering mitochondrial activity, whereas the non-bioactive BODIPY-DIF-3R localizes to the mitochondria but does not affect mitochondrial activity or cell proliferation. PMID:24682009

Quantitative measurement of mitochondrial membrane potential in cultured cells: calcium-induced de- and hyperpolarization of neuronal mitochondria

PubMed Central

Gerencser, Akos A; Chinopoulos, Christos; Birket, Matthew J; Jastroch, Martin; Vitelli, Cathy; Nicholls, David G; Brand, Martin D

2012-01-01

Mitochondrial membrane potential (ΔΨM) is a central intermediate in oxidative energy metabolism. Although ΔΨM is routinely measured qualitatively or semi-quantitatively using fluorescent probes, its quantitative assay in intact cells has been limited mostly to slow, bulk-scale radioisotope distribution methods. Here we derive and verify a biophysical model of fluorescent potentiometric probe compartmentation and dynamics using a bis-oxonol-type indicator of plasma membrane potential (ΔΨP) and the ΔΨM probe tetramethylrhodamine methyl ester (TMRM) using fluorescence imaging and voltage clamp. Using this model we introduce a purely fluorescence-based quantitative assay to measure absolute values of ΔΨM in millivolts as they vary in time in individual cells in monolayer culture. The ΔΨP-dependent distribution of the probes is modelled by Eyring rate theory. Solutions of the model are used to deconvolute ΔΨP and ΔΨM in time from the probe fluorescence intensities, taking into account their slow, ΔΨP-dependent redistribution and Nernstian behaviour. The calibration accounts for matrix:cell volume ratio, high- and low-affinity binding, activity coefficients, background fluorescence and optical dilution, allowing comparisons of potentials in cells or cell types differing in these properties. In cultured rat cortical neurons, ΔΨM is −139 mV at rest, and is regulated between −108 mV and −158 mV by concerted increases in ATP demand and Ca2+-dependent metabolic activation. Sensitivity analysis showed that the standard error of the mean in the absolute calibrated values of resting ΔΨM including all biological and systematic measurement errors introduced by the calibration parameters is less than 11 mV. Between samples treated in different ways, the typical equivalent error is ∼5 mV. PMID:22495585

Imaging Primary Mouse Sarcomas After Radiation Therapy Using Cathepsin-Activatable Fluorescent Imaging Agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cuneo, Kyle C.; Mito, Jeffrey K.; Javid, Melodi P.

2013-05-01

Purpose: Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. Methods and Materials: A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activatedmore » fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. Results: RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. Conclusions: In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.« less

Molecular Beacon-Based MicroRNA Imaging During Neurogenesis.

PubMed

Lee, Jonghwan; Kim, Soonhag

2016-01-01

The fluorescence monitoring system for examining endogenous microRNA (miRNA) activity in cellular level provides crucial information on not only understanding a critical role of miRNA involving a variety of biological processes, but also evaluating miRNA expression patterns in a noninvasive manner. In this protocol, we report the details of a new procedure for a molecular beacon-based miRNA monitoring system, which includes the illustration scheme for miRNA detection strategy, exogenous miRNA detection, and measurement of endogenous miRNA expression level during neurogenesis. The fluorescence signal of miR-124a beacon quenched by BHQ2 was gradually recovered as increasing concentration of the miR-124a in tube. The functional work of miR-124a beacon was examined in intracellular environment, allowing for the internalization of the miR-124a beacon by lipofectamine, which resulted in activated fluorescent signals of the miR-124a beacon in the HeLa cells after the addition of synthetic miR-124a. The endogenous miR-124a expression level was detected by miR-124a beacon system during neurogenesis, showing brighter fluorescence intensity in cytoplasmic area of P19 cells after induction of neuronal differentiation by retinoic acid. The molecular beacon based-miRNA detection technique could be applicable to the simultaneous visualization of a variety of miRNA expression patterns using different fluorescence dyes. For the study of examining endogenous miRNA expression level using miRNA-beacon system, if cellular differentiation step is already prepared, transfection step of miR-124a beacon into P19 cells, and acquisition of activated fluorescence signal measured by confocal microscope can be conducted approximately within 6 h.

Size of submicrometric and nanometric particles affect cellular uptake and biological activity of macrophages in vitro.

PubMed

Leclerc, L; Rima, W; Boudard, D; Pourchez, J; Forest, V; Bin, V; Mowat, P; Perriat, P; Tillement, O; Grosseau, P; Bernache-Assollant, D; Cottier, M

2012-08-01

Micrometric and nanometric particles are increasingly used in different fields and may exhibit variable toxicity levels depending on their physicochemical characteristics. The aim of this study was to determine the impact of the size parameter on cellular uptake and biological activity, working with well-characterized fluorescent particles. We focused our attention on macrophages, the main target cells of the respiratory system responsible for the phagocytosis of the particles. FITC fluorescent silica particles of variable submicronic sizes (850, 500, 250 and 150 nm) but with similar surface coating (COOH) were tailored and physico-chemically characterized. These particles were then incubated with the RAW 264.7 macrophage cell line. After microscopic observations (SEM, TEM, confocal), a quantitative evaluation of the uptake was carried out. Fluorescence detected after a quenching with trypan blue allows us to distinguish and quantify entirely engulfed fluorescent particles from those just adhering to the cell membrane. Finally, these data were compared to the in vitro toxicity assessed in terms of cell damage, inflammation and oxidative stress (evaluated by LDH release, TNF-α and ROS production respectively). Particles were well characterized (fluorescence, size distribution, zeta potential, agglomeration and surface groups) and easily visualized after cellular uptake using confocal and electron microscopy. The number of internalized particles was precisely evaluated. Size was found to be an important parameter regarding particles uptake and in vitro toxicity but this latter strongly depends on the particles doses employed.

Label-free density difference amplification-based cell sorting.

PubMed

Song, Jihwan; Song, Minsun; Kang, Taewook; Kim, Dongchoul; Lee, Luke P

2014-11-01

The selective cell separation is a critical step in fundamental life sciences, translational medicine, biotechnology, and energy harvesting. Conventional cell separation methods are fluorescent activated cell sorting and magnetic-activated cell sorting based on fluorescent probes and magnetic particles on cell surfaces. Label-free cell separation methods such as Raman-activated cell sorting, electro-physiologically activated cell sorting, dielectric-activated cell sorting, or inertial microfluidic cell sorting are, however, limited when separating cells of the same kind or cells with similar sizes and dielectric properties, as well as similar electrophysiological phenotypes. Here we report a label-free density difference amplification-based cell sorting (dDACS) without using any external optical, magnetic, electrical forces, or fluidic activations. The conceptual microfluidic design consists of an inlet, hydraulic jump cavity, and multiple outlets. Incoming particles experience gravity, buoyancy, and drag forces in the separation chamber. The height and distance that each particle can reach in the chamber are different and depend on its density, thus allowing for the separation of particles into multiple outlets. The separation behavior of the particles, based on the ratio of the channel heights of the inlet and chamber and Reynolds number has been systematically studied. Numerical simulation reveals that the difference between the heights of only lighter particles with densities close to that of water increases with increasing the ratio of the channel heights, while decreasing Reynolds number can amplify the difference in the heights between the particles considered irrespective of their densities.

Cellular Uptake and Localization of Polymyxins in Renal Tubular Cells Using Rationally Designed Fluorescent Probes

PubMed Central

Yun, Bo; Azad, Mohammad A. K.; Nowell, Cameron J.; Nation, Roger L.; Thompson, Philip E.; Roberts, Kade D.

2015-01-01

Polymyxins are cyclic lipopeptide antibiotics that serve as a last line of defense against Gram-negative bacterial superbugs. However, the extensive accumulation of polymyxins in renal tubular cells can lead to nephrotoxicity, which is the major dose-limiting factor in clinical use. In order to gain further insights into the mechanism of polymyxin-induced nephrotoxicity, we have rationally designed novel fluorescent polymyxin probes to examine the localization of polymyxins in rat renal tubular (NRK-52E) cells. Our design strategy focused on incorporating a dansyl fluorophore at the hydrophobic centers of the polymyxin core structure. To this end, four novel regioselectively labeled monodansylated polymyxin B probes (MIPS-9541, MIPS-9542, MIPS-9543, and MIPS-9544) were designed, synthesized, and screened for their antimicrobial activities and apoptotic effects against rat kidney proximal tubular cells. On the basis of the assessment of antimicrobial activities, cellular uptake, and apoptotic effects on renal tubular cells, incorporation of a dansyl fluorophore at either position 6 or 7 (MIPS-9543 and MIPS-9544, respectively) of the polymyxin core structure appears to be an appropriate strategy for generating representative fluorescent polymyxin probes to be utilized in intracellular imaging and mechanistic studies. Furthermore, confocal imaging experiments utilizing these probes showed evidence of partial colocalization of the polymyxins with both the endoplasmic reticulum and mitochondria in rat renal tubular cells. Our results highlight the value of these new fluorescent polymyxin probes and provide further insights into the mechanism of polymyxin-induced nephrotoxicity. PMID:26392495

An Automatic Segmentation Method Combining an Active Contour Model and a Classification Technique for Detecting Polycomb-group Proteinsin High-Throughput Microscopy Images.

PubMed

Gregoretti, Francesco; Cesarini, Elisa; Lanzuolo, Chiara; Oliva, Gennaro; Antonelli, Laura

2016-01-01

The large amount of data generated in biological experiments that rely on advanced microscopy can be handled only with automated image analysis. Most analyses require a reliable cell image segmentation eventually capable of detecting subcellular structures.We present an automatic segmentation method to detect Polycomb group (PcG) proteins areas isolated from nuclei regions in high-resolution fluorescent cell image stacks. It combines two segmentation algorithms that use an active contour model and a classification technique serving as a tool to better understand the subcellular three-dimensional distribution of PcG proteins in live cell image sequences. We obtained accurate results throughout several cell image datasets, coming from different cell types and corresponding to different fluorescent labels, without requiring elaborate adjustments to each dataset.

An Improved Flow Cytometry Method For Precise Quantitation Of Natural-Killer Cell Activity

NASA Technical Reports Server (NTRS)

Crucian, Brian; Nehlsen-Cannarella, Sandra; Sams, Clarence

2006-01-01

The ability to assess NK cell cytotoxicity using flow cytometry has been previously described and can serve as a powerful tool to evaluate effector immune function in the clinical setting. Previous methods used membrane permeable dyes to identify target cells. The use of these dyes requires great care to achieve optimal staining and results in a broad spectral emission that can make multicolor cytometry difficult. Previous methods have also used negative staining (the elimination of target cells) to identify effector cells. This makes a precise quantitation of effector NK cells impossible due to the interfering presence of T and B lymphocytes, and the data highly subjective to the variable levels of NK cells normally found in human peripheral blood. In this study an improved version of the standard flow cytometry assay for NK activity is described that has several advantages of previous methods. Fluorescent antibody staining (CD45FITC) is used to positively identify target cells in place of membranepermeable dyes. Fluorescent antibody staining of target cells is less labor intensive and more easily reproducible than membrane dyes. NK cells (true effector lymphocytes) are also positively identified by fluorescent antibody staining (CD56PE) allowing a simultaneous absolute count assessment of both NK cells and target cells. Dead cells are identified by membrane disruption using the DNA intercalating dye PI. Using this method, an exact NK:target ratio may be determined for each assessment, including quantitation of NK target complexes. Backimmunoscatter gating may be used to track live vs. dead Target cells via scatter properties. If desired, NK activity may then be normalized to standardized ratios for clinical comparisons between patients, making the determination of PBMC counts or NK cell percentages prior to testing unnecessary. This method provides an exact cytometric determination of NK activity that highly reproducible and may be suitable for routine use in the clinical setting.

Monitoring thioredoxin redox with a genetically encoded red fluorescent biosensor.

PubMed

Fan, Yichong; Makar, Merna; Wang, Michael X; Ai, Hui-Wang

2017-09-01

Thioredoxin (Trx) is one of the two major thiol antioxidants, playing essential roles in redox homeostasis and signaling. Despite its importance, there is a lack of methods for monitoring Trx redox dynamics in live cells, hindering a better understanding of physiological and pathological roles of the Trx redox system. In this work, we developed the first genetically encoded fluorescent biosensor for Trx redox by engineering a redox relay between the active-site cysteines of human Trx1 and rxRFP1, a redox-sensitive red fluorescent protein. We used the resultant biosensor-TrxRFP1-to selectively monitor perturbations of Trx redox in various mammalian cell lines. We subcellularly localized TrxRFP1 to image compartmentalized Trx redox changes. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor Trx and glutathione redox dynamics in live cells in response to chemical and physiologically relevant stimuli.

Fluorescence-encoded gold nanoparticles: library design and modulation of cellular uptake into dendritic cells.

PubMed

Rodriguez-Lorenzo, Laura; Fytianos, Kleanthis; Blank, Fabian; von Garnier, Christophe; Rothen-Rutishauser, Barbara; Petri-Fink, Alke

2014-04-09

In order to harness the unique properties of nanoparticles for novel clinical applications and to modulate their uptake into specific immune cells we designed a new library of homo- and hetero-functional fluorescence-encoded gold nanoparticles (Au-NPs) using different poly(vinyl alcohol) and poly(ethylene glycol)-based polymers for particle coating and stabilization. The encoded particles were fully characterized by UV-Vis and fluorescence spectroscopy, zeta potential and dynamic light scattering. The uptake by human monocyte derived dendritic cells in vitro was studied by confocal laser scanning microscopy and quantified by fluorescence-activated cell sorting and inductively coupled plasma atomic emission spectroscopy. We show how the chemical modification of particle surfaces, for instance by attaching fluorescent dyes, can conceal fundamental particle properties and modulate cellular uptake. In order to mask the influence of fluorescent dyes on cellular uptake while still exploiting its fluorescence for detection, we have created hetero-functionalized Au-NPs, which again show typical particle dependent cellular interactions. Our study clearly prove that the thorough characterization of nanoparticles at each modification step in the engineering process is absolutely essential and that it can be necessary to make substantial adjustments of the particles in order to obtain reliable cellular uptake data, which truly reflects particle properties. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of methanogenic activity in a thermophilic-dry anaerobic reactor: use of fluorescent in situ hybridization.

PubMed

Montero, B; García-Morales, J L; Sales, D; Solera, R

2009-03-01

Methanogenic activity in a thermophilic-dry anaerobic reactor was determined by comparing the amount of methane generated for each of the organic loading rates with the size of the total and specific methanogenic population, as determined by fluorescent in situ hybridization. A high correlation was evident between the total methanogenic activity and retention time [-0.6988Ln(x)+2.667] (R(2) 0.8866). The total methanogenic activity increased from 0.04x10(-8) mLCH(4) cell(-1)day(-1) to 0.38x10(-8) mLCH(4) cell(-1)day(-1) while the retention time decreased, augmenting the organic loading rates. The specific methanogenic activities of H(2)-utilizing methanogens and acetate-utilizing methanogens increased until they stabilised at 0.64x10(-8) mLCH(4) cell(-1)day(-1) and 0.33x10(-8) mLCH(4) cell(-1)day(-1), respectively. The methanogenic activity of H(2)-utilizing methanogens was higher than acetate-utilizing methanogens, indicating that maintaining a low partial pressure of hydrogen does not inhibit the acetoclastic methanogenesis or the anaerobic process.

Correlated Fluorescence-Atomic Force Microscopy Studies of the Clathrin Mediated Endocytosis in SKMEL Cells

NASA Astrophysics Data System (ADS)

Smith, Steve; Hor, Amy; Luu, Anh; Kang, Lin; Scott, Brandon; Bailey, Elizabeth; Hoppe, Adam

Clathrin-mediated endocytosis is one of the central pathways for cargo transport into cells, and plays a major role in the maintenance of cellular functions, such as intercellular signaling, nutrient intake, and turnover of plasma membrane in cells. The clathrin-mediated endocytosis process involves invagination and formation of clathrin-coated vesicles. However, the biophysical mechanisms of vesicle formation are still debated. We investigate clathrin vesicle formation mechanisms through the utilization of tapping-mode atomic force microscopy for high resolution topographical imaging in neutral buffer solution of unroofed cells exposing the inner membrane, combined with fluorescence imaging to definitively label intracellular constituents with specific fluorescent fusion proteins (actin filaments labeled with green phalloidin-antibody and clathrin coated vesicles with the fusion protein Tq2) in SKMEL (Human Melanoma) cells. Results from our work are compared against dynamical polarized total internal fluorescence (TIRF), super-resolution photo-activated localization microscopy (PALM) and transmission electron microscopy (TEM) to draw conclusions regarding the prominent model of vesicle formation in clathrin-mediated endocytosis. Funding provided by NSF MPS/DMR/BMAT award # 1206908.

Monitoring apoptosis of TK-GFP-expressing ACC-M cells induced by ACV using FRET technique

NASA Astrophysics Data System (ADS)

Xiong, Tao; Zhang, Zhihong; Lin, Juqiang; Yang, Jie; Zeng, Shaoqun; Luo, Qingming

2006-05-01

Apoptosis is an evolutionary conserved cellular process that plays an important role during development, but it is also involved in tissue homeostasis and in many diseases. To study the characteristics of suicide gene system of the herpes simplex virus thymidine kinase (HSV-tk) gene in tumor cells and explore the apoptosis phenomena in this system and its effect on the human adenoid cystic carcinoma line ACC-M cell, we detected apoptosis of CD3- (ECFP-CRS-DsRed) and TK-GFP-expressing ACC-M (ACC-M-TK-GFP-CD3) cells induced by acyclovir (ACV) using fluorescence resonance energy transfer (FRET) technique. CD3 is a FRET-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3 sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. FRET from ECFP to DsRed could be detected in normal ACC-M-TK-GFP-CD3 cells, and the FRET efficient was remarkably decreased and then disappeared during the cells apoptosis induced by ACV. It was due to the activated caspase-3 cleaved the CD3 fusion protein. In this study, the results suggested that the ACV-induced apoptosis of ACC-M-TK-GFP-CD3 cells was through caspase-3 pathway.

Monitoring apoptosis of TK-GFP-expressing ACC-M cells induced by ACV using FRET technique

NASA Astrophysics Data System (ADS)

Xiong, Tao; Zhang, Zhihong; Lin, Juqiang; Yang, Jie; Zeng, Shaoqun; Luo, Qingming

2006-09-01

Apoptosis is an evolutionary conserved cellular process that plays an important role during development, but it is also involved in tissue homeostasis and in many diseases. To study the characteristics of suicide gene system of the herpes simplex virus thymidine kinase (HSV-tk) gene in tumor cells and explore the apoptosis phenomena in this system and its effect on the human adenoid cystic carcinoma line ACC-M cell, we detected apoptosis of CD3- (ECFP-CRS-DsRed) and TK-GFP-expressing ACC-M (ACC-M-TK-GFP-CD3) cells induced by acyclovir (ACV) using fluorescence resonance energy transfer (FRET) technique. CD3 is a FRET-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3 sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. FRET from ECFP to DsRed could be detected in normal ACC-M-TK-GFP-CD3 cells, and the FRET efficient was remarkably decreased and then disappeared during the cells apoptosis induced by ACV. It was due to the activated caspase-3 cleaved the CD3 fusion protein. In this study, the results suggested that the AVC-induced apoptosis of ACC-M-TK-GFP-CD3 cells was through caspase-3 pathway.

Fluorescent recovery after photobleaching (FRAP) analysis of nuclear export rates identifies intrinsic features of nucleocytoplasmic transport.

PubMed

Cardarelli, Francesco; Tosti, Luca; Serresi, Michela; Beltram, Fabio; Bizzarri, Ranieri

2012-02-17

A quantitative description of carrier-mediated nuclear export in live cells is presented. To this end, we fused a prototypical leucine-rich nuclear export signal (NES) to GFP as a cargo model and expressed the fluorescent chimera in live CHO-K1 cells. By modeling FRAP data, we calculate the NES affinity for the export machinery and the maximum rate of nuclear export achievable at saturation of endogenous carriers. The measured active-export time through the Nuclear Pore Complex (NPC) is 18 ms, remarkably similar to the previously determined active-import rate. Also, our results reveal that active export/import and active export/passive diffusion fluxes are uncoupled, thus complementing previous reports on active import/passive diffusion uncoupling. These findings suggest differential gating at the NPC level.

Targeting Quiescence in Prostate Cancer

DTIC Science & Technology

actively dividing cancer cells causing primary tumor shrinkage, but leave behind quiescent cancer cells which may seed new, more aggressive and chemo...resistant cancers at a later date . During this first year of funding, we have successfully developed prostate cancer cell lines carrying fluorescent cell

Lenticular mitoprotection. Part A: Monitoring mitochondrial depolarization with JC-1 and artifactual fluorescence by the glycogen synthase kinase-3β inhibitor, SB216763.

PubMed

Brooks, Morgan M; Neelam, Sudha; Fudala, Rafal; Gryczynski, Ignacy; Cammarata, Patrick R

2013-01-01

Dissipation of the electrochemical gradient across the inner mitochondrial membrane results in mitochondrial membrane permeability transition (mMPT), a potential early marker for the onset of apoptosis. In this study, we demonstrate a role for glycogen synthase kinase-3β (GSK-3β) in regulating mMPT. Using direct inhibition of GSK-3β with the GSK-3β inhibitor SB216763, mitochondria may be prevented from depolarizing (hereafter referred to as mitoprotection). Cells treated with SB216763 showed an artifact of fluorescence similar to the green emission spectrum of the JC-1 dye. We demonstrate the novel use of spectral deconvolution to negate the interfering contributing fluorescence by SB216763, thus allowing an unfettered analysis of the JC-1 dye to determine the mitochondrial membrane potential. Secondary cultures of virally transfected human lens epithelial cells (HLE-B3) were exposed to acute hypoxic conditions (approximately 1% O₂) followed by exposure to atmospheric oxygen (approximately 21% O₂). The fluorescent dye JC-1 was used to monitor the extent of mitochondrial depolarization upon exposure of inhibitor treatment relative to the control cells (mock inhibition) in atmospheric oxygen. Annexin V-fluorescein isothiocyanate/propidium iodide staining was implemented to determine cell viability. Treatment of HLE-B3 cells with SB216763 (12 µM), when challenged by oxidative stress, suppressed mitochondrial depolarization relative to control cells as demonstrated with JC-1 fluorescent dye analysis. Neither the control nor the SB216763-treated HLE-B3 cells tested positive with annexin V-fluorescein isothiocyanate/propidium iodide staining under the conditions of the experiment. Inhibition of GSK-3β activity by SB216763 blocked mMPT relative to the slow but consistent depolarization observed with the control cells. We conclude that inhibition of GSK-3β activity by the GSK-3β inhibitor SB216763 provides positive protection against mitochondrial depolarization.

Analysis of the conductivity of plasmodesmata by microinjection.

PubMed

Kragler, Friedrich

2015-01-01

Pressure microinjection can be used to introduce fluorescent dyes and labeled macromolecules into single cells. The method allows measuring transport activity of macromolecules such as proteins and RNA molecules within and between cells. Routinely, plant mesophyll cells are injected with fluorescent dextran molecules of specific sizes to measure an increase of the size exclusion limit of plasmodesmata in the presence of a co-injected or expressed protein. The mobility of a macromolecule can also be addressed directly by injecting a recombinant protein that itself is labeled with fluorescent dye and following its transport to neighboring cells. This chapter describes a pressure microinjection protocol successfully applied to Nicotiana leaves. This protocol requires basic skills and experience in handling a microscope equipped with an imaging system, a micromanipulator, and a microinjection system attached to an upright microscope. Using this equipment, a trained person can inject approximately 10-20 mesophyll cells per hour.

Boron nitride nanotubes as vehicles for intracellular delivery of fluorescent drugs and probes.

PubMed

Niskanen, Jukka; Zhang, Issan; Xue, Yanming; Golberg, Dmitri; Maysinger, Dusica; Winnik, Françoise M

2016-01-01

To evaluate the response of cells to boron nitride nanotubes (BNNTs) carrying fluorescent probes or drugs in their inner channel by assessment of the cellular localization of the fluorescent cargo, evaluation of the in vitro release and biological activity of a drug (curcumin) loaded in BNNTs. Cells treated with curcumin-loaded BNNTs and stimulated with lipopolysaccharide were assessed for nitric oxide release and stimulation of IL-6 and TNF-α. The cellular trafficking of two cell-permeant dyes and a non-cell-permeant dye loaded within BNNTs was imaged. BNNTs loaded with up to 13 wt% fluorophores were internalized by cells and controlled release of curcumin triggered cellular pathways associated with the known anti-inflammatory effects of the drug. The overall findings indicate that BNNTs can function as nanocarriers of biologically relevant probes/drugs allowing one to examine/control their local intracellular localization and biochemical effects, leading the way to applications as intracellular nanosensors.

Optical painting and fluorescence activated sorting of single adherent cells labelled with photoswitchable Pdots

PubMed Central

Kuo, Chun-Ting; Thompson, Alison M.; Gallina, Maria Elena; Ye, Fangmao; Johnson, Eleanor S.; Sun, Wei; Zhao, Mengxia; Yu, Jiangbo; Wu, I-Che; Fujimoto, Bryant; DuFort, Christopher C.; Carlson, Markus A.; Hingorani, Sunil R.; Paguirigan, Amy L.; Radich, Jerald P.; Chiu, Daniel T.

2016-01-01

The efficient selection and isolation of individual cells of interest from a mixed population is desired in many biomedical and clinical applications. Here we show the concept of using photoswitchable semiconducting polymer dots (Pdots) as an optical ‘painting' tool, which enables the selection of certain adherent cells based on their fluorescence, and their spatial and morphological features, under a microscope. We first develop a Pdot that can switch between the bright (ON) and dark (OFF) states reversibly with a 150-fold contrast ratio on irradiation with ultraviolet or red light. With a focused 633-nm laser beam that acts as a ‘paintbrush' and the photoswitchable Pdots as the ‘paint', we select and ‘paint' individual Pdot-labelled adherent cells by turning on their fluorescence, then proceed to sort and recover the optically marked cells (with 90% recovery and near 100% purity), followed by genetic analysis. PMID:27118210

Quantitative imaging with fluorescent biosensors.

PubMed

Okumoto, Sakiko; Jones, Alexander; Frommer, Wolf B

2012-01-01

Molecular activities are highly dynamic and can occur locally in subcellular domains or compartments. Neighboring cells in the same tissue can exist in different states. Therefore, quantitative information on the cellular and subcellular dynamics of ions, signaling molecules, and metabolites is critical for functional understanding of organisms. Mass spectrometry is generally used for monitoring ions and metabolites; however, its temporal and spatial resolution are limited. Fluorescent proteins have revolutionized many areas of biology-e.g., fluorescent proteins can report on gene expression or protein localization in real time-yet promoter-based reporters are often slow to report physiologically relevant changes such as calcium oscillations. Therefore, novel tools are required that can be deployed in specific cells and targeted to subcellular compartments in order to quantify target molecule dynamics directly. We require tools that can measure enzyme activities, protein dynamics, and biophysical processes (e.g., membrane potential or molecular tension) with subcellular resolution. Today, we have an extensive suite of tools at our disposal to address these challenges, including translocation sensors, fluorescence-intensity sensors, and Förster resonance energy transfer sensors. This review summarizes sensor design principles, provides a database of sensors for more than 70 different analytes/processes, and gives examples of applications in quantitative live cell imaging.

Investigation of tryptophan-NADH interactions in live human cells using three-photon fluorescence lifetime imaging and Förster resonance energy transfer microscopy

NASA Astrophysics Data System (ADS)

Jyothikumar, Vinod; Sun, Yuansheng; Periasamy, Ammasi

2013-06-01

A method to investigate the metabolic activity of intracellular tryptophan (TRP) and coenzyme-NADH using three-photon (3P) fluorescence lifetime imaging (FLIM) and Förster resonance energy transfer (FRET) is presented. Through systematic analysis of FLIM data from tumorigenic and nontumorigenic cells, a statistically significant decrease in the fluorescence lifetime of TRP was observed in response to the increase in protein-bound NADH as cells were treated with glucose. The results demonstrate the potential use of 3P-FLIM-FRET as a tool for label-free screening of the change in metabolic flux occurring in human diseases or other clinical conditions.

Seeing the forest and trees: whole-body and whole-brain imaging for circadian biology.

PubMed

Ode, K L; Ueda, H R

2015-09-01

Recent advances in methods for making mammalian organs translucent have made possible whole-body fluorescent imaging with single-cell resolution. Because organ-clearing methods can be used to image the heterogeneous nature of cell populations, they are powerful tools to investigate the hierarchical organization of the cellular circadian clock, and how the clock synchronizes a variety of physiological activities. In particular, methods compatible with genetically encoded fluorescent reporters have the potential to detect circadian activity in different brain regions and the circadian-phase distribution across the whole body. In this review, we summarize the current methods and strategy for making organs translucent (removal of lipids, decolourization of haemoglobin and adjusting the refractive index of the specimen). We then discuss possible applications to circadian biology. For example, the coupling of circadian rhythms among different brain regions, brain activity in sleep-wake cycles and the role of migrating cells such as immune cells and cancer cells in chronopharmacology. © 2015 John Wiley & Sons Ltd.

Non-plaque-forming virions of Modified Vaccinia virus Ankara express viral genes.

PubMed

Lülf, Anna-Theresa; Freudenstein, Astrid; Marr, Lisa; Sutter, Gerd; Volz, Asisa

2016-12-01

In cell culture infections with vaccinia virus the number of counted virus particles is substantially higher than the number of plaques obtained by titration. We found that standard vaccine preparations of recombinant Modified Vaccinia virus Ankara produce only about 20-30% plaque-forming virions in fully permissive cell cultures. To evaluate the biological activity of the non-plaque-forming particles, we generated recombinant viruses expressing fluorescent reporter proteins under transcriptional control of specific viral early and late promoters. Live cell imaging and automated counting by fluorescent microscopy indicated that virtually all virus particles can enter cells and switch on viral gene expression. Although most of the non-plaque-forming infections are arrested at the level of viral early gene expression, we detected activation of late viral transcription in 10-20% of single infected cells. Thus, non-plaque-forming particles are biologically active, and likely contribute to the immunogenicity of vaccinia virus vaccines. Copyright © 2016 Elsevier Inc. All rights reserved.

A high-throughput quantitative expression analysis of cancer-related genes in human HepG2 cells in response to limonene, a potential anticancer agent.

PubMed

Hafidh, Rand R; Hussein, Saba Z; MalAllah, Mohammed Q; Abdulamir, Ahmed S; Abu Bakar, Fatimah

2017-11-14

Citrus bioactive compounds, as active anticancer agent, have been under focus by several studies worldwide. However, the underlying genes responsible for the anticancer potential have not been sufficiently highlighted. The current study investigated the gene expression profile of hepatocellular carcinoma, HepG2, cells after treatment with Limonene. The concentration that killed 50% of HepG2 cells was used to elucidate the genetic mechanisms of limonene anticancer activity. The apoptotic induction was detected by flow cytometry and confocal fluorescence microscope. Two of pro-apoptotic events, caspase-3 activation and phosphatidylserine translocation were manifested by confocal fluorescence microscopy. High-throughput real-time PCR was used to profile 1023 cancer-related genes in 16 different gene families related to the cancer development. In comparison to untreated cells, limonene increased the percentage of apoptotic cells up to 89.61%, by flow cytometry, and 48.2% by fluorescence microscopy. There was a significant limonene-driven differential gene expression of HepG2 cells in 15 different gene families. Limonene was shown to significantly (>2log) up-regulate and down-regulate 14 and 59 genes, respectively. The affected gene families, from most to least affected, were apoptosis induction, signal transduction, cancer genes augmentation, alteration in kinases expression, inflammation, DNA damage repair, and cell cycle proteins. The current study reveals that limonene could be a promising, cheap, and effective anticancer compound. The broad spectrum of limonene anticancer activity is interesting for anticancer drug development. Further research is needed to confirm the current findings and to examine the anticancer potential of limonene along with underlying mechanisms on different cell lines. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence.

PubMed

Li, Dong; Zheng, Wei; Qu, Jianan Y

2008-10-15

A time-resolved spectroscopic imaging system is built to study the fluorescence characteristics of nicotinamide adenine dinucleotide (NADH), an important metabolic coenzyme and endogenous fluorophore in cells. The system provides a unique approach to measure fluorescence signals in different cellular organelles and cytoplasm. The ratios of free over protein-bound NADH signals in cytosol and nucleus are slightly higher than those in mitochondria. The mitochondrial fluorescence contributes about 70% of overall cellular fluorescence and is not a completely dominant signal. Furthermore, NADH signals in mitochondria, cytosol, and the nucleus respond to the changes of cellular activity differently, suggesting that cytosolic and nuclear fluorescence may complicate the well-known relationship between mitochondrial fluorescence and cellular metabolism.

Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based Fluorescence Assay with λ Exonuclease Assistance.

PubMed

Zhang, Hua; Zhao, Zhen; Lei, Zhen; Wang, Zhenxin

2016-12-06

The phosphorylation of nucleic acid with 5'-OH termini catalyzed by polynucleotide kinase (PNK) involves several significant cellular events. Here a paper-based fluorescence assay with λ exonuclease assistance was reported for facile detection of PNK activity through monitoring the change of fluorescence intensity on paper surface. Cy5-labeled ssDNA was first immobilized on the surface of aldehyde group modified paper, and BHQ-labeled ssDNA was then employed to quench the fluorescence of the immobilized Cy5-labeled ssDNA with the help of an adaptor ssDNA. When PNK and λ exonuclease cleavage reaction were introduced, the fluorescence quenching effect on the paper surface was blocked because of the digestion of phosphorylated dsDNA by the coupled enzymes. By using this paper-based assay, PNK activity both in pure reaction buffer and in practical biosample have been successfully measured. Highly sensitive detection of PNK activity down to 0.0001 U mL -1 and lysate of about 50 cells is achieved. The inhibition of PNK activity has also been investigated and a satisfactory result is obtained.

Structural basis for activity of highly efficient RNA mimics of green fluorescent protein

PubMed Central

Warner, Katherine Deigan; Chen, Michael C.; Song, Wenjiao; Strack, Rita L.; Thorn, Andrea; Jaffrey, Samie R.; Ferré-D’Amaré, Adrian R.

2014-01-01

Green fluorescent protein (GFP) and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro evolved RNA mimic of GFP, which as genetically encoded fusions, makes possible live-cell, real-time imaging of biological RNAs, without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we have solved its co-crystal structure bound to its cognate exogenous chromophore, revealing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex, and an unpaired guanine. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs, and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure has guided the design of a miniaturized 'Baby Spinach', and provides the foundation for structure-driven design and tuning of fluorescent RNAs. PMID:25026079

Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy

PubMed Central

Mazo-Vargas, Anyimilehidi; Park, Heungwon; Aydin, Mert; Buchler, Nicolas E.

2014-01-01

Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15–20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression. PMID:25232010

Production of chimeric recombinant single domain antibody-green fluorescent fusion protein in Chinese hamster ovary cells.

PubMed

Bazl, M Rajabi; Rasaee, M J; Foruzandeh, M; Rahimpour, A; Kiani, J; Rahbarizadeh, F; Alirezapour, B; Mohammadi, M

2007-02-01

There is an increasing interest in the application of nanobodies such as VHH in the field of therapy and imaging. In the present study a stable genetically engineered cell line of Chinese hamster ovary (CHO) origin transfected using two sets of expression vectors was constructed in order to permit the cytoplasmic and extracellular expression of single domain antibody along with green fluorescent protein (GFP) as reporter gene. The quality of the constructs were examined both by the restriction map as well as sequence analysis. The gene transfection and protein expression was further examined by reverse transcription-polymerase chain reaction (RT-PCR). The transfected cells were grown in 200 microg/mL hygromycin containing media and the stable cell line obtained showed fluorescent activity for more than a period of 180 days. The production of fusion protein was also detected by fluorescent microscopy, fluorescent spectroscopy as well as by enzyme-linked immunosorbent assay (ELISA) analysis. This strategy allows a rapid production of recombinant fluobodies involving VHH, which can be used in various experiments such as imaging and detection in which a primary labeled antibody is required.

Direct imaging of APP proteolysis in living cells.

PubMed

Parenti, Niccoló; Del Grosso, Ambra; Antoni, Claudia; Cecchini, Marco; Corradetti, Renato; Pavone, Francesco S; Calamai, Martino

2017-01-01

Alzheimer's disease is a multifactorial disorder caused by the interaction of genetic, epigenetic and environmental factors. The formation of cytotoxic oligomers consisting of A β peptide is widely accepted as being one of the main key events triggering the development of Alzheimer's disease. A β peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP). Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro . By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal (mChAPPmGFP), respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β -secretase BACE1, or the α -secretase ADAM10, fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the proteolytic processing of APP in real time. In order to allow the discrimination between the α - and the β -secretase activity, we have created a variant of mChAPPmGFP with a mutation that inhibits the α -secretase cleavage without perturbing the β -secretase processing. Moreover, we obtained a quantitatively robust estimate of the changes in the red/green ratio for the above conditions by using a flow cytometer able to simultaneously excite and measure the red and green fluorescence. Our novel approach lay the foundation for a bioassay suitable to study the effect of drugs or particular conditions, to investigate in an unbiased way the the proteolytic processing of APP in single living cells in order, and to elucidate the causes of the variability and the factors driving the processing of APP.

A genetically encoded fluorescent sensor of ERK activity.

PubMed

Harvey, Christopher D; Ehrhardt, Anka G; Cellurale, Cristina; Zhong, Haining; Yasuda, Ryohei; Davis, Roger J; Svoboda, Karel

2008-12-09

The activity of the ERK has complex spatial and temporal dynamics that are important for the specificity of downstream effects. However, current biochemical techniques do not allow for the measurement of ERK signaling with fine spatiotemporal resolution. We developed a genetically encoded, FRET-based sensor of ERK activity (the extracellular signal-regulated kinase activity reporter, EKAR), optimized for signal-to-noise ratio and fluorescence lifetime imaging. EKAR selectively and reversibly reported ERK activation in HEK293 cells after epidermal growth factor stimulation. EKAR signals were correlated with ERK phosphorylation, required ERK activity, and did not report the activities of JNK or p38. EKAR reported ERK activation in the dendrites and nucleus of hippocampal pyramidal neurons in brain slices after theta-burst stimuli or trains of back-propagating action potentials. EKAR therefore permits the measurement of spatiotemporal ERK signaling dynamics in living cells, including in neuronal compartments in intact tissues.

Novel assay for direct fluorescent imaging of sialidase activity

NASA Astrophysics Data System (ADS)

Tomin, A.; Shkandina, T.; Bilyy, R.

2011-07-01

Here we describe a novel approach to sialidase activity estimation. Sialidases (EC 3.2.1.18, exo-α-sialidases), also known as neuraminidases, are the group of enzymes, which hydrolyze the glycoside bound between terminal sialic acid and subsequent carbohydrate residue in glycoproteins and glycolipids. Sialic acids are the group of monosaccharides with acidic properties, since they are acetylated or glycolylated derivates of neuraminic acid. Flu and some other viruses use neuraminidase activity to infect host cells. The level of sialylation was shown to be tightly connected with tumor cell invasiveness and metastatic potential, sialylation level also determines the clearance of aged or virus-infected cells. Thus, detection of sialidase activity is of primary importance for clinical diagnostics as well as life science research. The authors developed the assay for both visualization and estimation of sialidase activity in living cells. Previously known methods for sialidase activity detection required destruction of cellular material, or were low-sensitive, or provided no information on the activity localization in certain intracellular compartment. To overcome these problems, a fluorogenic neuraminidase substrate, 4-MUNA was utilized, and the method for detection of neuraminidase activity using fluorescent microscopy was proposed, it provided a high signal level and information on cellular localization of the studied enzyme. By using this approach the increase of sialidase activity on apoptotic cells was demonstrated in comparison to viable and primary necrotic cells.

Protein-Coupled Fluorescent Probe To Visualize Potassium Ion Transition on Cellular Membranes.

PubMed

Hirata, Tomoya; Terai, Takuya; Yamamura, Hisao; Shimonishi, Manabu; Komatsu, Toru; Hanaoka, Kenjiro; Ueno, Tasuku; Imaizumi, Yuji; Nagano, Tetsuo; Urano, Yasuteru

2016-03-01

K(+) is the most abundant metal ion in cells, and changes of [K(+)] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K(+)] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K(+), TLSHalo. TLSHalo is responsive to [K(+)] in the physiological range, with good selectivity over Na(+) and retains its K(+)-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K(+)] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K(+)] change induced by K(+) ionophores or by activation of a native Ca(2+)-dependent K(+) channel (BK channel). Further, we show that K(+) efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K(+) dynamics with high spatiotemporal resolution.

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements.

PubMed

Suzuki, Miho; Sakata, Ichiro; Sakai, Takafumi; Tomioka, Hiroaki; Nishigaki, Koichi; Tramier, Marc; Coppey-Moisan, Maïté

2015-12-15

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Rapid and sensitive detection of early esophageal squamous cell carcinoma with fluorescence probe targeting dipeptidylpeptidase IV

PubMed Central

Onoyama, Haruna; Kamiya, Mako; Kuriki, Yugo; Komatsu, Toru; Abe, Hiroyuki; Tsuji, Yosuke; Yagi, Koichi; Yamagata, Yukinori; Aikou, Susumu; Nishida, Masato; Mori, Kazuhiko; Yamashita, Hiroharu; Fujishiro, Mitsuhiro; Nomura, Sachiyo; Shimizu, Nobuyuki; Fukayama, Masashi; Koike, Kazuhiko; Urano, Yasuteru; Seto, Yasuyuki

2016-01-01

Early detection of esophageal squamous cell carcinoma (ESCC) is an important prognosticator, but is difficult to achieve by conventional endoscopy. Conventional lugol chromoendoscopy and equipment-based image-enhanced endoscopy, such as narrow-band imaging (NBI), have various practical limitations. Since fluorescence-based visualization is considered a promising approach, we aimed to develop an activatable fluorescence probe to visualize ESCCs. First, based on the fact that various aminopeptidase activities are elevated in cancer, we screened freshly resected specimens from patients with a series of aminopeptidase-activatable fluorescence probes. The results indicated that dipeptidylpeptidase IV (DPP-IV) is specifically activated in ESCCs, and would be a suitable molecular target for detection of esophageal cancer. Therefore, we designed, synthesized and characterized a series of DPP-IV-activatable fluorescence probes. When the selected probe was topically sprayed onto endoscopic submucosal dissection (ESD) or surgical specimens, tumors were visualized within 5 min, and when the probe was sprayed on biopsy samples, the sensitivity, specificity and accuracy reached 96.9%, 85.7% and 90.5%. We believe that DPP-IV-targeted activatable fluorescence probes are practically translatable as convenient tools for clinical application to enable rapid and accurate diagnosis of early esophageal cancer during endoscopic or surgical procedures. PMID:27245876

Characterization, catalyzed water oxidation and anticancer activities of a NIR BODIPY-Mn polymer

NASA Astrophysics Data System (ADS)

Lan, Ya-Quan; Xiao, Ke-Jing; Wu, Yun-Jie; Chen, Qiu-Yun

2017-04-01

To obtain near-IR absorbing biomaterials as fluorescence cellular imaging and anticancer agents for hypoxic cancer cell, a nano NIR fluorescence Mn(III/IV) polymer (PMnD) was spectroscopically characterized. The PMnD shows strong emission at 661 nm when excited with 643 nm. Furthermore, PMnD can catalyze water oxidation to generate dioxygen when irradiated by red LED light (10 W). In particular, the PMnD can enter into HepG-2 cells and mitochondria. Both anticancer activity and the inhibition of the expression of HIF-1α for PMnD were concentration dependent. Our results demonstrate that PMnD can be developed as mitochondria targeted imaging agents and new inhibitors for HIF-1 in hypoxic cancer cells.

Superresolution imaging in live Caulobacter crescentus cells using photoswitchable enhanced yellow fluorescent protein

NASA Astrophysics Data System (ADS)

Biteen, Julie S.; Thompson, Michael A.; Tselentis, Nicole K.; Shapiro, Lucy; Moerner, W. E.

2009-02-01

Recently, photoactivation and photoswitching were used to control single-molecule fluorescent labels and produce images of cellular structures beyond the optical diffraction limit (e.g., PALM, FPALM, and STORM). While previous live-cell studies relied on sophisticated photoactivatable fluorescent proteins, we show in the present work that superresolution imaging can be performed with fusions to the commonly used fluorescent protein EYFP. Rather than being photoactivated, however, EYFP can be reactivated with violet light after apparent photobleaching. In each cycle after initial imaging, only a sparse subset fluorophores is reactivated and localized, and the final image is then generated from the measured single-molecule positions. Because these methods are based on the imaging nanometer-sized single-molecule emitters and on the use of an active control mechanism to produce sparse sub-ensembles, we suggest the phrase "Single-Molecule Active-Control Microscopy" (SMACM) as an inclusive term for this general imaging strategy. In this paper, we address limitations arising from physiologically imposed upper boundaries on the fluorophore concentration by employing dark time-lapse periods to allow single-molecule motions to fill in filamentous structures, increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot. We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live Caulobacter crescentus cells with sub-40-nm resolution for the first time. Furthermore, we quantify the reactivation quantum yield of EYFP, and find this to be 1.6 x 10-6, on par with conventional photoswitchable fluorescent proteins like Dronpa. These studies show that EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells.

Detecting and Imaging of γ-Glutamytranspeptidase Activity in Serum, Live Cells, and Pathological Tissues with a High Signal-Stability Probe by Releasing a Precipitating Fluorochrome.

PubMed

Ou-Yang, Juan; Li, Yong-Fei; Wu, Ping; Jiang, Wen-Li; Liu, Hong-Wen; Li, Chun-Yan

2018-06-20

γ-Glutamytranspeptidase (GGT) is a significant tumor-related biomarker that overexpresses in several tumor cells. Accurate detection and imaging of GGT activity in serum, live cells, and pathological tissues hold great significance for cancer diagnosis, treatment, and management. Recently developed small molecule fluorescent probes for GGT tend to diffuse to the whole cytoplasm and then translocate out of live cells after enzymatic reaction, which make them fail to provide high spatial resolution and long-term imaging in biological systems. To address these problems, a novel fluorescent probe (HPQ-PDG) which releases a precipitating fluorochrome upon the catalysis of GGT is designed and synthesized. HPQ-PDG is able to detect GGT activity with high spatial resolution and good signal-stability. The large Stokes shift of the probe enables it to detect the activity of GGT in serum samples with high sensitivity. To our delight, the probe is used for imaging GGT activity in live cells with the ability of discriminating cancer cells from normal cells. What's more, we successfully apply it for pathological tissues imaging, with the results indicating that the potential application of HPQ-PDG in histopathological examination. All these results demonstrate the potential application of HPQ-PDG in the clinic.

Actin microfilaments participate in the regulation of the COL1A1 promoter activity in ROS17/2.8 cells under simulated microgravity

NASA Astrophysics Data System (ADS)

Dai, Zhongquan; Li, Yinghui; Ding, Bai; Zhang, Xiaoyou; Tan, Yingjun; Wan, Yumin

2006-01-01

IntroductionMicrogravity is thought to decrease osteoblastic activity and induce osteoporosis during spaceflight, but the mechanisms, particularly the attendant changes in gene expression, are not well understood. It is suspected that the cytoskeletal system is involved in the manifold changes of cell shape, function, and signaling under microgravity conditions. MethodsWe constructed cell lines stably transfected with pJI36EGFP and pJI23EGFP, which contained a 3.6 and a 2.3 kb fragment, respectively, of the α1(I) collagen gene (COL1A1) promoter fused with the enhanced green fluorescence protein (EGFP) reporter gene. We then developed a semi-quantitative analysis of EGFP fluorescence intensity to evaluate the effects of clinorotation and/or cytochalasin B on the activity of the COL1A1 promoter. Simultaneously, we assessed the collagen type I protein content versus total protein content in clinorotated or control osteoblasts, using immunocytochemistry and the Bradford method, respectively. ResultsThe fluorescence intensity analysis revealed that the expression of COL1A1-EGFP increased in GFP-ROS cells clinorotated for 24 or 48 h, as compared with stationary control cultures. We observed a similar trend in collagen type I content, as assessed by immunocytochemistry. We found that the osteoblast microfilaments tended to disassemble and show a reduction in stress fibers under space flight and clinorotation. Treatment with cytochalasin B in normal gravity resulted in a dose-dependent increase of EGFP fluorescence intensity, indicating that disruption of the actin system was associated with increased activity of the COL1A1 promoter. ConclusionOur study demonstrates that disrupting the actin cytoskeleton by treatment with cytochalasin B and real or simulated microgravity conditions led to altered COL1A1 promoter activity. Together, these results suggest that actin may participate in the regulation of the COL1A1 promoter activity under microgravity conditions.

A quantitative characterization of the yeast heterotrimeric G protein cycle

PubMed Central

Yi, Tau-Mu; Kitano, Hiroaki; Simon, Melvin I.

2003-01-01

The yeast mating response is one of the best understood heterotrimeric G protein signaling pathways. Yet, most descriptions of this system have been qualitative. We have quantitatively characterized the heterotrimeric G protein cycle in yeast based on direct in vivo measurements. We used fluorescence resonance energy transfer to monitor the association state of cyan fluorescent protein (CFP)-Gα and Gβγ-yellow fluorescent protein (YFP), and we found that receptor-mediated G protein activation produced a loss of fluorescence resonance energy transfer. Quantitative time course and dose–response data were obtained for both wild-type and mutant cells possessing an altered pheromone response. These results paint a quantitative portrait of how regulators such as Sst2p and the C-terminal tail of α-factor receptor modulate the kinetics and sensitivity of G protein signaling. We have explored critical features of the dynamics including the rapid rise and subsequent decline of active G proteins during the early response, and the relationship between the G protein activation dose–response curve and the downstream dose–response curves for cell-cycle arrest and transcriptional induction. Fitting the data to a mathematical model produced estimates of the in vivo rates of heterotrimeric G protein activation and deactivation in yeast. PMID:12960402

Cytoskeletal regulation of CD44 membrane organization and interactions with E-selectin.

PubMed

Wang, Ying; Yago, Tadayuki; Zhang, Nan; Abdisalaam, Salim; Alexandrakis, George; Rodgers, William; McEver, Rodger P

2014-12-19

Interactions of CD44 on neutrophils with E-selectin on activated endothelial cells mediate rolling under flow, a prerequisite for neutrophil arrest and migration into perivascular tissues. How CD44 functions as a rolling ligand despite its weak affinity for E-selectin is unknown. We examined the nanometer scale organization of CD44 on intact cells. CD44 on leukocytes and transfected K562 cells was cross-linked within a 1.14-nm spacer. Depolymerizing actin with latrunculin B reduced cross-linking. Fluorescence resonance energy transfer (FRET) revealed tight co-clustering between CD44 fused to yellow fluorescent protein (YFP) and CD44 fused to cyan fluorescent protein on K562 cells. Latrunculin B reduced FRET-reported co-clustering. Number and brightness analysis confirmed actin-dependent CD44-YFP clusters on living cells. CD44 lacking binding sites for ankyrin and for ezrin/radixin/moesin (ERM) proteins on its cytoplasmic domain (ΔANKΔERM) did not cluster. Unexpectedly, CD44 lacking only the ankyrin-binding site (ΔANK) formed larger but looser clusters. Fluorescence recovery after photobleaching demonstrated increased CD44 mobility by latrunculin B treatment or by deleting the cytoplasmic domain. ΔANKΔERM mobility increased only modestly, suggesting that the cytoplasmic domain engages the cytoskeleton by an additional mechanism. Ex vivo differentiated CD44-deficient neutrophils expressing exogenous CD44 rolled on E-selectin and activated Src kinases after binding anti-CD44 antibody. In contrast, differentiated neutrophils expressing ΔANK had impaired rolling and kinase activation. These data demonstrate that spectrin and actin networks regulate CD44 clustering and suggest that ankyrin enhances CD44-mediated neutrophil rolling and signaling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Cytoskeletal Regulation of CD44 Membrane Organization and Interactions with E-selectin*

PubMed Central

Wang, Ying; Yago, Tadayuki; Zhang, Nan; Abdisalaam, Salim; Alexandrakis, George; Rodgers, William; McEver, Rodger P.

2014-01-01

Interactions of CD44 on neutrophils with E-selectin on activated endothelial cells mediate rolling under flow, a prerequisite for neutrophil arrest and migration into perivascular tissues. How CD44 functions as a rolling ligand despite its weak affinity for E-selectin is unknown. We examined the nanometer scale organization of CD44 on intact cells. CD44 on leukocytes and transfected K562 cells was cross-linked within a 1.14-nm spacer. Depolymerizing actin with latrunculin B reduced cross-linking. Fluorescence resonance energy transfer (FRET) revealed tight co-clustering between CD44 fused to yellow fluorescent protein (YFP) and CD44 fused to cyan fluorescent protein on K562 cells. Latrunculin B reduced FRET-reported co-clustering. Number and brightness analysis confirmed actin-dependent CD44-YFP clusters on living cells. CD44 lacking binding sites for ankyrin and for ezrin/radixin/moesin (ERM) proteins on its cytoplasmic domain (ΔANKΔERM) did not cluster. Unexpectedly, CD44 lacking only the ankyrin-binding site (ΔANK) formed larger but looser clusters. Fluorescence recovery after photobleaching demonstrated increased CD44 mobility by latrunculin B treatment or by deleting the cytoplasmic domain. ΔANKΔERM mobility increased only modestly, suggesting that the cytoplasmic domain engages the cytoskeleton by an additional mechanism. Ex vivo differentiated CD44-deficient neutrophils expressing exogenous CD44 rolled on E-selectin and activated Src kinases after binding anti-CD44 antibody. In contrast, differentiated neutrophils expressing ΔANK had impaired rolling and kinase activation. These data demonstrate that spectrin and actin networks regulate CD44 clustering and suggest that ankyrin enhances CD44-mediated neutrophil rolling and signaling. PMID:25359776

Quantifying substrate uptake by individual cells of marine bacterioplankton by catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography.

PubMed

Sintes, Eva; Herndl, Gerhard J

2006-11-01

Catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography (MICRO-CARD-FISH) is increasingly being used to obtain qualitative information on substrate uptake by individual members of specific prokaryotic communities. Here we evaluated the potential for using this approach quantitatively by relating the measured silver grain area around cells taking up (3)H-labeled leucine to bulk leucine uptake measurements. The increase in the silver grain area over time around leucine-assimilating cells of coastal bacterial assemblages was linear during 4 to 6 h of incubation. By establishing standardized conditions for specific activity levels and concomitantly performing uptake measurements with the bulk community, MICRO-CARD-FISH can be used quantitatively to determine uptake rates on a single-cell level. Therefore, this approach allows comparisons of single-cell activities for bacterial communities obtained from different sites or growing under different ecological conditions.

Quantifying Substrate Uptake by Individual Cells of Marine Bacterioplankton by Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Microautoradiography▿

PubMed Central

Sintes, Eva; Herndl, Gerhard J.

2006-01-01

Catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography (MICRO-CARD-FISH) is increasingly being used to obtain qualitative information on substrate uptake by individual members of specific prokaryotic communities. Here we evaluated the potential for using this approach quantitatively by relating the measured silver grain area around cells taking up 3H-labeled leucine to bulk leucine uptake measurements. The increase in the silver grain area over time around leucine-assimilating cells of coastal bacterial assemblages was linear during 4 to 6 h of incubation. By establishing standardized conditions for specific activity levels and concomitantly performing uptake measurements with the bulk community, MICRO-CARD-FISH can be used quantitatively to determine uptake rates on a single-cell level. Therefore, this approach allows comparisons of single-cell activities for bacterial communities obtained from different sites or growing under different ecological conditions. PMID:16950912

Laser-Induced Fluorescence Detection in High-Throughput Screening of Heterogeneous Catalysts and Single Cells Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Hui

2001-01-01

Laser-induced fluorescence detection is one of the most sensitive detection techniques and it has found enormous applications in various areas. The purpose of this research was to develop detection approaches based on laser-induced fluorescence detection in two different areas, heterogeneous catalysts screening and single cell study. First, the author introduced laser-induced imaging (LIFI) as a high-throughput screening technique for heterogeneous catalysts to explore the use of this high-throughput screening technique in discovery and study of various heterogeneous catalyst systems. This scheme is based on the fact that the creation or the destruction of chemical bonds alters the fluorescence properties ofmore » suitably designed molecules. By irradiating the region immediately above the catalytic surface with a laser, the fluorescence intensity of a selected product or reactant can be imaged by a charge-coupled device (CCD) camera to follow the catalytic activity as a function of time and space. By screening the catalytic activity of vanadium pentoxide catalysts in oxidation of naphthalene, they demonstrated LIFI has good detection performance and the spatial and temporal resolution needed for high-throughput screening of heterogeneous catalysts. The sample packing density can reach up to 250 x 250 subunits/cm 2 for 40-μm wells. This experimental set-up also can screen solid catalysts via near infrared thermography detection. In the second part of this dissertation, the author used laser-induced native fluorescence coupled with capillary electrophoresis (LINF-CE) and microscope imaging to study the single cell degranulation. On the basis of good temporal correlation with events observed through an optical microscope, they have identified individual peaks in the fluorescence electropherograms as serotonin released from the granular core on contact with the surrounding fluid.« less

Seamless Combination of Fluorescence-Activated Cell Sorting and Hanging-Drop Networks for Individual Handling and Culturing of Stem Cells and Microtissue Spheroids.

PubMed

Birchler, Axel; Berger, Mischa; Jäggin, Verena; Lopes, Telma; Etzrodt, Martin; Misun, Patrick Mark; Pena-Francesch, Maria; Schroeder, Timm; Hierlemann, Andreas; Frey, Olivier

2016-01-19

Open microfluidic cell culturing devices offer new possibilities to simplify loading, culturing, and harvesting of individual cells or microtissues due to the fact that liquids and cells/microtissues are directly accessible. We present a complete workflow for microfluidic handling and culturing of individual cells and microtissue spheroids, which is based on the hanging-drop network concept: The open microfluidic devices are seamlessly combined with fluorescence-activated cell sorting (FACS), so that individual cells, including stem cells, can be directly sorted into specified culturing compartments in a fully automated way and at high accuracy. Moreover, already assembled microtissue spheroids can be loaded into the microfluidic structures by using a conventional pipet. Cell and microtissue culturing is then performed in hanging drops under controlled perfusion. On-chip drop size control measures were applied to stabilize the system. Cells and microtissue spheroids can be retrieved from the chip by using a parallelized transfer method. The presented methodology holds great promise for combinatorial screening of stem-cell and multicellular-spheroid cultures.

Chronology of Islet Differentiation Revealed By Temporal Cell Labeling

PubMed Central

Miyatsuka, Takeshi; Li, Zhongmei; German, Michael S.

2009-01-01

OBJECTIVE Neurogenin 3 plays a pivotal role in pancreatic endocrine differentiation. Whereas mouse models expressing reporters such as eGFP or LacZ under the control of the Neurog3 gene enable us to label cells in the pancreatic endocrine lineage, the long half-life of most reporter proteins makes it difficult to distinguish cells actively expressing neurogenin 3 from differentiated cells that have stopped transcribing the gene. RESEARCH DESIGN AND METHODS In order to separate the transient neurogenin 3 –expressing endocrine progenitor cells from the differentiating endocrine cells, we developed a mouse model (Ngn3-Timer) in which DsRed-E5, a fluorescent protein that shifts its emission spectrum from green to red over time, was expressed transgenically from the NEUROG3 locus. RESULTS In the Ngn3-Timer embryos, green-dominant cells could be readily detected by microscopy or flow cytometry and distinguished from green/red double-positive cells. When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h. The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas. CONCLUSIONS The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells. PMID:19478145

Mechanism study of low-energy laser irradiation-induced lung adenocarcinoma cell proliferation by FRET in living cell

NASA Astrophysics Data System (ADS)

Wang, Fang; Chen, Xiao-Chuan; Xing, Da

2004-07-01

Low-energy laser irradiation (LELI) has been shown to promote cell proliferation in various cell types, yet the mechanism of which has not been fully clarified. The Ras/Raf/MEK (mitogen-activated protein kinase)ERK kinase)/ERK (extracellular-signal-regulated kinase) signaling pathway is a network that govern proliferation, differentiation and cell survival. Recent studies suggested that Ras/Raf/MEK/ERK pathway is involved in the LELI-induced cell proliferation. Here, we utilized fluorescence resonance energy transfer (FRET) technique to investigate the effect of LELI on Ras/Raf signaling pathway in living cells. Raichu-Ras reporter plasmid was utilized which consisted of fusions of H-ras, the Ras-binding domain of Raf(RafRBD), a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP), so that intramolecular binding of GTP-Ras to RafRBD brings CFP close to YFP and increases FRET between CFP and YFP. Human lung adenocarcinoma cell line (ASTC-a-1) were transfected with the plasmid (pRaichu-Ras) and then were treated by LELI. The living cell imaging showed the increase of FRET at different time points after LELI at the dose of 1.8 J/cm2, which corresponds to the Ras/Raf activation assayed by Western Blotting. Furthermore, this dose of LELI enhanced the proliferation of ASTC-a-1 cells. Taken together, these in vivo imaging data provide direct evidences with temporal or spatial resolution that Ras/Raf/MEK/ pathway plays an important role in LELI-promoted cell proliferation.

Study and selection of in vivo protein interactions by coupling bimolecular fluorescence complementation and flow cytometry.

PubMed

Morell, Montse; Espargaro, Alba; Aviles, Francesc Xavier; Ventura, Salvador

2008-01-01

We present a high-throughput approach to study weak protein-protein interactions by coupling bimolecular fluorescent complementation (BiFC) to flow cytometry (FC). In BiFC, the interaction partners (bait and prey) are fused to two rationally designed fragments of a fluorescent protein, which recovers its function upon the binding of the interacting proteins. For weak protein-protein interactions, the detected fluorescence is proportional to the interaction strength, thereby allowing in vivo discrimination between closely related binders with different affinity for the bait protein. FC provides a method for high-speed multiparametric data acquisition and analysis; the assay is simple, thousands of cells can be analyzed in seconds and, if required, selected using fluorescence-activated cell sorting (FACS). The combination of both methods (BiFC-FC) provides a technically straightforward, fast and highly sensitive method to validate weak protein interactions and to screen and identify optimal ligands in biologically synthesized libraries. Once plasmids encoding the protein fusions have been obtained, the evaluation of a specific interaction, the generation of a library and selection of active partners using BiFC-FC can be accomplished in 5 weeks.

Fluorescence lifetime spectroscopy in multiple-scattering environments: an application to biotechnology

NASA Astrophysics Data System (ADS)

Cerussi, Albert E.; Gratton, Enrico; Fantini, Sergio

1999-07-01

Over the past few years, there has been significant research activity devoted to the application of fluorescence spectroscopy to strongly scattering media, where photons propagate diffusely. Much of this activity focused on fluorescence as a source of contrast enhancement in optical tomography. Our efforts have emphasized the quantitative recovery of fluorescence parameters for spectroscopy. Using a frequency-domain diffusion-based model, we have successfully recovered the lifetime, the absolute quantum yield, the fluorophore concentration, and the emission spectrum of the fluorophore, as well as the absorption and the reduced scattering coefficients at the emission wavelength of the medium in different measurements. In this contribution, we present a sensitive monitor of the binding between ethidium bromide and bovine cells in fresh milk. The spectroscopic contrast was the approximately tenfold increase in the ethidium bromide lifetime upon binding to DNA. The measurement clearly demonstrated that we could quantitatively measure the density of cells in the milk, which is an application vital to the tremendous economic burden of bovine subclinical mastitis detection. Furthermore, we may in principle use the spirit of this technique as a quantitative monitor of the binding of fluorescent drugs inside tissues. This is a first step towards lifetime spectroscopy in tissues.

Dynamic [Cl-]i measurement with chloride sensing quantum dots nanosensor in epithelial cells

NASA Astrophysics Data System (ADS)

Wang, Yuchi; Mao, Hua; Wong, Lid B.

2010-02-01

We have synthesized a chloride sensing quantum dots (QD) nanosensor, Cl-QD, for the dynamic measurements of chloride ion concentration in the millimolar range, a sensitivity that is applicable to most physiological intracellular chloride ion concentration ([Cl-]i) measurements in epithelial cells. The Cl-QD is synthesized by conjugating an anion receptor, 1-(2-mercapto-ethyl)-3-phenyl-thiourea (MEPTU) to a water soluble CdSe/ZnS QD at an emission wavelength of 620 nm. Upon binding of chloride ions to the Cl-QD, a photo-induced electron transfer mechanism caused the fluorescence of the QD to quench. This resulted in an inversely proportional relationship between the chloride ion concentration and the fluorescence intensity of the Cl-QD. We have utilized this Cl-QD to measure [Cl-]i in T84 and CF-PAC cultured cells, with either the C1C-2 or CFTR chloride channels being manipulated by pharmacological chloride channel activators and inhibitors. Activations of C1C-2 and CFTR chloride channels in T84 by the respective lubiprostone and genistein caused predictive increases in the fluorescence of the Cl-QD, i.e., a decrease of [Cl-]i. Conversely, glibenclamide, a chloride channel inhibitor, applied to the CF-PAC cells caused a predictable decrease in the fluorescence of Cl-QD due to the increase of [Cl-]i. These are the first data in using QD-based chloride ion sensors for dynamic measurements of intracellular chloride ion concentrations in epithelial cells.

Biological applications of confocal fluorescence polarization microscopy

NASA Astrophysics Data System (ADS)

Bigelow, Chad E.

Fluorescence polarization microscopy is a powerful modality capable of sensing changes in the physical properties and local environment of fluorophores. In this thesis we present new applications for the technique in cancer diagnosis and treatment and explore the limits of the modality in scattering media. We describe modifications to our custom-built confocal fluorescence microscope that enable dual-color imaging, optical fiber-based confocal spectroscopy and fluorescence polarization imaging. Experiments are presented that indicate the performance of the instrument for all three modalities. The limits of confocal fluorescence polarization imaging in scattering media are explored and the microscope parameters necessary for accurate polarization images in this regime are determined. A Monte Carlo routine is developed to model the effect of scattering on images. Included in it are routines to track the polarization state of light using the Mueller-Stokes formalism and a model for fluorescence generation that includes sampling the excitation light polarization ellipse, Brownian motion of excited-state fluorophores in solution, and dipole fluorophore emission. Results from this model are compared to experiments performed on a fluorophore-embedded polymer rod in a turbid medium consisting of polystyrene microspheres in aqueous suspension. We demonstrate the utility of the fluorescence polarization imaging technique for removal of contaminating autofluorescence and for imaging photodynamic therapy drugs in cell monolayers. Images of cells expressing green fluorescent protein are extracted from contaminating fluorescein emission. The distribution of meta-tetrahydroxypheny1chlorin in an EMT6 cell monolayer is also presented. A new technique for imaging enzyme activity is presented that is based on observing changes in the anisotropy of fluorescently-labeled substrates. Proof-of-principle studies are performed in a model system consisting of fluorescently labeled bovine serum albumin attached to sepharose beads. The action of trypsin and proteinase K on the albumin is monitored to demonstrate validity of the technique. Images of the processing of the albumin in J774 murine macrophages are also presented indicating large intercellular differences in enzyme activity. Future directions for the technique are also presented, including the design of enzyme probes specific for prostate specific antigen based on fluorescently-labeled dendrimers. A technique for enzyme imaging based on extracellular autofluorescence is also proposed.

Effect of azathioprine on Na(+)/H(+) exchanger activity in dendritic cells.

PubMed

Bhandaru, Madhuri; Pasham, Venkanna; Yang, Wenting; Bobbala, Diwakar; Rotte, Anand; Lang, Florian

2012-01-01

Azathioprine is a powerful immunosuppressive drug, which is partially effective by interfering with the maturation and function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs are stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS), paralleled by activation of the Na(+)/H(+) exchanger. The carrier is involved in the regulation of cytosolic pH, cell volume and migration. The present study explored whether azathioprine influences Na(+)/H(+) exchanger activity in DCs. DCs were isolated from murine bone marrow, cytosolic pH (pH(i)) was estimated utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM) fluorescence, Na(+)/H(+) exchanger activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNFα release utilizing ELISA, and migration utilizing transwell migration assays. Exposure of DCs to lipopolysaccharide (LPS, 1 μg/ml) led to a transient increase of Na(+)/H(+) exchanger activity, an effect paralleled by ROS formation, increased cell volume, TNFα production and stimulated migration. Azathioprine (10 μM) did not significantly alter the Na(+)/H(+) exchanger activity, cell volume and ROS formation prior to LPS exposure but significantly blunted the LPS-induced stimulation of Na(+)/H(+) exchanger activity, ROS formation, cell swelling, TNFα production and cell migration. In conclusion, azathioprine interferes with the activation of dendritic cell Na(+)/H(+) exchanger by bacterial lipopolysaccharides, an effect likely participating in the anti-inflammatory action of the drug. Copyright © 2012 S. Karger AG, Basel.

Nonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength

PubMed Central

Hou, Wenya; Kielhorn, Martin; Arai, Yoshiyuki; Nagai, Takeharu; Kessels, Michael M.; Qualmann, Britta; Heintzmann, Rainer

2016-01-01

Structured illumination microscopy (SIM) is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM (LSIM). To achieve the required nonlinear dependence in nonlinear SIM (NL-SIM) we exploited the photoswitching of the recently introduced fluorophore Kohinoor. It is particularly suitable due to its positive contrast photoswitching characteristics. Contrary to other reversibly photoswitchable fluorescent proteins which only have high photostability in living cells, Kohinoor additionally showed little degradation in fixed cells over many switching cycles. PMID:27783656

Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology.

PubMed

Duarte, José M; Barbier, Içvara; Schaerli, Yolanda

2017-11-17

Synthetic biologists increasingly rely on directed evolution to optimize engineered biological systems. Applying an appropriate screening or selection method for identifying the potentially rare library members with the desired properties is a crucial step for success in these experiments. Special challenges include substantial cell-to-cell variability and the requirement to check multiple states (e.g., being ON or OFF depending on the input). Here, we present a high-throughput screening method that addresses these challenges. First, we encapsulate single bacteria into microfluidic agarose gel beads. After incubation, they harbor monoclonal bacterial microcolonies (e.g., expressing a synthetic construct) and can be sorted according their fluorescence by fluorescence activated cell sorting (FACS). We determine enrichment rates and demonstrate that we can measure the average fluorescent signals of microcolonies containing phenotypically heterogeneous cells, obviating the problem of cell-to-cell variability. Finally, we apply this method to sort a pBAD promoter library at ON and OFF states.

Visualizing and quantifying protein secretion using a Renilla luciferase-GFP fusion protein.

PubMed

Liu, J; Wang, Y; Szalay, A A; Escher, A

2000-01-01

We have shown previously that an engineered form of Renilla luciferase (SRUC) can be secreted as a functional enzyme by mammalian cells, and that fusing wild-type Renilla luciferase with the green fluorescent protein from Aequorea victoria (GFP) yields a chimeric protein retaining light-emission properties similar to that of unfused Renilla luciferase and GFP. In the work presented here, SRUC was fused with GFP to determine whether it could be used to both visualize and quantify protein secretion in mammalian cells. Simian COS-7 and Chinese hamster ovary (CHO) cells were transiently transfected with gene constructs encoding a secreted or an intracellular version of a Renilla luciferase-GFP fusion protein. Renilla luciferase activity was measured from COS-7 cell lysates and culture media, and GFP activity was detected in CHO cells using fluorescence microscopy. Data indicated that the SRUC-GFP fusion protein was secreted as a chimeric protein that had both Renilla luciferase and GFP activity. This fusion protein could be a useful marker for the study of protein secretion in mammalian cells. Copyright 2000 John Wiley & Sons, Ltd.

Clostridium perfringens Iota Toxin: Binding Studies and Characterization of Cell Surface Receptor by Fluorescence-Activated Cytometry

PubMed Central

Stiles, Bradley G.; Hale, Martha L.; Marvaud, Jean-Christophe; Popoff, Michel R.

2000-01-01

The binding characteristics of iota toxin, a binary enterotoxin produced by Clostridium perfringens type E, were studied by fluorescence-activated cytometry. The proteolytically activated binding component of iota toxin, iota b (Ib), bound to various cell types when incubated at 4, 25, or 37°C for 10 min. The binding of Ib was inhibited by antisera against C. perfringens type E or Clostridium spiroforme culture supernatants, but not C. perfringens types C or D. Pretreatment of Vero cells with glycosidases or lectins did not affect Ib interactions, while pronase effectively prevented Ib binding to the cell surface. The Ib protomer (Ibp) bound to the cell surface, but trypsinization of Ibp was necessary for docking of the ADP-ribosylating component, iota a (Ia). Ia attached to cell-bound Ib within 10 min at 37°C, but surface levels of Ia decreased 90% after 30 min and were undetectable by 60 min. Detectable surface levels of Ib also diminished over time, and Western blot analysis suggested internalization or embedment of Ib into the membrane. PMID:10816501

Clostridium perfringens iota toxin: binding studies and characterization of cell surface receptor by fluorescence-activated cytometry.

PubMed

Stiles, B G; Hale, M L; Marvaud, J C; Popoff, M R

2000-06-01

The binding characteristics of iota toxin, a binary enterotoxin produced by Clostridium perfringens type E, were studied by fluorescence-activated cytometry. The proteolytically activated binding component of iota toxin, iota b (Ib), bound to various cell types when incubated at 4, 25, or 37 degrees C for 10 min. The binding of Ib was inhibited by antisera against C. perfringens type E or Clostridium spiroforme culture supernatants, but not C. perfringens types C or D. Pretreatment of Vero cells with glycosidases or lectins did not affect Ib interactions, while pronase effectively prevented Ib binding to the cell surface. The Ib protomer (Ibp) bound to the cell surface, but trypsinization of Ibp was necessary for docking of the ADP-ribosylating component, iota a (Ia). Ia attached to cell-bound Ib within 10 min at 37 degrees C, but surface levels of Ia decreased 90% after 30 min and were undetectable by 60 min. Detectable surface levels of Ib also diminished over time, and Western blot analysis suggested internalization or embedment of Ib into the membrane.

Visualization of cell death in mice with focal cerebral ischemia using fluorescent annexin A5, propidium iodide, and TUNEL staining.

PubMed

Bahmani, Peyman; Schellenberger, Eyk; Klohs, Jan; Steinbrink, Jens; Cordell, Ryan; Zille, Marietta; Müller, Jochen; Harhausen, Denise; Hofstra, Leo; Reutelingsperger, Chris; Farr, Tracy Deanne; Dirnagl, Ulrich; Wunder, Andreas

2011-05-01

To monitor stroke-induced brain damage and assess neuroprotective therapies, specific imaging of cell death after cerebral ischemia in a noninvasive manner is highly desirable. Annexin A5 has been suggested as a marker for imaging cell death under various disease conditions including stroke. In this study, C57BL6/N mice received middle cerebral artery occlusion (MCAO) and were injected intravenously with either active or inactive Cy5.5-annexin A5 48 hours after reperfusion. Some mice also received propidium iodide (PI), a cell integrity marker. Only in mice receiving active Cy5.5-annexin A5 were fluorescence intensities significantly higher over the hemisphere ipsilateral to MCAO than on the contralateral side. This was detected noninvasively and ex vivo 4 and 8 hours after injection. The majority of cells positive for fluorescent annexin A5 were also positive for PI and fragmented DNA as detected by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining. This study demonstrates the high specificity of annexin A5 for visualization of cell death in a mouse model of stroke. To our knowledge, this is the first study to compare the distribution of injected active and inactive annexin A5, PI, and TUNEL staining. It provides important information on the experimental and potential clinical applications of annexin A5-based imaging agents in stroke.

Visualization of cell death in mice with focal cerebral ischemia using fluorescent annexin A5, propidium iodide, and TUNEL staining

PubMed Central

Bahmani, Peyman; Schellenberger, Eyk; Klohs, Jan; Steinbrink, Jens; Cordell, Ryan; Zille, Marietta; Müller, Jochen; Harhausen, Denise; Hofstra, Leo; Reutelingsperger, Chris; Farr, Tracy Deanne; Dirnagl, Ulrich; Wunder, Andreas

2011-01-01

To monitor stroke-induced brain damage and assess neuroprotective therapies, specific imaging of cell death after cerebral ischemia in a noninvasive manner is highly desirable. Annexin A5 has been suggested as a marker for imaging cell death under various disease conditions including stroke. In this study, C57BL6/N mice received middle cerebral artery occlusion (MCAO) and were injected intravenously with either active or inactive Cy5.5-annexin A5 48 hours after reperfusion. Some mice also received propidium iodide (PI), a cell integrity marker. Only in mice receiving active Cy5.5-annexin A5 were fluorescence intensities significantly higher over the hemisphere ipsilateral to MCAO than on the contralateral side. This was detected noninvasively and ex vivo 4 and 8 hours after injection. The majority of cells positive for fluorescent annexin A5 were also positive for PI and fragmented DNA as detected by terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling (TUNEL) staining. This study demonstrates the high specificity of annexin A5 for visualization of cell death in a mouse model of stroke. To our knowledge, this is the first study to compare the distribution of injected active and inactive annexin A5, PI, and TUNEL staining. It provides important information on the experimental and potential clinical applications of annexin A5-based imaging agents in stroke. PMID:21245871

Phospholamban mutants compete with wild type for SERCA binding in living cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gruber, Simon J.; Haydon, Suzanne; Thomas, David D., E-mail: ddt@umn.edu

2012-04-06

Highlights: Black-Right-Pointing-Pointer PLB phosphorylation in HEK cells increased FRET between YFP-PLB and CFP-SERCA. Black-Right-Pointing-Pointer Competition: Expressing loss-of-function PLB mutants in the system decreased FRET. Black-Right-Pointing-Pointer The FRET assay could screen potential therapeutic PLB mutants to activate SERCA. -- Abstract: We have used fluorescent fusion proteins stably expressed in HEK cells to detect directly the interaction between the sarcoplasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLB) in living cells, in order to design PLB mutants for gene therapy. Ca{sup 2+} cycling in muscle cells depends strongly on SERCA. Heart failure (HF), which contributes to 12% of US deaths, typically exhibits decreased SERCAmore » activity, and several potential therapies for HF aim to increase SERCA activity. We are investigating the use of LOF-PLB mutants (PLB{sub M}) as gene therapy vectors to increase SERCA activity. Active SERCA1a and WT-PLB, tagged at their N termini with fluorescent proteins (CFP and YFP), were coexpressed in stable HEK cell lines, and fluorescence resonance energy transfer (FRET) was used to detect their interaction directly. Phosphorylation of PLB, induced by forskolin, caused an increase in FRET from CFP-SERCA to YFP-PLB, indicating that SERCA inhibition can be relieved without dissociation of the complex. This suggests that a LOF mutant might bind to SERCA with sufficient affinity to complete effectively with WT-PLB, thus relieving SERCA inhibition. Therefore, we transiently expressed a series of PLB{sub M} in the CFP-SERCA/YFP-PLB cell line, and found decreased FRET, implying competition between PLB{sub M} and WT-PLB for binding to SERCA. These results establish this FRET assay as a rapid and quantitative means of screening PLB{sub M} for optimization of gene therapy to activate SERCA, as needed for gene therapy in HF.« less

Cellular Uptake and Localization of Polymyxins in Renal Tubular Cells Using Rationally Designed Fluorescent Probes.

PubMed

Yun, Bo; Azad, Mohammad A K; Nowell, Cameron J; Nation, Roger L; Thompson, Philip E; Roberts, Kade D; Velkov, Tony; Li, Jian

2015-12-01

Polymyxins are cyclic lipopeptide antibiotics that serve as a last line of defense against Gram-negative bacterial superbugs. However, the extensive accumulation of polymyxins in renal tubular cells can lead to nephrotoxicity, which is the major dose-limiting factor in clinical use. In order to gain further insights into the mechanism of polymyxin-induced nephrotoxicity, we have rationally designed novel fluorescent polymyxin probes to examine the localization of polymyxins in rat renal tubular (NRK-52E) cells. Our design strategy focused on incorporating a dansyl fluorophore at the hydrophobic centers of the polymyxin core structure. To this end, four novel regioselectively labeled monodansylated polymyxin B probes (MIPS-9541, MIPS-9542, MIPS-9543, and MIPS-9544) were designed, synthesized, and screened for their antimicrobial activities and apoptotic effects against rat kidney proximal tubular cells. On the basis of the assessment of antimicrobial activities, cellular uptake, and apoptotic effects on renal tubular cells, incorporation of a dansyl fluorophore at either position 6 or 7 (MIPS-9543 and MIPS-9544, respectively) of the polymyxin core structure appears to be an appropriate strategy for generating representative fluorescent polymyxin probes to be utilized in intracellular imaging and mechanistic studies. Furthermore, confocal imaging experiments utilizing these probes showed evidence of partial colocalization of the polymyxins with both the endoplasmic reticulum and mitochondria in rat renal tubular cells. Our results highlight the value of these new fluorescent polymyxin probes and provide further insights into the mechanism of polymyxin-induced nephrotoxicity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Establishment of Sf9 Transformants Constitutively Expressing PBAN Receptor Variants: Application to Functional Evaluation

PubMed Central

Lee, Jae Min; Hull, J. Joe; Kawai, Takeshi; Tsuneizumi, Kazuhide; Kurihara, Masaaki; Tanokura, Masaru; Nagata, Koji; Nagasawa, Hiromichi; Matsumoto, Shogo

2012-01-01

To facilitate further evaluation of pheromone biosynthesis activating neuropeptide receptor (PBANR) functionality and regulation, we generated cultured insect cell lines constitutively expressing green fluorescent protein chimeras of the recently identified Bombyx mori PBANR (BommoPBANR) and Pseudaletia separata PBANR (PsesePBANR) variants. Fluorescent chimeras included the BommoPBANR-A, -B, and -C variants and the PsesePBANR-B and -C variants. Cell lines expressing non-chimeric BommoPBANR-B and -C variants were also generated. Functional evaluation of these transformed cell lines using confocal laser microscopy revealed that a Rhodamine Red-labeled PBAN derivative (RR-C10PBANR2K) specifically co-localized with all of the respective PBANR variants at the plasma membrane. Near complete internalization of the fluorescent RR-C10PBANR2K ligand 30 min after binding was observed in all cell lines except those expressing the BommoPBANR-A variant, in which the ligand/receptor complex remained at the plasma membrane. Fluorescent Ca2+ imaging further showed that the BommoPBANR-A cell line exhibited drastically different Ca2+ mobilization kinetics at a number of RR-C10PBANR2K concentrations including 10 μM. These observations demonstrate a clear functional difference between the BommoPBANR-A variant and the BommoPBANR-B and -C variants in terms of receptor regulation and activation of downstream effector molecules. We also found that, contrary to previous reports, ligand-induced internalization of BommoPBANR-B and BommoPBANR-C in cell lines stably expressing these variants occurred in the absence of extracellular Ca2+. PMID:22654874

Surface acoustic wave actuated cell sorting (SAWACS).

PubMed

Franke, T; Braunmüller, S; Schmid, L; Wixforth, A; Weitz, D A

2010-03-21

We describe a novel microfluidic cell sorter which operates in continuous flow at high sorting rates. The device is based on a surface acoustic wave cell-sorting scheme and combines many advantages of fluorescence activated cell sorting (FACS) and fluorescence activated droplet sorting (FADS) in microfluidic channels. It is fully integrated on a PDMS device, and allows fast electronic control of cell diversion. We direct cells by acoustic streaming excited by a surface acoustic wave which deflects the fluid independently of the contrast in material properties of deflected objects and the continuous phase; thus the device underlying principle works without additional enhancement of the sorting by prior labelling of the cells with responsive markers such as magnetic or polarizable beads. Single cells are sorted directly from bulk media at rates as fast as several kHz without prior encapsulation into liquid droplet compartments as in traditional FACS. We have successfully directed HaCaT cells (human keratinocytes), fibroblasts from mice and MV3 melanoma cells. The low shear forces of this sorting method ensure that cells survive after sorting.

Fluorescent Recovery after Photobleaching (FRAP) Analysis of Nuclear Export Rates Identifies Intrinsic Features of Nucleocytoplasmic Transport*

PubMed Central

Cardarelli, Francesco; Tosti, Luca; Serresi, Michela; Beltram, Fabio; Bizzarri, Ranieri

2012-01-01

A quantitative description of carrier-mediated nuclear export in live cells is presented. To this end, we fused a prototypical leucine-rich nuclear export signal (NES) to GFP as a cargo model and expressed the fluorescent chimera in live CHO-K1 cells. By modeling FRAP data, we calculate the NES affinity for the export machinery and the maximum rate of nuclear export achievable at saturation of endogenous carriers. The measured active-export time through the Nuclear Pore Complex (NPC) is 18 ms, remarkably similar to the previously determined active-import rate. Also, our results reveal that active export/import and active export/passive diffusion fluxes are uncoupled, thus complementing previous reports on active import/passive diffusion uncoupling. These findings suggest differential gating at the NPC level. PMID:22190681

Disulfide proteomics of rice cultured cells in response to OsRacl and probenazole-related immune signaling pathway in rice.

PubMed

Morino, Kazuko; Kimizu, Mayumi; Fujiwara, Masayuki

2016-01-01

Reactive oxygen species (ROS) production is an early event in the immune response of plants. ROS production affects the redox-based modification of cysteine residues in redox proteins, which contribute to protein functions such as enzymatic activity, protein-protein interactions, oligomerization, and intracellular localization. Thus, the sensitivity of cysteine residues to changes in the cellular redox status is critical to the immune response of plants. We used disulfide proteomics to identify immune response-related redox proteins. Total protein was extracted from rice cultured cells expressing constitutively active or dominant-negative OsRacl, which is a key regulator of the immune response in rice, and from rice cultured cells that were treated with probenazole, which is an activator of the plant immune response, in the presence of the thiol group-specific fluorescent probe monobromobimane (mBBr), which was a tag for reduced proteins in a differential display two-dimensional gel electrophoresis. The mBBr fluorescence was detected by using a charge-coupled device system, and total protein spots were detected using Coomassie brilliant blue staining. Both of the protein spots were analyzed by gel image software and identified using MS spectrometry. The possible disulfide bonds were identified using the disulfide bond prediction software. Subcellular localization and bimolecular fluorescence complementation analysis were performed in one of the identified proteins: Oryza sativa cold shock protein 2 (OsCSP2). We identified seven proteins carrying potential redox-sensitive cysteine residues. Two proteins of them were oxidized in cultured cells expressing DN-OsRac1, which indicates that these two proteins would be inactivated through the inhibition of OsRac1 signaling pathway. One of the two oxidized proteins, OsCSP2, contains 197 amino acid residues and six cysteine residues. Site-directed mutagenesis of these cysteine residues revealed that a Cys 140 mutation causes mislocalization of a green fluorescent protein fusion protein in the root cells of rice. Bimolecular fluorescence complementation analysis revealed that OsCSP2 is localized in the nucleus as a homo dimer in rice root cells. The findings of the study indicate that redox-sensitive cysteine modification would contribute to the immune response in rice.

Diving under a microscope--a new simple and versatile in vitro diving device for fluorescence and confocal microscopy allowing the controls of hydrostatic pressure, gas pressures, and kinetics of gas saturation.

PubMed

Wang, Qiong; Belhomme, Marc; Guerrero, François; Mazur, Aleksandra; Lambrechts, Kate; Theron, Michaël

2013-06-01

How underwater diving effects the function of the arterial wall and the activities of endothelial cells is the focus of recent studies on decompression sickness. Here we describe an in vitro diving system constructed to achieve real-time monitoring of cell activity during simulated dives under fluorescent microscopy and confocal microscopy. A 1-mL chamber with sapphire windows on both sides and located on the stage of an inverted microscope was built to allow in vitro diving simulation of isolated cells or arteries in which activities during diving are monitored in real-time via fluorescent microscopy and confocal microscopy. Speed of compression and decompression can range from 20 to 2000 kPa/min, allowing systemic pressure to range up to 6500 kPa. Diving temperature is controlled at 37°C. During air dive simulation oxygen partial pressure is optically monitored. Perfusion speed can range from 0.05 to 10 mL/min. The system can support physiological viability of in vitro samples for real-time monitoring of cellular activity during diving. It allows regulations of pressure, speeds of compression and decompression, temperature, gas saturation, and perfusion speed. It will be a valuable tool for hyperbaric research.

Internalization of aggregated photosensitizers by tumor cells: subcellular time-resolved fluorescence spectroscopy on derivatives of pyropheophorbide-a ethers and chlorin e6 under femtosecond one- and two-photon excitations.

PubMed

Kelbauskas, L; Dietel, W

2002-12-01

Amphiphilic sensitizers self-associate in aqueous environments and form aggregated species that exhibit no or only negligible photodynamic activity. However, amphiphilic photosensitizers number among the most potent agents of photodynamic therapy. The processes by which these sensitizers are internalized into tumor cells have yet to be fully elucidated and thus remain the subject of debate. In this study the uptake of photosensitizer aggregates into tumor cells was examined directly using subcellular time-resolved fluorescence spectroscopy with a high temporal resolution (20-30 ps) and high sensitivity (time-correlated single-photon counting). The investigations were performed on selected sensitizers that exhibit short fluorescence decay times (< 50 ps) in aggregated form. Derivatives of pyropheophorbide-a ether and chlorin e6 with varying lipophilicity were used for the study. The characteristic fluorescence decay times and spectroscopic features of the sensitizer aggregates measured in aqueous solution also could be observed in A431 human endothelial carcinoma cells administered with these photosensitizers. This shows that tumor cells can internalize sensitizers in aggregated form. Uptake of aggregates and their monomerization inside cells were demonstrated directly for the first time by means of fluorescence lifetime imaging with a high temporal resolution. Internalization of the aggregates seems to be endocytosis mediated. The degree of their monomerization in tumor cells is strongly influenced by the lipophilicity of the compounds.

Quenching of Superoxide Radicals by Green Fluorescent Protein

PubMed Central

Bou-Abdallah, Fadi; Chasteen, N. Dennis; Lesser, Michael P.

2006-01-01

Green fluorescent protein (GFP) is a widely used in vivo molecular marker. These proteins are particularly resistant, and maintain function, under a variety of cellular conditions such as pH extremes and elevated temperatures. Green fluorescent proteins are also abundant in several groups of marine invertebrates including reef-forming corals. While molecular oxygen is required for the post-translational maturation of the protein, mature GFPs are found in corals where hyperoxia and reactive oxygen species (ROS) occur due to the photosynthetic activity of algal symbionts. In vitro spin trapping electron paramagnetic resonance and spectrophotometric assays of superoxide dismutase (SOD)-like enzyme activity show that wild type GFP from the hydromedusa, Aequorea victoria, quenches superoxide radicals (O2•−) and exhibits SOD-like activity by competing with cytochrome c for reaction with O2•−. When exposed to high amounts of O2•− the SOD-like activity and protein structure of GFP are altered without significant changes to the fluorescent properties of the protein. Because of the distribution of fluorescent proteins in both the epithelial and gastrodermal cells of reef-forming corals we propose that GFP, and possibly other fluorescent proteins, can provide supplementary antioxidant protection. PMID:17023114

Synthesis and spectroscopic characterization of fluorescent 4-aminoantipyrine analogues: Molecular docking and in vitro cytotoxicity studies

NASA Astrophysics Data System (ADS)

Premnath, D.; Mosae Selvakumar, P.; Ravichandiran, P.; Tamil Selvan, G.; Indiraleka, M.; Jannet Vennila, J.

2016-01-01

Two substituted aromatic carbonyl compounds (compounds 1 and 2) of 4-aminoantipyrine were synthesized by condensation of fluorine substituted benzoyl chlorides and 4-aminoantipyrine. The structures of synthesized derivatives were established on the basis of UV-Vis, IR, and Mass, 1H, 13C NMR and Fluorescence spectroscopy. Both compounds showed significant fluorescence emission and two broad emission bands were observed in the region at 340 nm and 450 nm on excitation at 280 nm. Theoretically to prove that the molecule has anticancer activity against cervical cancer cells, the compounds were analyzed for molecular docking interactions with HPV16-E7 target protein by Glide protocol. Furthermore, 4-aminoantipyrine derivatives were evaluated for their in vitro cytotoxic activity against human cervical cancer cells (SiHa) by MTT assay. Compound 1 showed two fold higher activity (IC50 = 0.912 μM) over compound 2, and its activity was similar to that of Pazopanib, suggesting that although the two compounds were chemically very similar the difference in substituent on the phenyl moiety caused changes in properties.

Becton-Dickson Model 420 Fluorescence-Activated Cell Sorter (FACS).

DTIC Science & Technology

1986-05-01

respectively) have been associated with certain autoimmune or immunodeficient diseases. The effects of UDMH on Lyt. antigens were previously evaluated...measured in cells from feline leukemia virus (FeLV)-infected cats and normal cat cells. The measurements are performed using the calcium-specific dye...ucavd as a stimulator, which allows for quantitation of " . phagocytosis activity of the cells. c) Quantitation of IL-2 receptor site on feline and murine

Patterning pallet arrays for cell selection based on high-resolution measurements of fluorescent biosensors

PubMed Central

Shadpour, Hamed; Zawistowski, Jon S.; Herman, Annadele; Hahn, Klaus; Allbritton, Nancy L.

2011-01-01

Pallet arrays enable cells to be separated while they remain adherent to a surface and provide a much greater range of cell selection criteria relative to that of current technologies. However there remains a need to further broaden cell selection criteria to include dynamic intracellular signaling events. To demonstrate the feasibility of measuring cellular protein behavior on the arrays using high resolution microscopy, the surfaces of individual pallets were modified to minimize the impact of scattered light at the pallet edges. The surfaces of the three-dimensional pallets on an array were patterned with a coating such as fibronectin using a customized stamping tool. Micropatterns of varying shape and size were printed in designated regions on the pallets in single or multiple steps to demonstrate the reliability and precision of patterning molecules on the pallet surface. Use of a fibronectin matrix stamped at the center of each pallet permitted the localization of H1299 and mouse embryonic fibroblast (MEF) cells to the pallet centers and away from the edges. Compared to pallet arrays with fibronection coating the entire top surface, arrays with a central fibronectin pattern increased the percentage of cells localized to the pallet center by 3-4 fold. Localization of cells to the pallet center also enabled the physical separation of cells from optical artifacts created by the rough pallet side walls. To demonstrate the measurement of dynamic intracellular signaling on the arrays, fluorescence measurements of high spatial resolution were performed using a RhoA GTPase biosensor. This biosensor utilized fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to measure localized RhoA activity in cellular ruffles at the cell periphery. These results demonstrated the ability to perform spatially resolved measurements of fluorescence-based sensors on the pallet arrays. Thus, the patterned pallet arrays should enable novel cell separations in which cell selection is based on complex cellular signaling properties. PMID:21621038

Multiparameter fluorescence imaging for quantification of TH-1 and TH-2 cytokines at the single-cell level

NASA Astrophysics Data System (ADS)

Fekkar, Hakim; Benbernou, N.; Esnault, S.; Shin, H. C.; Guenounou, Moncef

1998-04-01

Immune responses are strongly influenced by the cytokines following antigenic stimulation. Distinct cytokine-producing T cell subsets are well known to play a major role in immune responses and to be differentially regulated during immunological disorders, although the characterization and quantification of the TH-1/TH-2 cytokine pattern in T cells remained not clearly defined. Expression of cytokines by T lymphocytes is a highly balanced process, involving stimulatory and inhibitory intracellular signaling pathways. The aim of this study was (1) to quantify the cytokine expression in T cells at the single cell level using optical imaging, (2) and to analyze the influence of cyclic AMP- dependent signal transduction pathway in the balance between the TH-1 and TH-2 cytokine profile. We attempted to study several cytokines (IL-2, IFN-(gamma) , IL-4, IL-10 and IL-13) in peripheral blood mononuclear cells. Cells were prestimulated in vitro using phytohemagglutinin and phorbol ester for 36h, and then further cultured for 8h in the presence of monensin. Cells were permeabilized and then simple-, double- or triple-labeled with the corresponding specific fluorescent monoclonal antibodies. The cell phenotype was also determined by analyzing the expression of each of CD4, CD8, CD45RO and CD45RA with the cytokine expression. Conventional images of cells were recorded with a Peltier- cooled CCD camera (B/W C5985, Hamamatsu photonics) through an inverted microscope equipped with epi-fluorescence (Diaphot 300, Nikon). Images were digitalized using an acquisition video interface (Oculus TCX Coreco) in 762 by 570 pixels coded in 8 bits (256 gray levels), and analyzed thereafter in an IBM PC computer based on an intel pentium processor with an adequate software (Visilog 4, Noesis). The first image processing step is the extraction of cell areas using an edge detection and a binary thresholding method. In order to reduce the background noise of fluorescence, we performed an opening procedure of the original image using a structuring element. The opened image was therefore subtracted from the original one, and the gray intensities were subsequently measured. Fluorescence intensities are mapped in MD representation using Matlab software. Consequently, quantitative comparative expression of intracellular cytokines and cell membrane markers was achieved. Using this technique, we showed that CD4+ and CD8+T lymphocytes expressed a large panel of cytokines, and that protein kinase A (PKA) activation pathway induced a polarization of activated human T cells to the TH-2 type profile. Data also showed different sensitivities of CD45 RO/CD45RA lymphocytes to the activation of PKA, thus suggesting the implication of memory CD4+- and CD8+-T cells in the T cell specific immune and inflammatory processes and their control by PKA activation pathway. Finally, this method represents a powerful tool for the detection and quantification of intracellular cytokine expression and the analysis of the functional properties of T lymphocytes during immune responses.

Synthesis and characterization of Her2-NLP peptide conjugates targeting circulating breast cancer cells: cellular uptake and localization by fluorescent microscopic imaging.

PubMed

Cai, Huawei; Singh, Ajay N; Sun, Xiankai; Peng, Fangyu

2015-01-01

To synthesize a fluorescent Her2-NLP peptide conjugate consisting of Her2/neu targeting peptide and nuclear localization sequence peptide (NLP) and assess its cellular uptake and intracellular localization for radionuclide cancer therapy targeting Her2/neu-positive circulating breast cancer cells (CBCC). Fluorescent Cy5.5 Her2-NLP peptide conjugate was synthesized by coupling a bivalent peptide sequence, which consisted of a Her2-binding peptide (NH2-GSGKCCYSL) and an NLP peptide (CGYGPKKKRKVGG) linked by a polyethylene glycol (PEG) chain with 6 repeating units, with an activated Cy5.5 ester. The conjugate was separated and purified by HPLC and then characterized by Maldi-MS. The intracellular localization of fluorescent Cy5.5 Her2-NLP peptide conjugate was assessed by fluorescent microscopic imaging using a confocal microscope after incubation of Cy5.5-Her2-NLP with Her2/neu positive breast cancer cells and Her2/neu negative control breast cancer cells, respectively. Fluorescent signals were detected in cytoplasm of Her2/neu positive breast cancer cells (SKBR-3 and BT474 cell lines), but not or little in cytoplasm of Her2/neu negative breast cancer cells (MDA-MB-231), after incubation of the breast cancer cells with Cy5.5-Her2-NLP conjugates in vitro. No fluorescent signals were detected within the nuclei of Her2/neu positive SKBR-3 and BT474 breast cancer cells, neither Her2/neu negative MDA-MB-231 cells, incubated with the Cy5.5-Her2-NLP peptide conjugates, suggesting poor nuclear localization of the Cy5.5-Her2-NLP conjugates localized within the cytoplasm after their cellular uptake and internalization by the Her2/neu positive breast cancer cells. Her2-binding peptide (KCCYSL) is a promising agent for radionuclide therapy of Her2/neu positive breast cancer using a β(-) or α emitting radionuclide, but poor nuclear localization of the Her2-NLP peptide conjugates may limit its use for eradication of Her2/neu-positive CBCC using I-125 or other Auger electron emitting radionuclide.

DNA-hosted copper nanoclusters/graphene oxide based fluorescent biosensor for protein kinase activity detection.

PubMed

Wang, Mengke; Lin, Zihan; Liu, Qing; Jiang, Shan; Liu, Hua; Su, Xingguang

2018-07-05

A novel fluorescent biosensor for protein kinase activity (PKA) detection was designed by applying double-strands DNA-hosted copper nanoclusters (dsDNA-CuNCs) and graphene oxide (GO). One DNA strand of the dsDNA consisted of two domains, one domain can hybridize with another complementary DNA strand to stabilize the fluorescent CuNCs and another domain was adenosine 5'-triphosphate (ATP) aptamer. ATP aptamer of the dsDNA-CuNCs would be spontaneously absorbed onto the GO surface through π-π stacking interactions. Thus GO can efficiently quench the fluorescence (FL) of dsDNA-CuNCs through fluorescence resonance energy transfer (FRET). In the present of ATP, ATP specifically combined with ATP aptamer to form ATP-ATP aptamer binding complexes, which had much less affinity to GO, resulting in the fluorescence recovery of the system. Nevertheless, in the presence of PKA, ATP could be translated into ADP and ADP could not combine with ATP aptamer resulting in the fluorescence quenching of dsDNA-CuNCs again. According to the change of the fluorescence signal, PKA activity could be successfully monitored in the range of 0.1-5.0 U mL -1 with a detection limit (LOD) of 0.039 U mL -1 . Besides, the inhibitory effect of H-89 on PKA activity was studied. The sensor was performed for PKA activity detection in cell lysates with satisfactory results. Copyright © 2018 Elsevier B.V. All rights reserved.

Sequentially Programmable and Cellularly Selective Assembly of Fluorescent Polymerized Vesicles for Monitoring Cell Apoptosis

PubMed Central

Peng, Shu; Pan, Yu‐Chen; Wang, Yaling; Xu, Zhe; Chen, Chao

2017-01-01

Abstract The introduction of controlled self‐assembly into living organisms opens up desired biomedical applications in wide areas including bioimaging/assays, drug delivery, and tissue engineering. Besides the enzyme‐activated examples reported before, controlled self‐assembly under integrated stimuli, especially in the form of sequential input, is unprecedented and ultimately challenging. This study reports a programmable self‐assembling strategy in living cells under sequentially integrated control of both endogenous and exogenous stimuli. Fluorescent polymerized vesicles are constructed by using cholinesterase conversion followed by photopolymerization and thermochromism. Furthermore, as a proof‐of‐principle application, the cell apoptosis involved in the overexpression of cholinesterase in virtue of the generated fluorescence is monitored, showing potential in screening apoptosis‐inducing drugs. The approach exhibits multiple advantages for bioimaging in living cells, including specificity to cholinesterase, red emission, wash free, high signal‐to‐noise ratio. PMID:29201625

Sequentially Programmable and Cellularly Selective Assembly of Fluorescent Polymerized Vesicles for Monitoring Cell Apoptosis.

PubMed

Peng, Shu; Pan, Yu-Chen; Wang, Yaling; Xu, Zhe; Chen, Chao; Ding, Dan; Wang, Yongjian; Guo, Dong-Sheng

2017-11-01

The introduction of controlled self-assembly into living organisms opens up desired biomedical applications in wide areas including bioimaging/assays, drug delivery, and tissue engineering. Besides the enzyme-activated examples reported before, controlled self-assembly under integrated stimuli, especially in the form of sequential input, is unprecedented and ultimately challenging. This study reports a programmable self-assembling strategy in living cells under sequentially integrated control of both endogenous and exogenous stimuli. Fluorescent polymerized vesicles are constructed by using cholinesterase conversion followed by photopolymerization and thermochromism. Furthermore, as a proof-of-principle application, the cell apoptosis involved in the overexpression of cholinesterase in virtue of the generated fluorescence is monitored, showing potential in screening apoptosis-inducing drugs. The approach exhibits multiple advantages for bioimaging in living cells, including specificity to cholinesterase, red emission, wash free, high signal-to-noise ratio.

Detection of early changes in lung cell cytology by flow-systems analysis techniques. [Rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steinkamp, J.A.; Wilson, J.S.; Svitra, Z.V.

1980-03-01

Ongoing experiments designed to develop automated flow-analysis methods for assaying damage to pulmonary lavage cells in experimental animals exposed by inhalation to environmental pollutants are summarized. Pulmonary macrophages were characterized on their ability to phagocytize polystyrene latex fluorescent spheres. Lung cells consisting primarily of macrophages and leukocytes were analyzed for fluorescence (phagocytosis of spheres) and size using flow cytometric methods. Studies also concentrated on combining phagocytosis with other cellular parameters (DNA content, cell viability, and B-glucuronidase activity). As baseline studies are completed in normal animals, experimental animals will be exposed to gaseous and particulate environmental pollutants. (ERB

Optical switch probes and optical lock-in detection (OLID) imaging microscopy: high-contrast fluorescence imaging within living systems.

PubMed

Yan, Yuling; Marriott, M Emma; Petchprayoon, Chutima; Marriott, Gerard

2011-02-01

Few to single molecule imaging of fluorescent probe molecules can provide information on the distribution, dynamics, interactions and activity of specific fluorescently tagged proteins during cellular processes. Unfortunately, these imaging studies are made challenging in living cells because of fluorescence signals from endogenous cofactors. Moreover, related background signals within multi-cell systems and intact tissue are even higher and reduce signal contrast even for ensemble populations of probe molecules. High-contrast optical imaging within high-background environments will therefore require new ideas on the design of fluorescence probes, and the way their fluorescence signals are generated and analysed to form an image. To this end, in the present review we describe recent studies on a new family of fluorescent probe called optical switches, with descriptions of the mechanisms that underlie their ability to undergo rapid and reversible transitions between two distinct states. Optical manipulation of the fluorescent and non-fluorescent states of an optical switch probe generates a modulated fluorescence signal that can be isolated from a larger unmodulated background by using OLID (optical lock-in detection) techniques. The present review concludes with a discussion on select applications of synthetic and genetically encoded optical switch probes and OLID microscopy for high-contrast imaging of specific proteins and membrane structures within living systems.

Monitoring of thermal dose during ablation therapy using quantum dot-mediated fluorescence thermometry.

PubMed

Bensalah, Karim; Tuncel, Altug; Hanson, Willard; Stern, Joshua; Han, Bumsoo; Cadeddu, Jeffrey

2010-12-01

The objective of this study was to demonstrate the feasibility of quantum dot (QD)-mediated fluorescence thermometry to monitor thermal dose in an in-vitro thermal ablation zone generated by laser-heated gold nanoshells (LGNS). Hyperthermic cell death of human prostate cancer cell line (PC-3) was determined after various heating settings and correlated to the thermal conditions using an Arrhenius model prior to LGNS ablation. PC-3 cells with gold nanoshells (GNS) and QDs were exposed to a near-infrared laser and QD excitation light. When the cells were heated by GNS, local temperature was measured using the temperature-dependent fluorescence intensity of QDs. Using the predetermined Arrhenius model, the thermal dose (i.e., cell death of PC-3 cells) by LGNS was estimated with local temperatures measured with QD-mediated thermometry. The estimated thermal dose was confirmed with calcein-acetoxy-methylester viability assay. For PC-3 cell line, the activation energy and frequency factor of the Arrhenius model were 86.78 kcal/mol and 6.35 × 10(55) Hz, respectively. During LGNS ablation of PC-3 cells, QD-mediated temperature measurement showed that the temperature of the laser spot increased rapidly to ∼58 °C ± 4 °C. The estimated thermal dose showed that cell death reached to ∼90% in 120 seconds. The death cell zone observed after staining corresponded to a peak area of the temperature profile generated after analysis of the QD fluorescence intensity. This study shows that the QD fluorescence thermometry can accurately monitor the PC-3 cell death by LGNS ablation. This approach holds promises for a better monitoring of thermal ablation procedures in clinical practice.

Revealing the sequence of interactions of PuroA peptide with Candida albicans cells by live-cell imaging

NASA Astrophysics Data System (ADS)

Shagaghi, Nadin; Bhave, Mrinal; Palombo, Enzo A.; Clayton, Andrew H. A.

2017-03-01

To determine the mechanism(s) of action of antimicrobial peptides (AMPs) it is desirable to provide details of their interaction kinetics with cellular, sub-cellular and molecular targets. The synthetic peptide, PuroA, displays potent antimicrobial activities which have been attributed to peptide-induced membrane destabilization, or intracellular mechanisms of action (DNA-binding) or both. We used time-lapse fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM) to directly monitor the localization and interaction kinetics of a FITC- PuroA peptide on single Candida albicans cells in real time. Our results reveal the sequence of events leading to cell death. Within 1 minute, FITC-PuroA was observed to interact with SYTO-labelled nucleic acids, resulting in a noticeable quenching in the fluorescence lifetime of the peptide label at the nucleus of yeast cells, and cell-cycle arrest. A propidium iodide (PI) influx assay confirmed that peptide translocation itself did not disrupt the cell membrane integrity; however, PI entry occurred 25-45 minutes later, which correlated with an increase in fractional fluorescence of pores and an overall loss of cell size. Our results clarify that membrane disruption appears to be the mechanism by which the C. albicans cells are killed and this occurs after FITC-PuroA translocation and binding to intracellular targets.

Detection of protease activity by fluorescent protein FRET sensors: from computer simulation to live cells

NASA Astrophysics Data System (ADS)

Goryashchenko, Alexander S.; Khrenova, Maria G.; Savitsky, Alexander P.

2018-04-01

Förster resonance energy transfer (FRET) sensors are widely used for the detection of protease activity in vitro and in vivo. Usually they consist of a FRET pair connected with a polypeptide linker containing a specific cleavage site for the relevant protease. Use of the fluorescent proteins as components of the FRET pair allows genetic encoding of such sensors and solves the problem of their delivery into live cells and animals. There are several ways to improve the properties of such sensors, mainly to increase FRET efficiency and therefore the dynamic range. One of the ways to achieve this is to use a non-fluorescent chromoprotein as an acceptor. Molecular dynamic simulations may assist in the construction of linker structures connecting donor and acceptor molecules. Estimation of the orientation factor κ 2 can be obtained by methods based on quantum theory and combined quantum mechanics/molecular mechanics approaches. The linker can be structured by hydrophobic interactions, bringing it into a closed conformation that shortens the distance between donor and acceptor and, consequently, increases FRET efficiency. We analyzed the effects of different linker structures on the detection of caspase-3 activity using a non-fluorescent acceptor. Also we have constructed the Tb3+- TagRFP sensor in which a complex of the terbium ion and terbium-binding peptide is used as a donor. This allowed us to use the unique property of lanthanide ions—fluorescence lifetime up to milliseconds—to perform measurements with time delay and exclude the nanosecond-order fluorescence. Using our systems as a starting point, by changing the recognition site in the linker it is possible to perform imaging of different protease activity in vitro or in vivo.

Receptor trafficking via the perinuclear recycling compartment accompanied by cell division is necessary for permanent neurotensin cell sensitization and leads to chronic mitogen-activated protein kinase activation.

PubMed

Toy-Miou-Leong, Mireille; Cortes, Catherine Llorens; Beaudet, Alain; Rostène, William; Forgez, Patricia

2004-03-26

Most G protein-coupled receptors are internalized after interaction with their respective ligand, a process that subsequently contributes to cell desensitization, receptor endocytosis, trafficking, and finally cell resensitization. Although cellular mechanisms leading to cell desensitization have been widely studied, those responsible for cell resensitization are still poorly understood. We examined here the traffic of the high affinity neurotensin receptor (NT1 receptor) following prolonged exposure to high agonist concentration. Fluorescence and confocal microscopy of Chinese hamster ovary, human neuroblastoma (CHP 212), and murine neuroblastoma (N1E-115) cells expressing green fluorescent protein-tagged NT1 receptor revealed that under prolonged treatment with saturating concentrations of neurotensin (NT) agonist, NT1 receptor and NT transiently accumulated in the perinuclear recycling compartment (PNRC). During this cellular event, cell surface receptors remained markedly depleted as detected by both confocal microscopy and (125)I-NT binding assays. In dividing cells, we observed that following prolonged NT agonist stimulation, NT1 receptors were removed from the PNRC, accumulated in dispersed vesicles inside the cytoplasm, and subsequently reappeared at the cell surface. This NT binding recovery allowed for constant cell sensitization and led to a chronic activation of mitogen-activated protein kinases p42 and p44. Under these conditions, the constant activation of NT1 receptor generates an oncogenic regulation. These observations support the potent role for neuropeptides, such as NT, in cancer progression.

Antitumor Activity of Ethanolic Extract of Dendrobium formosum in T-Cell Lymphoma: An In Vitro and In Vivo Study

PubMed Central

Prasad, Ritika; Koch, Biplob

2014-01-01

Dendrobium, a genus of orchid, was found to possess useful therapeutic activities like anticancer, hypoglycaemic, antimicrobial, immunomodulatory, hepatoprotective, antioxidant, and neuroprotective activities. The study was aimed to evaluate the anticancer property of the ethanolic extract of Dendrobium formosum on Dalton's lymphoma. In vitro cytotoxicity was determined by MTT assay, apoptosis was determined by fluorescence microscopy, and cell cycle progression was analysed using flow cytometry; in vivo antitumor activity was performed in Dalton's lymphoma bearing mice. The IC50 value of ethanolic extract was obtained at 350 μg/mL in Dalton's lymphoma cells. Fluorescence microscopy analysis showed significant increase in apoptotic cell death in dose- and time-dependent manner which was further confirmed through the resulting DNA fragmentation. Further, flow cytometry analysis showed that the ethanolic extract arrests the cells in G2/M phase of the cell cycle. The in vivo anticancer activity study illustrates significant increase in the survival time of Dalton's lymphoma bearing mice on treatment with ethanolic extract when compared to control. These results substantiate the antitumor properties of ethanolic extract of Dendrobium formosum and suggest an alternative in treatment of cancer. Further studies are required regarding the isolation and characterization of bioactive components along with the analysis of molecular mechanism involved. PMID:24959588

Efficient, non-toxic anion transport by synthetic carriers in cells and epithelia

NASA Astrophysics Data System (ADS)

Li, Hongyu; Valkenier, Hennie; Judd, Luke W.; Brotherhood, Peter R.; Hussain, Sabir; Cooper, James A.; Jurček, Ondřej; Sparkes, Hazel A.; Sheppard, David N.; Davis, Anthony P.

2016-01-01

Transmembrane anion transporters (anionophores) have potential for new modes of biological activity, including therapeutic applications. In particular they might replace the activity of defective anion channels in conditions such as cystic fibrosis. However, data on the biological effects of anionophores are scarce, and it remains uncertain whether such molecules are fundamentally toxic. Here, we report a biological study of an extensive series of powerful anion carriers. Fifteen anionophores were assayed in single cells by monitoring anion transport in real time through fluorescence emission from halide-sensitive yellow fluorescent protein. A bis-(p-nitrophenyl)ureidodecalin shows especially promising activity, including deliverability, potency and persistence. Electrophysiological tests show strong effects in epithelia, close to those of natural anion channels. Toxicity assays yield negative results in three cell lines, suggesting that promotion of anion transport may not be deleterious to cells. We therefore conclude that synthetic anion carriers are realistic candidates for further investigation as treatments for cystic fibrosis.

Optical visualisation of thermogenesis in stimulated single-cell brown adipocytes.

PubMed

Kriszt, Rókus; Arai, Satoshi; Itoh, Hideki; Lee, Michelle H; Goralczyk, Anna G; Ang, Xiu Min; Cypess, Aaron M; White, Andrew P; Shamsi, Farnaz; Xue, Ruidan; Lee, Jung Yeol; Lee, Sung-Chan; Hou, Yanyan; Kitaguchi, Tetsuya; Sudhaharan, Thankiah; Ishiwata, Shin'ichi; Lane, E Birgitte; Chang, Young-Tae; Tseng, Yu-Hua; Suzuki, Madoka; Raghunath, Michael

2017-05-03

The identification of brown adipose deposits in adults has led to significant interest in targeting this metabolically active tissue for treatment of obesity and diabetes. Improved methods for the direct measurement of heat production as the signature function of brown adipocytes (BAs), particularly at the single cell level, would be of substantial benefit to these ongoing efforts. Here, we report the first application of a small molecule-type thermosensitive fluorescent dye, ERthermAC, to monitor thermogenesis in BAs derived from murine brown fat precursors and in human brown fat cells differentiated from human neck brown preadipocytes. ERthermAC accumulated in the endoplasmic reticulum of BAs and displayed a marked change in fluorescence intensity in response to adrenergic stimulation of cells, which corresponded to temperature change. ERthermAC fluorescence intensity profiles were congruent with mitochondrial depolarisation events visualised by the JC-1 probe. Moreover, the averaged fluorescence intensity changes across a population of cells correlated well with dynamic changes such as thermal power, oxygen consumption, and extracellular acidification rates. These findings suggest ERthermAC as a promising new tool for studying thermogenic function in brown adipocytes of both murine and human origins.

Near-infrared emitting fluorescent nanocrystals-labeled natural killer cells as a platform technology for the optical imaging of immunotherapeutic cells-based cancer therapy

NASA Astrophysics Data System (ADS)

Taik Lim, Yong; Cho, Mi Young; Noh, Young-Woock; Chung, Jin Woong; Chung, Bong Hyun

2009-11-01

This study describes the development of near-infrared optical imaging technology for the monitoring of immunotherapeutic cell-based cancer therapy using natural killer (NK) cells labeled with fluorescent nanocrystals. Although NK cell-based immunotherapeutic strategies have drawn interest as potent preclinical or clinical methods of cancer therapy, there are few reports documenting the molecular imaging of NK cell-based cancer therapy, primarily due to the difficulty of labeling of NK cells with imaging probes. Human natural killer cells (NK92MI) were labeled with anti-human CD56 antibody-coated quantum dots (QD705) for fluorescence imaging. FACS analysis showed that the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 have no effect on the cell viability. The effect of anti-human CD56 antibody-coated QD705 labeling on the NK92MI cell function was investigated by measuring interferon gamma (IFN- γ) production and cytolytic activity. Finally, the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 showed a therapeutic effect similar to that of unlabeled NK92MI cells. Images of intratumorally injected NK92MI cells labeled with anti-human CD56 antibody-coated could be acquired using near-infrared optical imaging both in vivo and in vitro. This result demonstrates that the immunotherapeutic cells labeled with fluorescent nanocrystals can be a versatile platform for the effective tracking of injected therapeutic cells using optical imaging technology, which is very important in cell-based cancer therapies.

Simultaneous Fluorescent Gram Staining and Activity Assessment of Activated Sludge Bacteria

PubMed Central

Forster, Scott; Snape, Jason R.; Lappin-Scott, Hilary M.; Porter, Jonathan

2002-01-01

Wastewater treatment is one of the most important commercial biotechnological processes, and yet the component bacterial populations and their associated metabolic activities are poorly understood. The novel fluorescent dye hexidium iodide allows assessment of Gram status by differential absorption through bacterial cell walls. Differentiation between gram-positive and gram-negative wastewater bacteria was achieved after flow cytometric analysis. This study shows that the relative proportions of gram-positive and gram-negative bacterial cells identified by traditional microscopy and hexidium iodide staining were not significantly different. Dual staining of cells for Gram status and activity proved effective in analyzing mixtures of cultured bacteria and wastewater populations. Levels of highly active organisms at two wastewater treatment plants, both gram positive and gram negative, ranged from 1.5% in activated sludge flocs to 16% in the activated sludge fluid. Gram-positive organisms comprised <5% of the total bacterial numbers but accounted for 19 and 55% of the highly active organisms within flocs at the two plants. Assessment of Gram status and activity within activated sludge samples over a 4-day period showed significant differences over time. This method provides a rapid, quantitative measure of Gram status linked with in situ activity within wastewater systems. PMID:12324319

Simultaneous fluorescent gram staining and activity assessment of activated sludge bacteria.

PubMed

Forster, Scott; Snape, Jason R; Lappin-Scott, Hilary M; Porter, Jonathan

2002-10-01

Wastewater treatment is one of the most important commercial biotechnological processes, and yet the component bacterial populations and their associated metabolic activities are poorly understood. The novel fluorescent dye hexidium iodide allows assessment of Gram status by differential absorption through bacterial cell walls. Differentiation between gram-positive and gram-negative wastewater bacteria was achieved after flow cytometric analysis. This study shows that the relative proportions of gram-positive and gram-negative bacterial cells identified by traditional microscopy and hexidium iodide staining were not significantly different. Dual staining of cells for Gram status and activity proved effective in analyzing mixtures of cultured bacteria and wastewater populations. Levels of highly active organisms at two wastewater treatment plants, both gram positive and gram negative, ranged from 1.5% in activated sludge flocs to 16% in the activated sludge fluid. Gram-positive organisms comprised <5% of the total bacterial numbers but accounted for 19 and 55% of the highly active organisms within flocs at the two plants. Assessment of Gram status and activity within activated sludge samples over a 4-day period showed significant differences over time. This method provides a rapid, quantitative measure of Gram status linked with in situ activity within wastewater systems.

Side population cells in the human vocal fold.

PubMed

Yamashita, Masaru; Hirano, Shigeru; Kanemaru, Shin-ichi; Tsuji, Shunichiro; Suehiro, Atsushi; Ito, Juichi

2007-11-01

The regenerative processes of the vocal fold, or the existence of stem cells in the folds, are unknown. Side population (SP) cells are defined as cells that have the ability to exclude the DNA binding dye, Hoechst 33342. They are regarded as a cell population enriched with stem cells and can be isolated from non-SP cells by a fluorescence-activated cell sorter. This study was designed to determine whether SP cells exist in the human vocal fold, as a first step in elucidating the regenerative mechanisms of the vocal fold. Seven human excised larynges were used in this study. Two were used for fluorescence-activated cell sorter analysis, and 5 were subjected to immunohistochemical analysis with antibodies against an adenosine triphosphate binding cassette transporter family member, ABCG2, which is expressed in SP cells. The number of SP cells in the human vocal fold was about 0.2% of the total number of cells. ABCG2-positive cells were identified in both the epithelium and subepithelial tissue throughout the entire vocal fold. This preliminary study demonstrated the existence of SP cells in the human vocal fold. Further studies are warranted to clarify how these cells work in the vocal fold, particularly in the regenerative process.

Quantitative In Vivo Fluorescence Cross-Correlation Analyses Highlight the Importance of Competitive Effects in the Regulation of Protein-Protein Interactions

PubMed Central

Sadaie, Wakako; Harada, Yoshie; Matsuda, Michiyuki

2014-01-01

Computer-assisted simulation is a promising approach for clarifying complicated signaling networks. However, this approach is currently limited by a deficiency of kinetic parameters determined in living cells. To overcome this problem, we applied fluorescence cross-correlation spectrometry (FCCS) to measure dissociation constant (Kd) values of signaling molecule complexes in living cells (in vivo Kd). Among the pairs of fluorescent molecules tested, that of monomerized enhanced green fluorescent protein (mEGFP) and HaloTag-tetramethylrhodamine was most suitable for the measurement of in vivo Kd by FCCS. Using this pair, we determined 22 in vivo Kd values of signaling molecule complexes comprising the epidermal growth factor receptor (EGFR)–Ras–extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway. With these parameters, we developed a kinetic simulation model of the EGFR-Ras-ERK MAP kinase pathway and uncovered a potential role played by stoichiometry in Shc binding to EGFR during the peak activations of Ras, MEK, and ERK. Intriguingly, most of the in vivo Kd values determined in this study were higher than the in vitro Kd values reported previously, suggesting the significance of competitive bindings inside cells. These in vivo Kd values will provide a sound basis for the quantitative understanding of signal transduction. PMID:24958104

Photostick: a method for selective isolation of target cells from culture† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4sc03676j Click here for additional data file.

PubMed Central

Chien, Miao-Ping; Werley, Christopher A.; Farhi, Samouil L.

2015-01-01

Sorting of target cells from a heterogeneous pool is technically difficult when the selection criterion is complex, e.g. a dynamic response, a morphological feature, or a combination of multiple parameters. At present, mammalian cell selections are typically performed either via static fluorescence (e.g. fluorescence activated cell sorter), via survival (e.g. antibiotic resistance), or via serial operations (flow cytometry, laser capture microdissection). Here we present a simple protocol for selecting cells based on any static or dynamic property that can be identified by video microscopy and image processing. The “photostick” technique uses a cell-impermeant photochemical crosslinker and digital micromirror array-based patterned illumination to immobilize selected cells on the culture dish. Other cells are washed away with mild protease treatment. The crosslinker also labels the selected cells with a fluorescent dye and a biotin for later identification. The photostick protocol preserves cell viability, permits genetic profiling of selected cells, and can be performed with complex functional selection criteria such as neuronal firing patterns. PMID:25705368

An Ultrasensitive Bacterial Motor Revealed by Monitoring Signaling Proteins in Single Cells

NASA Astrophysics Data System (ADS)

Cluzel, Philippe; Surette, Michael; Leibler, Stanislas

2000-03-01

Understanding biology at the single-cell level requires simultaneous measurements of biochemical parameters and behavioral characteristics in individual cells. Here, the output of individual flagellar motors in Escherichia coli was measured as a function of the intracellular concentration of the chemotactic signaling protein. The concentration of this molecule, fused to green fluorescent protein, was monitored with fluorescence correlation spectroscopy. Motors from different bacteria exhibited an identical steep input-output relation, suggesting that they actively contribute to signal amplification in chemotaxis. This experimental approach can be extended to quantitative in vivo studies of other biochemical networks.

Detection of focal adhesion kinase activation at membrane microdomains by fluorescence resonance energy transfer.

PubMed

Seong, Jihye; Ouyang, Mingxing; Kim, Taejin; Sun, Jie; Wen, Po-Chao; Lu, Shaoying; Zhuo, Yue; Llewellyn, Nicholas M; Schlaepfer, David D; Guan, Jun-Lin; Chien, Shu; Wang, Yingxiao

2011-07-26

Proper subcellular localization of focal adhesion kinase (FAK) is crucial for many cellular processes. It remains, however, unclear how FAK activity is regulated at subcellular compartments. To visualize the FAK activity at different membrane microdomains, we develop a fluorescence resonance energy transfer (FRET)-based FAK biosensor, and target it into or outside of detergent-resistant membrane (DRM) regions at the plasma membrane. Here we show that, on cell adhesion to extracellular matrix proteins or stimulation by platelet-derived growth factor (PDGF), the FRET responses of DRM-targeting FAK biosensor are stronger than that at non-DRM regions, suggesting that FAK activation can occur at DRM microdomains. Further experiments reveal that the PDGF-induced FAK activation is mediated and maintained by Src activity, whereas FAK activation on cell adhesion is independent of, and in fact essential for the Src activation. Therefore, FAK is activated at membrane microdomains with distinct activation mechanisms in response to different physiological stimuli. © 2011 Macmillan Publishers Limited. All rights reserved.

Evanescent field microscopy techniques for studying dynamics at the surface of living cells

NASA Astrophysics Data System (ADS)

Sund, Susan E.

This thesis presents two distinct optical microscopy techniques for applications in cell biophysics: (a)the extension to living cells of an established technique, total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) for the first time in imaging mode; and (b)the novel development of polarized total internal reflection fluorescence (p- TIRF) to study membrane orientation in living cells. Although reversible chemistry is crucial to dynamical processes in living cells, relatively little is known about the relevant chemical kinetic rates in vivo. TIR/FRAP, an established technique which can measure reversible biomolecular kinetic rates at surfaces, is extended here to measure kinetic parameters of microinjected rhodamine actin at the cytofacial surface of the plasma membrane of living cultured smooth muscle cells. For the first time, spatial imaging (with a CCD camera) is used in conjunction with TIR/FRAP. TIR/FRAP imaging allows production of spatially resolved images of kinetic data, and calculation of correlation distances, cell-wide gradients, and kinetic parameter dependence on initial fluorescence intensity. In living cells, membrane curvature occurs both in easily imaged large scale morphological features, and also in less visualizable submicroscopic regions of activity such as endocytosis, exocytosis, and cell surface ruffling. A fluorescence microscopic method, p-TIRF, is introduced here to visualize such regions. The method is based on fluorescence of the oriented membrane probe diI- C18-(3) (diI) excited by evanescent field light polarized either perpendicular or parallel to the plane of the substrate coverslip. The excitation efficiency from each polarization depends on the membrane orientation, and thus the ratio of the observed fluorescence excited by these two polarizations vividly shows regions of microscopic and submicroscopic curvature of the membrane. A theoretical background of the technique and experimental verifications are presented in samples of protein solutions, model lipid bilayers, and living cells. Sequential digital images of the polarized TIR fluorescence ratios show spatially-resolved time- course maps of membrane orientations on diI labeled macrophages from which low visibility membrane structures can be identified and quantified. The TIR images are sharpened and contrast-enhanced by deconvoluting them with an experimentally-measured point spread function.

Chloroplast-cytoplasmic interrelations involved in chloroplast development in Chlamydomonas reinhardi y-1: effect of selective depletion of chloroplast translates

PubMed Central

1980-01-01

Chlamydomonas reinhardi y-1 cells grown in the dark in the presence of chloramphenicol (CD cells) are depleted of photosynthetic membranes and 70S translates. These cells were found to be unable to synthesize chlorophyll in the light until chloroplast protein synthesis was resumed. On the other hand, CD cells acquired the capacity to partially green in the presence of cycloheximide. This greening was characterized by the development of photosynthetic activity, as demonstrated by light- dependent oxygen evolution of whole cells and by measurements of ribulose-1,5-bisphosphate carboxylase and fluorescence kinetics. The chlorophyll synthesized de novo during greening in the absence of 80S ribosomal activity was organized in chlorophyll-protein complexes, as ascertained by low-temperature fluorescence-emission spectra. The morphology of these cells appeared to be normal. A model has been proposed as a working hypothesis, which could account for the phenomena described above and previously reported data pertaining to chloroplast development. PMID:7419587

Enzyme-activatable imaging probe reveals enhanced neutrophil elastase activity in tumors following photodynamic therapy

PubMed Central

Modi, Kshitij D.; Foster, Thomas H.

2013-01-01

Abstract. We demonstrate the use of an enzyme-activatable fluorogenic probe, Neutrophil Elastase 680 FAST (NE680), for in vivo imaging of neutrophil elastase (NE) activity in tumors subjected to photodynamic therapy (PDT). NE protease activity was assayed in SCC VII and EMT6 tumors established in C3H and BALB/c mice, respectively. Four nanomoles of NE680 was injected intravenously immediately following PDT irradiation. 5 h following administration of NE680, whole-mouse fluorescence imaging was performed. At this time point, levels of NE680 fluorescence were at least threefold greater in irradiated versus unirradiated SCC VII and EMT6 tumors sensitized with Photofrin. To compare possible photosensitizer-specific differences in therapy-induced elastase activity, EMT6 tumors were also subjected to 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH)-PDT. NE levels measured in HPPH-PDT-treated tumors were twofold higher than in unirradiated controls. Ex vivo labeling of host cells using fluorophore-conjugated antibodies and confocal imaging were used to visualize Gr1+ cells in Photofrin-PDT-treated EMT6 tumors. These data were compared with recently reported analysis of Gr1+ cell accumulation in EMT6 tumors subjected to HPPH-PDT. The population density of infiltrating Gr1+ cells in treated versus unirradiated drug-only control tumors suggests that the differential in NE680 fold enhancement observed in Photofrin versus HPPH treatment may be attributed to the significantly increased inflammatory response induced by Photofrin-PDT. The in vivo imaging of NE680, which is a fluorescent reporter of NE extracellular release caused by neutrophil activation, demonstrates that PDT results in increased NE levels in treated tumors, and the accumulation of the cleaved probe tracks qualitatively with the intratumor Gr1+ cell population. PMID:23897439

Clustered localization of STAT3 during the cell cycle detected by super-resolution fluorescence microscopy

NASA Astrophysics Data System (ADS)

Gao, Jing; Chen, Junling; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Tong, Ti; Wang, Hongda

2017-06-01

Signal transducer and activator of transcription 3 (STAT3) plays a key role in various cellular processes such as cell proliferation, differentiation, apoptosis and immune responses. In particular, STAT3 has emerged as a potential molecular target for cancer therapy. The functional role and standard activation mechanism of STAT3 have been well studied, however, the spatial distribution of STAT3 during the cell cycle is poorly known. Therefore, it is indispensable to study STAT3 spatial arrangement and nuclear-cytoplasimic localization at the different phase of cell cycle in cancer cells. By direct stochastic optical reconstruction microscopy imaging, we find that STAT3 forms various number and size of clusters at the different cell-cycle stage, which could not be clearly observed by conventional fluorescent microscopy. STAT3 clusters get more and larger gradually from G1 to G2 phase, during which time transcription and other related activities goes on consistently. The results suggest that there is an intimate relationship between the clustered characteristic of STAT3 and the cell-cycle behavior. Meanwhile, clustering would facilitate STAT3 rapid response to activating signals due to short distances between molecules. Our data might open a new door to develop an antitumor drug for inhibiting STAT3 signaling pathway by destroying its clusters.

Toward noninvasive microspectrofluorometry of skin lesions for diagnostic and prognostic evaluation of cell metabolism and organelle interactions

NASA Astrophysics Data System (ADS)

Hirschberg, Joseph G.; Schachtschabel, Astrid; Kohen, Elli; Kohen, Cahide; Schachtschabel, Dietrich O.

1995-02-01

The basic principle of this approach relies on microspectrofluorometric observations of upheavals in the cell's energy metabolism and cell-to-cell metabolic communication in human and mouse melanoma cells. A striking feature is the definition of a highly active nuclear energy metabolism in M8255 human melanoma cells which is characterized by an intense fluorescence response associated with NAD(P) reduction by substrates of glycolysis or the hexose monophosphate shunt. Changes are also expected in the steady state levels of reduced/oxidized NAD(P) in the nuclear, cytoplasmic and mitochondrial compartments, which are probably dependent on ATP levels and distribution (as determined by cell metabolism and eventually the presence of ATP traps). A topographic scanning of skin lesions, either under metabolic steady state conditions or in the presence of permeating substrates, can lead to the recognition of characteristic patterns associated with pigmented and nonpigmented, malignant and nonmalignant skin lesions. The method is, in a way, an extension of microscopic transillumination techniques which have led to the identification of specific patterns associated with such lesions. However, here, a new dimension is added by introduction of fluorescence evaluations. This can represent the first step in a multiparameter approach to the non-invasive in situ fluorescence scan of dermatological lesions by inclusion of: (1) fluorescence excitation and emission spectra; (2) new fluorescence probes of cytoplasmic organelles and nuclear components. Primary emphasis should be placed on the highly active nuclear energy metabolism, which can be triggered to maximum levels when the role of mitochondria as the `cells's policeman' with regard to metabolic control is suppressed by use of topically cytotoxic agents such as the `antipsoriatic' anthralin and dicarboxylic acids used in the local treatment of melanoma. Fluorescence excitation spectroscopy may be of particular advantage in studies with the new highly sensitive cyanine nucleic acid dyes and their dimers, but caution should be exerted in the use of such compounds because of cytotoxicity (e.g., limiting it to cellular studies used in the interpretation of dermatological findings). Parallel cellular and non-invasive dermatological studies will help to define the most specific set of parameters to be used in diagnostic and prognostic evaluations of skin lesions.

Time-resolved nanosecond fluorescence lifetime imaging and picosecond infrared spectroscopy of combretastatin A-4 in solution and in cellular systems

NASA Astrophysics Data System (ADS)

Bisby, Roger H.; Botchway, Stanley W.; Greetham, Greg M.; Hadfield, John A.; McGown, Alan T.; Parker, Anthony W.; Scherer, Kathrin M.; Towrie, Mike

2012-08-01

Fluorescence lifetime images of intrinsic fluorescence obtained with two-photon excitation at 630 nm are shown following uptake of a series of E-combretastatins into live cells, including human umbilical vein endothelial cells (HUVECs) that are the target for the anticancer activity of combretastatins. Images show distribution of the compounds within the cell cytoplasm and in structures identified as lipid droplets by comparison with images obtained following Nile red staining of the same cells. The intracellular fluorescent lifetimes are generally longer than in fluid solution as a consequence of the high viscosity of the cellular environment. Following incubation, the intracellular concentrations of a fluorinated derivative of E-combretastatin A-4 in HUVECs are between two and three orders of magnitude higher than the concentration in the surrounding medium. Evidence is presented to indicate that at moderate laser powers (up to 6 mW), it is possible to isomerize up to 25% of the combretastatin within the femtolitre focal volume of the femtosecond laser beam. This suggests that it may be possible to activate the E-combretastatin (with low cellular toxicity) to the Z-isomer with high anticancer drug activity using two-photon irradiation. The isomerization of Z- and E-combretastatins by 266 nm irradiation has been probed by ultrafast time-resolved infrared spectroscopy. Results for the E-isomer show a rapid loss of excess vibrational energy in the excited state with a lifetime of 7 ps, followed by a slower process with a lifetime of 500 ps corresponding to the return to the ground state as also determined from the fluorescence lifetime. In contrast, the Z-isomer, whilst also appearing to undergo a rapid cooling of the initial excited state, has a much shorter overall excited state lifetime of 14 ps. DedicationThis paper is dedicated to the memory of Professor Christopher G Morgan (1949-2011). He was a valued colleague and friend at the University of Salford and made significant contributions to the development and applications of fluorescence lifetime imaging.

Immunomicrospheres - Reagents for cell labeling and separation

NASA Technical Reports Server (NTRS)

Rembaum, A.; Dreyer, W. J.

1980-01-01

Immunomicrospheres are specially designed microscopic particles that have antibodies or similar molecules chemically bound to their surfaces. The antibody-coated microspheres react in a highly specific way with target cells, viruses, or other antigenic agents. Immunomicrospheres may be synthesized so that they incorporate compounds that are highly radioactive, intensely fluorescent, magnetic, electron opaque, highly colored, or pharmacologically active. These various types of microspheres may be coated with pure, highly specific monoclonal antibodies obtained by the new hybridoma cell cloning techniques or with conventional antibody preparations. Some of the many present and potential applications for these new reagents are (1) new types of radioimmune or immunofluorescent assays, (2) improved fluorescence microscopy, (3) separation of cells on the basis of the fluorescent, electrophoretic, or magnetic properties of bound immunomicrospheres, (4) markers for use in several types of electron or standard light microscopy, and (5) delivery of lethal compouds to specific undesirable living cells. The combination of the various new types of synthetic microspheres and the newly available homogeneous antibodies offers new opportunities in research, diagnosis, and therapy.

Kinetics of T-cell receptor-dependent antigen recognition determined in vivo by multi-spectral normalized epifluorescence laser scanning

NASA Astrophysics Data System (ADS)

Favicchio, Rosy; Zacharakis, Giannis; Oikonomaki, Katerina; Zacharopoulos, Athanasios; Mamalaki, Clio; Ripoll, Jorge

2012-07-01

Detection of multiple fluorophores in conditions of low signal represents a limiting factor for the application of in vivo optical imaging techniques in immunology where fluorescent labels report for different functional characteristics. A noninvasive in vivo Multi-Spectral Normalized Epifluorescence Laser scanning (M-SNELS) method was developed for the simultaneous and quantitative detection of multiple fluorophores in low signal to noise ratios and used to follow T-cell activation and clonal expansion. Colocalized DsRed- and GFP-labeled T cells were followed in tandem during the mounting of an immune response. Spectral unmixing was used to distinguish the overlapping fluorescent emissions representative of the two distinct cell populations and longitudinal data reported the discrete pattern of antigen-driven proliferation. Retrieved values were validated both in vitro and in vivo with flow cytometry and significant correlation between all methodologies was achieved. Noninvasive M-SNELS successfully quantified two colocalized fluorescent populations and provides a valid alternative imaging approach to traditional invasive methods for detecting T cell dynamics.

Gold Functionalized Mesoporous Silica Nanoparticle Mediated Protein and DNA Codelivery to Plant Cells Via the Biolistic Method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin-Ortigosa, Susana; Valenstein, Justin S.; Lin, Victor S.-Y.

2012-09-11

The synthesis and characterization of a gold nanoparticle functionalized mesoporous silica nanoparticle (Au-MSN) platform for codelivery of proteins and plasmid DNA to plant tissues using a biolistic particle delivery system is reported. The in vitro uptake and release profiles of fluorescently labeled bovine serum albumin (BSA) and enhanced green fluorescent protein (eGFP) are investigated. As a proof-of-concept demonstration, Au-MSN with large average pore diameters (10 nm) are shown to deliver and subsequently release proteins and plasmid DNA to the same cell after passing through the plant cell wall upon bombardment. Release of fluorescent eGFP indicates the delivery of active, non-denaturedmore » proteins to plant cells. This advance represents the first example of biolistic-mediated codelivery of proteins and plasmid DNA to plant cells via gold-functionalized MSN and provides a powerful tool for both fundamental and applied research of plant sciences.« less

Dynamic detection of caspase-3 activation during photosensitization by fluorescence resonance energy transfer

NASA Astrophysics Data System (ADS)

Wu, Yunxia; Xing, Da; Chen, Qun; Chen, Tongsheng; Tang, Yonghong; Wan, Qingling

2005-04-01

Apoptosis is one of the important modes in PDT-induced cell death. Activation of caspase-3 is considered to be the final step in many apoptosis pathways. In this study, we used SCAT3, a fluorescence resonance energy transfer (FRET) probe containing caspase-3 substrate, to study the dynamics of caspase-3 activation in living ASTC-a-1 cells expressing stably SCAT3. The FRET analysis results indicated that caspase-3 activation in response to tumor necrosis factor-α or PDT resulted in cleavage of the linker peptide and subsequent disruption of the FRET signal. The SCAT3 was cleaved immediately after PDT treatment, but that for TNF-a treatment was delayed two hours. Our experimental results suggested that the different apoptotic pathways induced by TNF-α or PDT caused different cleavage kinetics of SCAT3. This study shows that FRET technique based on GFPs could be used to study the mechanism of PDT-induced apoptosis in living cells.

Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

PubMed

Mas, Abraham; Amenós, Montse; Lois, L Maria

2016-01-01

Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected.

Dynamic nuclear protein interactions investigated using fluorescence lifetime and fluorescence fluctuation spectroscopy

NASA Astrophysics Data System (ADS)

Siegel, Amanda P.; Hays, Nicole M.; Day, Richard N.

2012-03-01

The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for Förster resonance energy transfer (FRET) microscopy and fluorescence fluctuation spectroscopy (FFS) provide important tools for monitoring dynamic protein interactions inside living cells. Fluorescence lifetime imaging microscopy (FLIM) quantitatively maps changes in the spatial distribution of donor FP lifetimes that result from FRET with acceptor FPs. FFS probes dynamic protein associations through its capacity to monitor localized protein diffusion. Here, we use FRET-FLIM combined with FFS in living cells to investigate changes in protein mobility due to protein-protein interactions involving transcription factors and chromatin modifying proteins that function in anterior pituitary gene regulation. The heterochromatin protein 1 alpha (HP1α) plays a key role in the establishment and maintenance of heterochromatin through its interactions with histone methyltransferases. Recent studies, however, also highlight the importance of HP1α as a positive regulator of active transcription in euchromatin. Intriguingly, we observed that the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) interacts with HP1α in regions of pericentromeric heterochromatin in mouse pituitary cells. These observations prompted us to investigate the relationship between HP1α dynamic interactions in pituitary specific gene regulation.

Simultaneous imaging of temporal changes of NF-κB activity and viable tumor cells in Huh7/NF-κB-tk-luc2/rfp tumor-bearing mice.

PubMed

Wang, Wei-Hsun; Chiang, I-Tsang; Liu, Yu-Chang; Hsu, Fei-Ting; Chen, Hong-Wen; Chen, Chuan-Lin; Lee, Yi-Jang; Lin, Wuu-Jyh; Hwang, Jeng-Jong

2013-01-01

Few studies have reported that the effect of sorafenib on advanced human hepatocellular carcinoma (HCC) is taking place via the inhibition of NF-κB signal transduction. Here we constructed a human HCC Huh7 stable clone with NF-κB-responsive element to drive dual reporter genes, herpes simplex virus thymidine kinase (tk) and firefly luciferase (luc2), and co-transfected with a third red fluorescent protein (rfp) gene, renamed as Huh7/NF-κB-tk-luc2/rfp cells, and combined with bioluminescent imaging (BLI) and red fluorescent protein imaging (RFPI) to monitor the effect of sorafenib on NF-κB activation and tumor inhibition. The results show that sorafenib could suppress the NF-κB-DNA binding activity, and the expression of downstream effector proteins. Notably, the relative photon fluxes obtained from RFPI and BLI, which represent the viable tumor cells and cells with NF-κB activation, decreased after sorafenib treatment by 50 to 65%, and 87.5 to >90%, respectively, suggesting that NF-κB activation is suppressed in viable HCC cells by sorafenib. Simultaneous molecular imaging of the temporal change of NF-κB activity and of viable cells in the same Huh7/NF-κB-tk-luc2/rfp tumors of the animal may reflect the real status of NF-κB activity and the viable tumor cells at the time of imaging.

Constitutive mRNA expression and protein activity levels of nine ABC efflux transporters in seven permanent cell lines derived from different tissues of rainbow trout (Oncorhynchus mykiss).

PubMed

Fischer, Stephan; Loncar, Jovica; Zaja, Roko; Schnell, Sabine; Schirmer, Kristin; Smital, Tvrtko; Luckenbach, Till

2011-01-25

Permanent fish cell lines have become common model systems for determining ecotoxicological effects of pollutants. For these cell lines little is known on the cellular active transport mechanisms that control the amount of a compound entering the cell, such as the MXR (multixenobiotic resistance) system mediated by ATP binding cassette (ABC) transport proteins. Therefore, for toxic evaluation of chemicals with those cells information on MXR is important. We here present data on constitutive mRNA expression and protein activity levels of a series of ABC efflux transporters in seven permanent cell lines derived from liver (RTL-W1; R1) and liver hepatoma (RTH-149), gill (RTgill-W1), gonad (RTG-2), gut (RTgutGC) and brain (RTbrain) of rainbow trout (Oncorhynchus mykiss). In addition to known transporters abcb1 (designated here abcb1a), abcb11, abcc1-3, abcc5 and abcg2, we quantified expression levels of a newly identified abcb1 isoform (abcb1b) and abcc4, previously unknown in trout. Quantitative real time PCR (qPCR) indicated that mRNA of the examined ABC transporters was constitutively expressed in all cell lines. Transporter mRNA expression patterns were similar in all cell lines, with expression levels of abcc transporters being 80 to over 1000 fold higher than for abcg2, abcb1a/b and abcb11 (abcc1-5>abcg2>abcb1a/b, 11). Transporter activity in the cell lines was determined by measuring uptake of transporter type specific fluorescent substrates in the presence of activity inhibitors. The combination of the ABCB1 and ABCC transporter substrate calcein-AM with inhibitors cyclosporine A, PSC833 and MK571 resulted in a concentration-dependent fluorescence increase of up to 3-fold, whereas reversin 205 caused a slight, but not concentration-dependent fluorescence increase. Accumulation of the dyes Hoechst 33342 and 2',7'-dichlorodihydrofluorescein diacetate was basically unchanged in the presence of Ko134 and taurocholate, respectively, indicating low Abcg2 and Abcb11 activities, in accordance with low abcg2 and abcb11 transcript levels. Our data indicate that transporter expression and activity patterns in the different trout cell lines are irrespective of the tissue of origin, but are determined by factors of cell cultivation. 2010 Elsevier B.V. All rights reserved.

New fluorescent bile acids: synthesis, chemical characterization, and disastereoselective uptake by Caco-2 cells of 3-deoxy 3-NBD-amino deoxycholic and ursodeoxycholic acid.

PubMed

Májer, Ferenc; Salomon, Johanna J; Sharma, Ruchika; Etzbach, Simona V; Najib, Mohd Nadzri Mohd; Keaveny, Ray; Long, Aideen; Wang, Jun; Ehrhardt, Carsten; Gilmer, John F

2012-03-01

Deoxycholic acid (DCA), a secondary bile acid (BA), and ursodeoxycholic acid (UDCA), a tertiary BA, cause opposing effects in vivo and in cell suspensions. Fluorescent analogues of DCA and UDCA could help investigate important questions about their cellular interactions and distribution. We have prepared a set of isomeric 3α- and 3β-amino analogues of UDCA and DCA and derivatised these with the discrete fluorophore, 4-nitrobenzo-2-oxa-1,3-diazol (NBD), forming the corresponding four fluorescent adducts. These absorb in the range 465-470 nm and fluoresce at approx. 535 nm. In order to determine the ability of the new fluorescent bile acids to mimic the parents, their uptake was studied using monolayers of Caco-2 cells, which are known to express multiple proteins of the organic anion-transporting peptide (OATP) subfamily of transporters. Cellular uptake was monitored over time at 4 and 37°C to distinguish between passive and active transport. All four BA analogues were taken up but in a strikingly stereo- and structure-specific manner, suggesting highly discriminatory interactions with transporter protein(s). The α-analogues of DCA and to a lesser extent UDCA were actively transported, whereas the β-analogues were not. The active transport process was saturable, with Michaelis-Menten constants for 3α-NBD DCA (5) being K(m)=42.27±12.98 μM and V(max)=2.8 ± 0.4 nmol/(mg protein*min) and for 3α-NBD UDCA (3) K(m)=28.20 ± 7.45 μM and V(max)=1.8 ± 0.2 nmol/(mg protein*min). These fluorescent bile acids are promising agents for investigating questions of bile acid biology and for detection of bile acids and related organic anion transport processes. Copyright © 2012 Elsevier Ltd. All rights reserved.

Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy

NASA Astrophysics Data System (ADS)

Alfonso-García, Alba; Smith, Tim D.; Datta, Rupsa; Luu, Thuy U.; Gratton, Enrico; Potma, Eric O.; Liu, Wendy F.

2016-04-01

Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.

Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases

PubMed Central

González-Vera, Juan A.; Morris, May C.

2015-01-01

Probing the dynamic activities of protein kinases in real-time in living cells constitutes a major challenge that requires specific and sensitive tools tailored to meet the particular demands associated with cellular imaging. The development of genetically-encoded and synthetic fluorescent biosensors has provided means of monitoring protein kinase activities in a non-invasive fashion in their native cellular environment with high spatial and temporal resolution. Here, we review existing technologies to probe different dynamic features of protein kinases and discuss limitations where new developments are required to implement more performant tools, in particular with respect to infrared and near-infrared fluorescent probes and strategies which enable improved signal-to-noise ratio and controlled activation of probes. PMID:28248276

A monolithic glass chip for active single-cell sorting based on mechanical phenotyping.

PubMed

Faigle, Christoph; Lautenschläger, Franziska; Whyte, Graeme; Homewood, Philip; Martín-Badosa, Estela; Guck, Jochen

2015-03-07

The mechanical properties of biological cells have long been considered as inherent markers of biological function and disease. However, the screening and active sorting of heterogeneous populations based on serial single-cell mechanical measurements has not been demonstrated. Here we present a novel monolithic glass chip for combined fluorescence detection and mechanical phenotyping using an optical stretcher. A new design and manufacturing process, involving the bonding of two asymmetrically etched glass plates, combines exact optical fiber alignment, low laser damage threshold and high imaging quality with the possibility of several microfluidic inlet and outlet channels. We show the utility of such a custom-built optical stretcher glass chip by measuring and sorting single cells in a heterogeneous population based on their different mechanical properties and verify sorting accuracy by simultaneous fluorescence detection. This offers new possibilities of exact characterization and sorting of small populations based on rheological properties for biological and biomedical applications.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mouledous, Lionel; Merker, Soren; Neasta, Jeremie

*: Mu opioid (MOP) receptor activation can be functionally modulated by stimulation of Neuropeptide FF 2 (NPFF{sub 2}) G protein-coupled receptors. Fluorescence recovery after photobleaching experiments have shown that activation of the NPFF{sub 2} receptor dramatically reduces the fraction of MOP receptors confined in microdomains of the plasma membrane of SH-SY5Y neuroblastoma cells. The aim of the present work was to assess if the direct observation of receptor compartmentation by fluorescence techniques in living cells could be related to indirect estimation of receptor partitioning in lipid rafts after biochemical fractionation of the cell. Our results show that MOP receptor distributionmore » in lipid rafts is highly dependent upon the method of purification, questioning the interpretation of previous data regarding MOP receptor compartmentation. Moreover, the NPFF analogue 1DMe does not modify the distribution profile of MOP receptors, clearly demonstrating that membrane fractionation data do not correlate with direct measurement of receptor compartmentation in living cells.« less

Reporter-Based Isolation of Developmental Myogenic Progenitors

PubMed Central

Kheir, Eyemen; Cusella, Gabriella; Messina, Graziella; Cossu, Giulio; Biressi, Stefano

2018-01-01

The formation and activity of mammalian tissues entail finely regulated processes, involving the concerted organization and interaction of multiple cell types. In recent years the prospective isolation of distinct progenitor and stem cell populations has become a powerful tool in the hands of developmental biologists and has rendered the investigation of their intrinsic properties possible. In this protocol, we describe how to purify progenitors with different lineage history and degree of differentiation from embryonic and fetal skeletal muscle by fluorescence-activated cell sorting (FACS). The approach takes advantage of a panel of murine strains expressing fluorescent reporter genes specifically in the myogenic progenitors. We provide a detailed description of the dissection procedures and of the enzymatic dissociation required to maximize the yield of mononucleated cells for subsequent FACS-based purification. The procedure takes ~6–7 h to complete and allows for the isolation and the subsequent molecular and phenotypic characterization of developmental myogenic progenitors. PMID:29674978

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM).

PubMed

Wang, Yilin; Kanchanawong, Pakorn

2016-12-01

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and > 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Endothelial microparticles interact with and support the proliferation of T cells.

PubMed

Wheway, Julie; Latham, Sharissa L; Combes, Valery; Grau, Georges E R

2014-10-01

Endothelial cells closely interact with circulating lymphocytes. Aggression or activation of the endothelium leads to an increased shedding of endothelial cell microparticles (MP). Endothelial MP (EMP) are found in high plasma levels in numerous immunoinflammatory diseases, such as atherosclerosis, sepsis, multiple sclerosis, and cerebral malaria, supporting their role as effectors and markers of vascular dysfunction. Given our recently described role for human brain microvascular endothelial cells (HBEC) in modulating immune responses, we investigated how HBEC-derived MP could interact with and support the proliferation of T cells. Like their mother cells, EMP expressed molecules important for Ag presentation and T cell costimulation, that is, β2-microglobulin, MHC II, CD40, and ICOSL. HBEC were able to take up fluorescently labeled Ags with EMP also containing fluorescent Ags, suggestive of Ag carryover from HBEC to EMP. In cocultures, fluorescently labeled EMP from resting or cytokine-stimulated HBEC formed conjugates with both CD4(+) and CD8(+) subsets, with higher proportions of T cells binding EMP from cytokine-stimulated cells. The increased binding of EMP from cytokinestimulated HBEC to T cells was VCAM-1 and ICAM-1 dependent. Finally, in CFSE T cell proliferation assays using anti-CD3 mAb or T cell mitogens, EMP promoted the proliferation of CD4(+) T cells and that of CD8(+) T cells in the absence of exogenous stimuli and in the T cell mitogenic stimulation. Our findings provide novel evidence that EMP can enhance T cell activation and potentially ensuing Ag presentation, thereby pointing toward a novel role for MP in neuroimmunological complications of infectious diseases. Copyright © 2014 by The American Association of Immunologists, Inc.

Pivotal role of glutathione depletion in plasma-induced endothelial oxidative stress during sepsis.

PubMed

Huet, Olivier; Cherreau, Christaine; Nicco, Carole; Dupic, Laurent; Conti, Marc; Borderie, Didier; Pene, Frédéric; Vicaut, Eric; Benhamou, Dan; Mira, Jean-Paul; Duranteau, Jacques; Batteux, Frédéric

2008-08-01

Plasma from septic shock patients can induce production of reactive oxygen species (ROS) by human umbilical vein endothelial cells (HUVEC) in vitro. How endothelial cells defend themselves against ROS under increased oxidative stress has not yet been examined. This study investigates the antioxidant defenses of HUVEC exposed to plasma obtained from either septic shock patients or healthy volunteers. Prospective, observational study. Medical intensive care unit in a university hospital. Twenty-five patients with septic shock and 10 healthy volunteers. Blood samples were collected within the first 24 hrs of septic shock. In vitro HUVEC production of ROS was studied by spectrofluorimetry using 2',7'-dichlorodihydrofluorescein diacetate fluorescent dye. Reactive nitrogen species were also assessed. Intracellular reduced glutathione (GSH) levels were measured using monochlorobimane fluorescent dye. Activity of catalase and superoxide dismutase in HUVEC were also measured. Cell death was assessed using YOPRO fluorescent dye and the MTT assay. On admission, the septic shock population's mean age was 55 yrs old, the mean Sequential Organ Failure Assessment score was 12, mean simplified acute physiology score was 50, and intensive care unit mortality rate was 45%. Evaluation of HUVEC antioxidant defenses showed a significantly decreased GSH level, increased catalase activity, and unchanged superoxide dismutase activity. ROS levels and cell death were significantly reduced when cells were pretreated with N-acetylcysteine or GSH, but no changes in reactive nitrogen species were observed. This study demonstrates that plasma-induced ROS production by HUVEC is associated with an intracellular decrease in reduced GSH. Both ROS levels and cell death decreased when N-acetylcysteine or GSH were added before exposing the cells to plasma. These data suggest a pivotal role of alterations in GSH in damage caused by sepsis-generated ROS in endothelial cell.

Detection of Subclinical Arthritis in Mice by a Thrombin Receptor-Derived Imaging Agent.

PubMed

Friedman, Beth; Whitney, Michael A; Savariar, Elamprakash N; Caneda, Christa; Steinbach, Paul; Xiong, Qing; Hingorani, Dina V; Crisp, Jessica; Adams, Stephen R; Kenner, Michael; Lippert, Csilla N; Nguyen, Quyen T; Guma, Monica; Tsien, Roger Y; Corr, Maripat

2018-01-01

Functional imaging of synovitis could improve both early detection of rheumatoid arthritis (RA) and long-term outcomes. Given the intersection of inflammation with coagulation protease activation, this study was undertaken to examine coagulation protease activities in arthritic mice with a dual-fluorescence ratiometric activatable cell-penetrating peptide (RACPP) that has a linker, norleucine (Nle)-TPRSFL, with a cleavage site for thrombin. K/BxN-transgenic mice with chronic arthritis and mice with day 1 passive serum-transfer arthritis were imaged in vivo for Cy5:Cy7 emission ratiometric fluorescence from proteolytic cleavage and activation of RACPP NleTPRSFL . Joint thickness in mice with serum-transfer arthritis was measured from days 0 to 10. The cleavage-evoked release of Cy5-tagged tissue-adhesive fragments enabled microscopic correlation with immunohistochemistry for inflammatory markers. Thrombin dependence of ratiometric fluorescence was tested by ex vivo application of RACPP NleTPRSFL and argatroban to cryosections obtained from mouse hind paws on day 1 of serum-transfer arthritis. In chronic arthritis, RACPP NleTPRSFL fluorescence ratios of Cy5:Cy7 emission were significantly higher in diseased swollen ankles of K/BxN-transgenic mice than in normal mouse ankles. A high ratio of RACPP NleTPRSFL fluorescence in mouse ankles and toes on day 1 of serum-transfer arthritis correlated with subsequent joint swelling. Foci of high ratiometric fluorescence localized to inflammation, as demarcated by immune reactivity for citrullinated histones, macrophages, mast cells, and neutrophils, in soft tissue on day 1 of serum-transfer arthritis. Ex vivo application of RACPP NleTPRSFL to cryosections obtained from mice on day 1 of serum-transfer arthritis produced ratiometric fluorescence that was inhibited by argatroban. RACPP NleTPRSFL activation detects established experimental arthritis, and the detection of inflammation by RACPP NleTPRSFL on day 1 of serum-transfer arthritis correlates with disease progression. © 2017, American College of Rheumatology.

MiRAR-miRNA Activity Reporter for Living Cells.

PubMed

Turk, Matthew A; Chung, Christina Z; Manni, Emad; Zukowski, Stephanie A; Engineer, Anish; Badakhshi, Yasaman; Bi, Yumin; Heinemann, Ilka U

2018-06-19

microRNA (miRNA) activity and regulation are of increasing interest as new therapeutic targets. Traditional approaches to assess miRNA levels in cells rely on RNA sequencing or quantitative PCR. While useful, these approaches are based on RNA extraction and cannot be applied in real-time to observe miRNA activity with single-cell resolution. We developed a green fluorescence protein (GFP)-based reporter system that allows for a direct, real-time readout of changes in miRNA activity in live cells. The miRNA activity reporter (MiRAR) consists of GFP fused to a 3′ untranslated region containing specific miRNA binding sites, resulting in miRNA activity-dependent GFP expression. Using qPCR, we verified the inverse relationship of GFP fluorescence and miRNA levels. We demonstrated that this novel optogenetic reporter system quantifies cellular levels of the tumor suppressor miRNA let-7 in real-time in single Human embryonic kidney 293 (HEK 293) cells. Our data shows that the MiRAR can be applied to detect changes in miRNA levels upon disruption of miRNA degradation pathways. We further show that the reporter could be adapted to monitor another disease-relevant miRNA, miR-122. With trivial modifications, this approach could be applied across the miRNome for quantification of many specific miRNA in cell cultures, tissues, or transgenic animal models.

Antifungal mechanism of an antimicrobial peptide, HP (2--20), derived from N-terminus of Helicobacter pylori ribosomal protein L1 against Candida albicans.

PubMed

Lee, Dong Gun; Park, Yoonkyung; Kim, Hee Nam; Kim, Hyung Keun; Kim, Pyoung Il; Choi, Bo Hwa; Hahm, Kyung-Soo

2002-03-08

The antifungal activity and mechanism of HP (2-20), a peptide derived from the N-terminus sequence of Helicobacter pylori Ribosomal Protein L1 were investigated. HP (2--20) displayed a strong antifungal activity against various fungi, and the antifungal activity was inhibited by Ca(2+) and Mg(2+) ions. In order to investigate the antifungal mechanism(s) of HP (2-20), fluorescence activated flow cytometry was performed. As determined by propidium iodide staining, Candida albicans treated with HP (2-20) showed a higher fluorescence intensity than untreated cells and was similar to melittin-treated cells. The effect on fungal cell membranes was examined by investigating the change in membrane dynamics of C. albicans using 1,6-diphenyl-1,3,5-hexatriene as a membrane probe and by testing the membrane disrupting activity using liposome (PC/PS; 3:1, w/w) and by treating protoplasts of C. albicans with the peptide. The action of peptide against fungal cell membrane was further examined by the potassium-release test, and HP (2-20) was able to increase the amount of K(+) released from the cells. The result suggests that HP (2-20) may exert its antifungal activity by disrupting the structure of cell membrane via pore formation or directly interacts with the lipid bilayers in a salt-dependent manner.

Calcein AM release-based cytotoxic cell assay for fish leucocytes.

PubMed

Iwanowicz, Luke R; Densmore, Christine L; Ottinger, Christopher A

2004-02-01

A non-specific cytotoxic cell assay for fish is presented that is based on the release of the activated fluorochrome calcein AM from lysed carp epithelioma papulosum cyprini (EPC) cells. To establish the suitability of treating EPC cells with calcein AM the uptake and spontaneous release of the calcein AM by the EPC cells was evaluated. Incubation of 5 microM calcein AM in culture medium with 1x10(5)EPC cells well(-1)for a minimum of 3 h provided sufficient labelling. Spontaneous release of fluorescence from the labelled EPC cells during 10 h of post labelling incubation ranged from 30 to 39% of the total observed fluorescence. Cytotoxic activity of trout leucocytes was evaluated at three leucocyte to target cell ratios (10:1, 2:1 and 1:1) following incubation (4, 6, 8, and 10 h) with calcein AM-labelled EPC cells at 15 degrees C. In some instances, the monoclonal antibody specific for the NCC surface receptor NCCRP-1 (MAb5C.6) was included in the cultures. The activity of NCC cells was significantly inhibited in the presence of 0.25 microg well(-1)of MAb5C.6 relative to no antibody (P

Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

NASA Technical Reports Server (NTRS)

Mozdziak, P. E.; Pulvermacher, P. M.; Schultz, E.; Schell, K.

2000-01-01

BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. Copyright 2000 Wiley-Liss, Inc.

A novel fluorescent assay for sucrose transporters.

PubMed

Gora, Peter J; Reinders, Anke; Ward, John M

2012-04-04

We have developed a novel assay based on the ability of type I sucrose uptake transporters (SUTs) to transport the fluorescent coumarin β-glucoside, esculin. Budding yeast (Saccharomyces cerevisiae) is routinely used for the heterologous expression of SUTs and does not take up esculin. When type I sucrose transporters StSUT1 from potato or AtSUC2 from Arabidopsis were expressed in yeast, the cells were able to take up esculin and became brightly fluorescent. We tested a variety of incubation times, esculin concentrations, and buffer pH values and found that for these transporters, a 1 hr incubation at 0.1 to 1 mM esculin at pH 4.0 produced fluorescent cells that were easily distinguished from vector controls. Esculin uptake was assayed by several methods including fluorescence microscopy, spectrofluorometry and fluorescence-activiated cell sorting (FACS). Expression of the type II sucrose transporter OsSUT1 from rice did not result in increased esculin uptake under any conditions tested. Results were reproduced successfully in two distinct yeast strains, SEY6210 (an invertase mutant) and BY4742. The esculin uptake assay is rapid and sensitive and should be generally useful for preliminary tests of sucrose transporter function by heterologous expression in yeast. This assay is also suitable for selection of yeast showing esculin uptake activity using FACS.

[Natural killer cell cytotoxic activity in critical pediatric patients with suspected hemophagocytic syndrome].

PubMed

Martínez, I; Fernández, L; Valentín, J; Castillo, C; Chamorro, C; Pérez-Martínez, A

2015-05-01

To determine the role of natural killer (NK) cytotoxic activity in patients with suspected hemophagocytic lymphohistiocytosis syndrome (HLH). A prospective study was conducted from September 2008 to February 2014. The study was carried out in the Hematological Oncology Laboratory of Hospital Infantil Universitario Niño Jesús, Madrid (Spain). We analyzed 30 peripheral blood samples from intensive care patients with suspected HLH. There were 18 males and 12 females, with a mean age of 4.7 years (range 0.2-22). NK cell cytotoxicity was compared with healthy controls according to age and sex. In vitro NK cell cytotoxicity against the K562 cell line was determined by time-resolved fluorescence (Europium-TDA) under resting conditions, after interleukin 15 stimulation, and following block with Fas ligand antibody. NK cell cytotoxicity. A total of 20 patients showed a significant decrease of NK cell activity compared with controls (P=.001). Nine of these patients were diagnosed with primary HLH. A total of 10 patients were diagnosed with secondary HLH. Cytotoxic activity was normal in 10 subjects. None of them were diagnosed with HLH. Interleukin 15 stimulation increased NK cell cytotoxicity in secondary HLH, and blocking Fas ligand on NK cells decreased cytotoxic activity in primary HLH patients (P=.001). In our experience, NK cell cytotoxic activity measured by time-resolved fluorescence is a simple and useful clinical diagnostic test for HLH. Interleukin 15 stimulation and Fas ligand blocking on NK cells could help differentiate between primary and secondary HLH. Copyright © 2014 Elsevier España, S.L.U. and SEMICYUC. All rights reserved.

Detection of tripeptidyl peptidase I activity in living cells by fluorogenic substrates.

PubMed

Steinfeld, Robert; Fuhrmann, Jens C; Gärtner, Jutta

2006-09-01

Tripeptidyl peptidase I (TPP-I) is a lysosomal peptidase with unclear physiological function. TPP-I deficiency is associated with late-infantile neuronal ceroid lipofuscinosis (NCL), a fatal neurodegenerative disease of childhood that is characterized by loss of neurons and photoreceptor cells. We have developed two novel fluorogenic substrates, [Ala-Ala-Phe]2-rhodamine 110 and [Arg-Nle-Nle]2-rhodamine 110, that are cleaved by TPP-I in living cells. Fluorescence of liberated rhodamine 110 was detected by flow cytometry and was dependent on the level of TPP-I expression. Rhodamine-related fluorescence could be suppressed by preincubation with a specific inhibitor of TPP-I. When investigated by fluorescent confocal microscopy, rhodamine signals colocalized with lysosomal markers. Thus, cleavage of these rhodamide-derived substrates is a marker for mature enzymatically active TPP-I. In addition, TPP-I-induced cleavage of [Ala-Ala-Phe]2-rhodamine 110 could be visualized in primary neurons. We conclude that [Ala-Ala-Phe]2-rhodamine 110 and [Arg-Nle-Nle]2-rhodamine 110 are specific substrates for determining TPP-I activity and intracellular localization in living cells. Further, these substrates could be a valuable tool for studying the neuronal pathology underlying classical late-infantile NCL. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Preferential and Increased Uptake of Hydroxyl-Terminated PAMAM Dendrimers by Activated Microglia in Rabbit Brain Mixed Glial Culture.

PubMed

Alnasser, Yossef; Kambhampati, Siva P; Nance, Elizabeth; Rajbhandari, Labchan; Shrestha, Shiva; Venkatesan, Arun; Kannan, Rangaramanujam M; Kannan, Sujatha

2018-04-27

Polyamidoamine (PAMAM) dendrimers are multifunctional nanoparticles with tunable physicochemical features, making them promising candidates for targeted drug delivery in the central nervous system (CNS). Systemically administered dendrimers have been shown to localize in activated glial cells, which mediate neuroinflammation in the CNS. These dendrimers delivered drugs specifically to activated microglia, producing significant neurological improvements in multiple brain injury models, including in a neonatal rabbit model of cerebral palsy. To gain further insight into the mechanism of dendrimer cell uptake, we utilized an in vitro model of primary glial cells isolated from newborn rabbits to assess the differences in hydroxyl-terminated generation 4 PAMAM dendrimer (D4-OH) uptake by activated and non-activated glial cells. We used fluorescently-labelled D4-OH (D-Cy5) as a tool for investigating the mechanism of dendrimer uptake. D4-OH PAMAM dendrimer uptake was determined by fluorescence quantification using confocal microscopy and flow cytometry. Our results indicate that although microglial cells in the mixed cell population demonstrate early uptake of dendrimers in this in vitro system, activated microglia take up more dendrimer compared to resting microglia. Astrocytes showed delayed and limited uptake. We also illustrated the differences in mechanism of uptake between resting and activated microglia using different pathway inhibitors. Both resting and activated microglia primarily employed endocytotic pathways, which are enhanced in activated microglial cells. Additionally, we demonstrated that hydroxyl terminated dendrimers are taken up by primary microglia using other mechanisms including pinocytosis, caveolae, and aquaporin channels for dendrimer uptake.

Catalytic properties of the Gas family β-(1,3)-glucanosyltransferases active in fungal cell-wall biogenesis as determined by a novel fluorescent assay.

PubMed

Mazáň, Marián; Ragni, Enrico; Popolo, Laura; Farkaš, Vladimír

2011-09-01

BGTs [β-(1,3)-glucanosyltransglycosylases; EC 2.4.1.-] of the GH72 (family 72 of glycosylhydrolases) are GPI (glycosylphosphatidylinositol)-anchored proteins that play an important role in the biogenesis of fungal cell walls. They randomly cleave glycosidic linkages in β-(1,3)-glucan chains and ligate the polysaccharide portions containing newly formed reducing ends to C(3)(OH) at non-reducing ends of other β-(1,3)-glucan molecules. We have developed a sensitive fluorescence-based method for the assay of transglycosylating activity of GH72 enzymes. In the new assay, laminarin [β-(1,3)-glucan] is used as the glucanosyl donor and LamOS (laminarioligosaccharides) fluorescently labelled with SR (sulforhodamine) serve as the acceptors. The new fluorescent assay was employed for partial biochemical characterization of the heterologously expressed Gas family proteins from the yeast Saccharomyces cerevisiae. All the Gas enzymes specifically used laminarin as the glucanosyl donor and a SR-LamOS of DP (degree of polymerization) ≥5 as the acceptors. Gas proteins expressed in distinct stages of the yeast life cycle showed differences in their pH optima. Gas1p and Gas5p, which are expressed during vegetative growth, had the highest activity at pH 4.5 and 3.5 respectively, whereas the sporulation-specific Gas2p and Gas4p were most active between pH 5 and 6. The novel fluorescent assay provides a suitable tool for the screening of potential glucanosyltransferases or their inhibitors.

Photoinduction of cyclosis-mediated interactions between distant chloroplasts.

PubMed

Bulychev, Alexander A; Komarova, Anna V

2015-01-01

Communications between chloroplasts and other organelles based on the exchange of metabolites, including redox active substances, are recognized as a part of intracellular regulation, chlororespiration, and defense against oxidative stress. Similar communications may operate between spatially distant chloroplasts in large cells where photosynthetic and respiratory activities are distributed unevenly under fluctuating patterned illumination. Microfluorometry of chlorophyll fluorescence in vivo in internodal cells of the alga Chara corallina revealed that a 30-s pulse of localized light induces a transient increase (~25%) in F' fluorescence of remote cell parts exposed to dim background light at a 1.5-mm distance on the downstream side from the illuminated spot in the plane of unilateral cytoplasmic streaming but has no effect on F' at equal distance on the upstream side. An abrupt arrest of cytoplasmic streaming for about 30s by triggering the action potential extended either the ascending or descending fronts of the F' fluorescence response, depending on the exact moment of streaming cessation. The response of F' fluorescence to localized illumination of a distant cell region was absent in dark-adapted internodes, when the localized light was applied within the first minute after switching on continuous background illumination of the whole cell, but it appeared in full after longer exposures to continuous background light. These results and the elimination of the F' response by methyl viologen known to redirect electron transport pathways beyond photosystem I indicate the importance of photosynthetic induction and the stromal redox state for long-distance communications of chloroplasts in vivo. Copyright © 2015 Elsevier B.V. All rights reserved.

Endothelial microparticles interact with and support the proliferation of T cells

PubMed Central

Wheway, Julie; Latham, Sharissa L; Combes, Valery; Grau, Georges ER

2014-01-01

Endothelial cells (EC) closely interact with circulating lymphocytes. Aggression or activation of the endothelium leads to an increased shedding of EC microparticles (MP). Endothelial MP (EMP) are found in high plasma levels in numerous immunoinflammatory diseases, e.g. atherosclerosis, sepsis, multiple sclerosis and cerebral malaria, supporting their role as effectors and markers of vascular dysfunction. Given our recently described role for human brain microvascular endothelial cells (HBEC) in modulating immune responses we investigated how HBEC-derived MP could interact with and support the proliferation of T cells. Like their mother cells, EMP expressed molecules important for antigen presentation and T cell co-stimulation, i.e., β2-microglobulin, MHC II, CD40 and ICOSL. HBEC were able to take up fluorescently labeled antigens with EMP also containing fluorescent antigens suggestive of antigen carryover from HBEC to EMP. In co-cultures, fluorescently labeled EMP from resting or cytokine-stimulated HBEC formed conjugates with both CD4+ and CD8+ subsets, with higher proportions of T cells binding EMP from cytokine stimulated cells. The increased binding of EMP from cytokine stimulated HBEC to T cells was VCAM-1 and ICAM-1-dependent. Finally, in CFSE T cell proliferation assays using anti-CD3 mAb or T cell mitogens, EMP promoted the proliferation of CD4+ T cells and that of CD8+ T cells in the absence of exogenous stimuli and in the T cell mitogenic stimulation. Our findings provide novel evidence that EMP can enhance T cell activation and potentially ensuing antigen presentation, thereby pointing towards a novel role for MP in neuro-immunological complications of infectious diseases. PMID:25187656

Isolation of intact RNA from murine CD4+ T cells after intracellular cytokine staining and fluorescence-activated cell sorting.

PubMed

Kunnath-Velayudhan, Shajo; Porcelli, Steven A

2018-05-01

Intracellular cytokine staining (ICS) is a powerful method for identifying functionally distinct lymphocyte subsets, and for isolating these by fluorescence activated cell sorting (FACS). Although transcriptomic analysis of cells sorted on the basis of ICS has many potential applications, this is rarely performed because of the difficulty in isolating intact RNA from cells processed using standard fixation and permeabilization buffers for ICS. To address this issue, we compared three buffers shown previously to preserve RNA in nonhematopoietic cells subjected to intracellular staining for their effects on RNA isolated from T lymphocytes processed for ICS. Our results showed that buffers containing the recombinant ribonuclease inhibitor RNasin or high molar concentrations of salt yielded intact RNA from fixed and permeabilized T cells. As proof of principle, we successfully used the buffer containing RNasin to isolate intact RNA from CD4 + T cells that were sorted by FACS on the basis of specific cytokine production, thus demonstrating the potential of this approach for coupling ICS with transcriptomic analysis. Copyright © 2018 Elsevier B.V. All rights reserved.

The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2

PubMed Central

Kerr, Niall; Holmes, Fiona E.; Hobson, Sally-Ann; Vanderplank, Penny; Leard, Alan; Balthasar, Nina; Wynick, David

2015-01-01

The neuropeptide galanin has diverse roles in the central and peripheral nervous systems, by activating the G protein-coupled receptors Gal1, Gal2 and the less studied Gal3 (GalR1–3 gene products). There is a wealth of data on expression of Gal1–3 at the mRNA level, but not at the protein level due to the lack of specificity of currently available antibodies. Here we report the generation of knock-in mice expressing Gal1 or Gal2 receptor fluorescently tagged at the C-terminus with, respectively, mCherry or hrGFP (humanized Renilla green fluorescent protein). In dorsal root ganglia (DRG) neurons expressing the highest levels of Gal1-mCherry, localization to the somatic cell membrane was detected by live-cell fluorescence and immunohistochemistry, and that fluorescence decreased upon addition of galanin. In spinal cord, abundant Gal1-mCherry immunoreactive processes were detected in the superficial layers of the dorsal horn, and highly expressing intrinsic neurons of the lamina III/IV border showed both somatic cell membrane localization and outward transport of receptor from the cell body, detected as puncta within cell processes. In brain, high levels of Gal1-mCherry immunofluorescence were detected within thalamus, hypothalamus and amygdala, with a high density of nerve endings in the external zone of the median eminence, and regions with lesser immunoreactivity included the dorsal raphe nucleus. Gal2-hrGFP mRNA was detected in DRG, but live-cell fluorescence was at the limits of detection, drawing attention to both the much lower mRNA expression than to Gal1 in mice and the previously unrecognized potential for translational control by upstream open reading frames (uORFs). PMID:26292267

Target-cancer-cell-specific activatable fluorescence imaging probes: rational design and in vivo applications.

PubMed

Kobayashi, Hisataka; Choyke, Peter L

2011-02-15

Conventional imaging methods, such as angiography, computed tomography (CT), magnetic resonance imaging (MRI), and radionuclide imaging, rely on contrast agents (iodine, gadolinium, and radioisotopes, for example) that are "always on." Although these indicators have proven clinically useful, their sensitivity is lacking because of inadequate target-to-background signal ratio. A unique aspect of optical imaging is that fluorescence probes can be designed to be activatable, that is, only "turned on" under certain conditions. These probes are engineered to emit signal only after binding a target tissue; this design greatly increases sensitivity and specificity in the detection of disease. Current research focuses on two basic types of activatable fluorescence probes. The first developed were conventional enzymatically activatable probes. These fluorescent molecules exist in the quenched state until activated by enzymatic cleavage, which occurs mostly outside of the cells. However, more recently, researchers have begun designing target-cell-specific activatable probes. These fluorophores exist in the quenched state until activated within targeted cells by endolysosomal processing, which results when the probe binds specific receptors on the cell surface and is subsequently internalized. In this Account, we present a review of the rational design and in vivo applications of target-cell-specific activatable probes. In engineering these probes, researchers have asserted control over a variety of factors, including photochemistry, pharmacological profile, and biological properties. Their progress has recently allowed the rational design and synthesis of target-cell-specific activatable fluorescence imaging probes, which can be conjugated to a wide variety of targeting molecules. Several different photochemical mechanisms have been utilized, each of which offers a unique capability for probe design. These include self-quenching, homo- and hetero-fluorescence resonance energy transfer (FRET), H-dimer formation, and photon-induced electron transfer (PeT). In addition, the repertoire is further expanded by the option for reversibility or irreversibility of the signal emitted through these mechanisms. Given the wide range of photochemical mechanisms and properties, target-cell-specific activatable probes have considerable flexibility and can be adapted to specific diagnostic needs. A multitude of cell surface molecules, such as overexpressed growth factor receptors, are directly related to carcinogenesis and thus provide numerous targets highly specific for cancer. This discussion of the chemical, pharmacological, and biological basis of target-cell-specific activatable imaging probes, and methods for successfully designing them, underscores the systematic, rational basis for further developing in vivo cancer imaging.

Insulin Induces an Increase in Cytosolic Glucose Levels in 3T3-L1 Cells with Inhibited Glycogen Synthase Activation

PubMed Central

Chowdhury, Helena H.; Kreft, Marko; Jensen, Jørgen; Zorec, Robert

2014-01-01

Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ) to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway. PMID:25279585

Berberine Induces Toxicity in HeLa Cells through Perturbation of Microtubule Polymerization by Binding to Tubulin at a Unique Site.

PubMed

Raghav, Darpan; Ashraf, Shabeeba M; Mohan, Lakshmi; Rathinasamy, Krishnan

2017-05-23

Berberine has been used traditionally for its diverse pharmacological actions. It exhibits remarkable anticancer activities and is currently under clinical trials. In this study, we report that the anticancer activity of berberine could be partly due to its inhibitory actions on tubulin and microtubule assembly. Berberine inhibited the proliferation of HeLa cells with an IC 50 of 18 μM and induced significant depolymerization of interphase and mitotic microtubules. At its IC 50 , berberine exerted a moderate G2/M arrest and mitotic block as detected by fluorescence-activated cell sorting analysis and fluorescence microscopy, respectively. In a wound closure assay, berberine inhibited the migration of HeLa cells at concentrations lower than its IC 50 , indicating its excellent potential as an anticancer agent. In vitro studies with tubulin isolated from goat brain indicated that berberine binds to tubulin at a single site with a K d of 11 μM. Berberine inhibited the assembly of tubulin into microtubules and also disrupted the preformed microtubules polymerized in the presence of glutamate and paclitaxel. Competition experiments indicated that berberine could partially displace colchicine from its binding site. Results from fluorescence resonance energy transfer, computational docking, and molecular dynamics simulations suggest that berberine forms a stable complex with tubulin and binds at a novel site 24 Å from the colchicine site on the β-tubulin. Data obtained from synchronous fluorescence analysis of the tryptophan residues of tubulin and from the Fourier transform infrared spectroscopy studies revealed that binding of berberine alters the conformation of the tubulin heterodimer, which could be the molecular mechanism behind the depolymerizing effects on tubulin assembly.

A Cell Number Counting Factor Regulates Akt/Protein Kinase B To Regulate Dictyostelium discoideum Group Size

PubMed Central

Gao, Tong; Knecht, David; Tang, Lei; Hatton, R. Diane; Gomer, Richard H.

2004-01-01

Little is known about how individual cells can organize themselves to form structures of a given size. During development, Dictyostelium discoideum aggregates in dendritic streams and forms groups of ∼20,000 cells. D. discoideum regulates group size by secreting and simultaneously sensing a multiprotein complex called counting factor (CF). If there are too many cells in a stream, the associated high concentration of CF will decrease cell-cell adhesion and increase cell motility, causing aggregation streams to break up. The pulses of cyclic AMP (cAMP) that mediate aggregation cause a transient translocation of Akt/protein kinase B (Akt/PKB) to the leading edge of the plasma membrane and a concomitant activation of the kinase activity, which in turn stimulates motility. We found that countin− cells (which lack bioactive CF) and wild-type cells starved in the presence of anticountin antibodies (which block CF activity) showed a decreased level of cAMP-stimulated Akt/PKB membrane translocation and kinase activity compared to parental wild-type cells. Recombinant countin has the bioactivity of CF, and a 1-min treatment of cells with recombinant countin potentiated Akt/PKB translocation to membranes and Akt/PKB activity. Western blotting of total cell lysates indicated that countin does not affect the total level of Akt/PKB. Fluorescence microscopy of cells expressing an Akt/PKB pleckstrin homology domain-green fluorescent protein (PH-GFP) fusion protein indicated that recombinant countin and anti-countin antibodies do not obviously alter the distribution of Akt/PKB PH-GFP when it translocates to the membrane. Our data indicate that CF increases motility by potentiating the cAMP-stimulated activation and translocation of Akt/PKB. PMID:15470246

Mitochondrial Fragmentation in Aspergillus fumigatus as Early Marker of Granulocyte Killing Activity

PubMed Central

Ruf, Dominik; Brantl, Victor; Wagener, Johannes

2018-01-01

The host's defense against invasive mold infections relies on diverse antimicrobial activities of innate immune cells. However, studying these mechanisms in vitro is complicated by the filamentous nature of such pathogens that typically form long, branched, multinucleated and compartmentalized hyphae. Here we describe a novel method that allows for the visualization and quantification of the antifungal killing activity exerted by human granulocytes against hyphae of the opportunistic pathogen Aspergillus fumigatus. The approach relies on the distinct impact of fungal cell death on the morphology of mitochondria that were visualized with green fluorescent protein (GFP). We show that oxidative stress induces complete fragmentation of the tubular mitochondrial network which correlates with cell death of affected hyphae. Live cell microscopy revealed a similar and non-reversible disruption of the mitochondrial morphology followed by fading of fluorescence in Aspergillus hyphae that were killed by human granulocytes. Quantitative microscopic analysis of fixed samples was subsequently used to estimate the antifungal activity. By utilizing this assay, we demonstrate that lipopolysaccharides as well as human serum significantly increase the killing efficacy of the granulocytes. Our results demonstrate that evaluation of the mitochondrial morphology can be utilized to assess the fungicidal activity of granulocytes against A. fumigatus hyphae. PMID:29868488

Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements.

PubMed

Wolfe, Kelly L; Liu, Rui Hai

2007-10-31

A cellular antioxidant activity (CAA) assay for quantifying the antioxidant activity of phytochemicals, food extracts, and dietary supplements has been developed. Dichlorofluorescin is a probe that is trapped within cells and is easily oxidized to fluorescent dichlorofluorescein (DCF). The method measures the ability of compounds to prevent the formation of DCF by 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-generated peroxyl radicals in human hepatocarcinoma HepG2 cells. The decrease in cellular fluorescence when compared to the control cells indicates the antioxidant capacity of the compounds. The antioxidant activities of selected phytochemicals and fruit extracts were evaluated using the CAA assay, and the results were expressed in micromoles of quercetin equivalents per 100 micromol of phytochemical or micromoles of quercetin equivalents per 100 g of fresh fruit. Quercetin had the highest CAA value, followed by kaempferol, epigallocatechin gallate (EGCG), myricetin, and luteolin among the pure compounds tested. Among the selected fruits tested, blueberry had the highest CAA value, followed by cranberry > apple = red grape > green grape. The CAA assay is a more biologically relevant method than the popular chemistry antioxidant activity assays because it accounts for some aspects of uptake, metabolism, and location of antioxidant compounds within cells.

Sensitive detection of proteasomal activation using the Deg-On mammalian synthetic gene circuit.

PubMed

Zhao, Wenting; Bonem, Matthew; McWhite, Claire; Silberg, Jonathan J; Segatori, Laura

2014-04-08

The ubiquitin proteasome system (UPS) has emerged as a drug target for diverse diseases characterized by altered proteostasis, but pharmacological agents that enhance UPS activity have been challenging to establish. Here we report the Deg-On system, a genetic inverter that translates proteasomal degradation of the transcriptional regulator TetR into a fluorescent signal, thereby linking UPS activity to an easily detectable output, which can be tuned using tetracycline. We demonstrate that this circuit responds to modulation of UPS activity in cell culture arising from the inhibitor MG-132 and activator PA28γ. Guided by predictive modelling, we enhanced the circuit's signal sensitivity and dynamic range by introducing a feedback loop that enables self-amplification of TetR. By linking UPS activity to a simple and tunable fluorescence output, these genetic inverters will enable a variety of applications, including screening for UPS activating molecules and selecting for mammalian cells with different levels of proteasome activity.

A new aggregation-induced emission fluorescent probe for rapid detection of nitroreductase and its application in living cells

NASA Astrophysics Data System (ADS)

Xu, Gaoping; Tang, Yonghe; Ma, Yanyan; Xu, An; Lin, Weiying

2018-01-01

The biological activity of nitroreductase (NTR) is closely related to biological hypoxia status in organisms. The development of effective methods for monitoring the activity of NTR is of great significance for medical diagnosis and tumor research. Toward this goal, we have developed a new aggregation-induced emission (AIE) fluorescence NTR probe TPE-HY used the tetraphenylethene as the fluorophore, and used the nitro group as the NTR recognition site. The probe TPE-HY has many excellent properties, including rapid response, AIE characteristics, high sensitivity and selectivity, and low cytotoxicity. Importantly, the probe TPE-HY is successfully applied to monitor endogenous NTR in living HeLa cells.

Uptake of Fluorescent Gentamicin by Peripheral Vestibular Cells after Systemic Administration

PubMed Central

Liu, Jianping; Kachelmeier, Allan; Dai, Chunfu; Li, Hongzhe; Steyger, Peter S.

2015-01-01

Objective In addition to cochleotoxicity, systemic aminoglycoside pharmacotherapy causes vestibulotoxicity resulting in imbalance and visual dysfunction. The underlying trafficking routes of systemically-administered aminoglycosides from the vasculature to the vestibular sensory hair cells are largely unknown. We investigated the trafficking of systemically-administered gentamicin into the peripheral vestibular system in C56Bl/6 mice using fluorescence-tagged gentamicin (gentamicin-Texas-Red, GTTR) imaged by scanning laser confocal microscopy to determine the cellular distribution and intensity of GTTR fluorescence in the three semicircular canal cristae, utricular, and saccular maculae at 5 time points over 4 hours. Results Low intensity GTTR fluorescence was detected at 0.5 hours as both discrete puncta and diffuse cytoplasmic fluorescence. The intensity of cytoplasmic fluorescence peaked at 3 hours, while punctate fluorescence was plateaued after 3 hours. At 0.5 and 1 hour, higher levels of diffuse GTTR fluorescence were present in transitional cells compared to hair cells and supporting cells. Sensory hair cells typically exhibited only diffuse cytoplasmic fluorescence at all time-points up to 4 hours in this study. In contrast, non-sensory cells rapidly exhibited both intense fluorescent puncta and weaker, diffuse fluorescence throughout the cytosol. The numbers and size of fluorescent puncta in dark cells and transitional cells increased over time. There is no preferential GTTR uptake by the five peripheral vestibular organs’ sensory cells. Control vestibular tissues exposed to Dulbecco’s phosphate-buffered saline or hydrolyzed Texas Red had negligible fluorescence. Conclusions All peripheral vestibular cells rapidly take up systemically-administered GTTR, reaching peak intensity 3 hours after injection. Sensory hair cells exhibited only diffuse fluorescence, while non-sensory cells displayed both diffuse and punctate fluorescence. Transitional cells may act as a primary pathway for trafficking of systemic GTTR from the vasculature to endolymph prior to entering hair cells. PMID:25793391

Computer simulations of the energy dissipation rate in a fluorescence-activated cell sorter: Implications to cells.

PubMed

Mollet, Mike; Godoy-Silva, Ruben; Berdugo, Claudia; Chalmers, Jeffrey J

2008-06-01

Fluorescence activated cell sorting, FACS, is a widely used method to sort subpopulations of cells to high purities. To achieve relatively high sorting speeds, FACS instruments operate by forcing suspended cells to flow in a single file line through a laser(s) beam(s). Subsequently, this flow stream breaks up into individual drops which can be charged and deflected into multiple collection streams. Previous work by Ma et al. (2002) and Mollet et al. (2007; Biotechnol Bioeng 98:772-788) indicates that subjecting cells to hydrodynamic forces consisting of both high extensional and shear components in micro-channels results in significant cell damage. Using the fluid dynamics software FLUENT, computer simulations of typical fluid flow through the nozzle of a BD FACSVantage indicate that hydrodynamic forces, quantified using the scalar parameter energy dissipation rate, are similar in the FACS nozzle to levels reported to create significant cell damage in micro-channels. Experimental studies in the FACSVantage, operated under the same conditions as the simulations confirmed significant cell damage in two cell lines, Chinese Hamster Ovary cells (CHO) and THP1, a human acute monocytic leukemia cell line.

Near-infrared fluorescent peptide probes for imaging of tumor in vivo and their biotoxicity evaluation.

PubMed

Liu, Liwei; Lin, Guimiao; Yin, Feng; Law, Wing-Cheung; Yong, Ken-Tye

2016-04-01

Optical imaging techniques are becoming increasingly urgent for the early detection and monitoring the progression of tumor development. However, tumor vasculature imaging has so far been largely unexplored because of the lack of suitable optical probes. In this study, we demonstrated the preparation of near-infrared (NIR) fluorescent RGD peptide probes for noninvasive imaging of tumor vasculature during tumor angiogenesis. The peptide optical probes combined the advantages of NIR emission and RGD peptide, which possesses minimal biological absorption and specially targets the integrin, which highly expressed on activated tumor endothelial cells. In vivo optical imaging of nude mice bearing pancreatic tumor showed that systemically delivered NIR probes enabled us to visualize the tumors at 24 hours post-injection. In addition, we have performed in vivo toxicity study on the prepared fluorescent RGD peptide probes formulation. The blood test results and histological analysis demonstrated that no obvious toxicity was found for the mice treated with RGD peptide probes for two weeks. These studies suggest that the NIR fluorescent peptide probes can be further designed and employed for ultrasensitive fluorescence imaging of angiogenic tumor vasculature, as well as imaging of other pathophysiological processes accompanied by activation of endothelial cells. © 2016 Wiley Periodicals, Inc.

Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells

NASA Astrophysics Data System (ADS)

Jahn, Karolina; Buschmann, Volker; Hille, Carsten

2015-09-01

In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

Fluorescent magnetic bead-based mast cell biosensor for electrochemical detection of allergens in foodstuffs.

PubMed

Jiang, Donglei; Zhu, Pei; Jiang, Hui; Ji, Jian; Sun, Xiulan; Gu, Wenshu; Zhang, Genyi

2015-08-15

In this study, a novel electrochemical rat basophilic leukemia cell (RBL-2H3) cell sensor, based on fluorescent magnetic beads, has been developed for the detection and evaluation of different allergens in foodstuffs. Fluorescein isothiocyanate (FITC) was successfully fused inside the SiO2 layer of SiO2 shell-coated Fe3O4 nanoparticles, which was superior to the traditional Fe3O4@SiO2@FITC modification process. The as-synthesized fluorescent magnetic beads were then encapsulated with lipidosome to form cationic magnetic fluorescent nanoparticles (CMFNPs) for mast cell magnetofection. The CMFNPs were then characterized by SEM, TEM, VSM, FTIR, and XRD analyses, and transfected into RBL-2H3 cells through a highly efficient, lipid-mediated magnetofection procedure. Magnetic glassy carbon electrode (MGCE), which possesses excellent reproducibility and regeneration qualities, was then employed to adsorb the CMFNP-transfected RBL-2H3 cells activated by an allergen antigen for electrochemical assay. Results show that the exposure of model antigen-dinitrophenol-bovine serum albumin (DNP-BSA) to anti-DNP IgE-sensitized mast cells induced a robust and long-lasting electrochemical impedance signal in a dose-dependent manner. The detection limit was identified at 3.3×10(-4) ng/mL. To demonstrate the utility of this mast cell-based biosensor for detection of real allergens in foodstuffs, Anti-Pen a1 IgE and Anti-PV IgE-activated cells were employed to quantify both shrimp allergen tropomyosin (Pen a 1) and fish allergen parvalbumin (PV). Results show high detection accuracy for these targets, with a limit of 0.03 μg/mL (shrimp Pen a 1) and 0.16 ng/mL (fish PV), respectively. To this effect, we conclude the proposed method is a facile, highly sensitive, innovative electrochemical method for the evaluation of food allergens. Copyright © 2015 Elsevier B.V. All rights reserved.

3D Tracking of individual growth factor receptors on polarized cells

NASA Astrophysics Data System (ADS)

Werner, James; Stich, Dominik; Cleyrat, Cedric; Phipps, Mary; Wadinger-Ness, Angela; Wilson, Bridget

We have been developing methods for following 3D motion of selected biomolecular species throughout mammalian cells. Our approach exploits a custom designed confocal microscope that uses a unique spatial filter geometry and active feedback 200 times/second to follow fast 3D motion. By exploiting new non-blinking quantum dots as fluorescence labels, individual molecular trajectories can be observed for several minutes. We also will discuss recent instrument upgrades, including the ability to perform spinning disk fluorescence microscopy on the whole mammalian cell performed simultaneously with 3D molecular tracking experiments. These instrument upgrades were used to quantify 3D heterogeneous transport of individual growth factor receptors (EGFR) on live human renal cortical epithelial cells.

A novel pH sensitive water soluble fluorescent nanomicellar sensor for potential biomedical applications.

PubMed

Georgiev, Nikolai I; Bryaskova, Rayna; Tzoneva, Rumiana; Ugrinova, Iva; Detrembleur, Christophe; Miloshev, Stoyan; Asiri, Abdullah M; Qusti, Abdullah H; Bojinov, Vladimir B

2013-11-01

Herein we report on the synthesis and sensor activity of a novel pH sensitive probe designed as highly water-soluble fluorescent micelles by grafting of 1,8-naphthalimide-rhodamine bichromophoric FRET system (RNI) to the PMMA block of a well-defined amphiphilic diblock copolymer-poly(methyl methacrylate)-b-poly(methacrylic acid) (PMMA48-b-PMAA27). The RNI-PMMA48-b-PMAA27 adduct is capable of self-assembling into micelles with a hydrophobic PMMA core, containing the anchored fluorescent probe, and a hydrophilic shell composed of PMAA block. Novel fluorescent micelles are able to serve as a highly sensitive pH probe in water and to internalize successfully HeLa and HEK cells. Furthermore, they showed cell specificity and significantly higher photostability than that of a pure organic dye label such as BODIPY. The valuable properties of the newly prepared fluorescent micelles indicate the high potential of the probe for future biological and biomedical applications. Copyright © 2013 Elsevier Ltd. All rights reserved.

Highly Enhanced Fluorescence of CdSeTe Quantum Dots Coated with Polyanilines via In-Situ Polymerization and Cell Imaging Application.

PubMed

Xue, Jingjing; Chen, Xinyi; Liu, Shanglin; Zheng, Fenfen; He, Li; Li, Lingling; Zhu, Jun-Jie

2015-09-02

The polyaniline (PAN)-coated CdSeTe quantum dots (QDs) were prepared by in situ polymerization of aniline on the surface of CdSeTe QDs. The PAN-coated CdSeTe QDs has a tremendously enhanced fluorescence (∼40 times) and improved biocompatibility compared to the uncoated CdSeTe QDs. The fluorescence intensity of the PAN-coated CdSeTe QDs can be adjusted by controlling the construction parameters of the PAN shell. The kinetics of the in situ controllable polymerization process was studied by varying the temperature, and the apparent activation energy of polymerization was estimated. With the same method, a series of the PAN derivatives were also tested to coat the CdSeTe QDs in this study. All the QDs showed a significant enhancement of the fluorescence intensity and better biocompatibility. The significantly enhanced fluorescence can provide highly amplified signal for luminescence-based cell imaging.

Current density reversibly alters metabolic spatial structure of exoelectrogenic anode biofilms

NASA Astrophysics Data System (ADS)

Sun, Dan; Cheng, Shaoan; Zhang, Fang; Logan, Bruce E.

2017-07-01

Understanding how current densities affect electrogenic biofilm activity is important for wastewater treatment as current densities can substantially decrease at COD concentrations greater than those suitable for discharge to the environment. We examined the biofilm's response, in terms of viability and enzymatic activity, to different current densities using microbial electrolysis cells with a lower (0.7 V) or higher (0.9 V) added voltage to alter current production. Viability was assessed using florescent dyes, with dead cells identified on the basis of dye penetration due to a compromised cell outer-membrane (red), and live cells (intact membrane) fluorescing green. Biofilms operated with 0.7 V produced 2.4 ± 0.2 A m-2, and had an inactive layer near the electrode and a viable layer at the biofilm-solution interface. The lack of cell activity near the electrode surface was confirmed by using an additional dye that fluoresces only with enzymatic activity. Adding 0.9 V increased the current by 61%, and resulted in a single, more homogeneous and active biofilm layer. Switching biofilms between these two voltages produced outcomes associated with the new current rather than the previous biofilm conditions. These findings suggest that maintaining higher current densities will be needed to ensure long-term viability electrogenic biofilms.

Reduced background autofluorescence for cell imaging using nanodiamonds and lanthanide chelates.

PubMed

Cordina, Nicole M; Sayyadi, Nima; Parker, Lindsay M; Everest-Dass, Arun; Brown, Louise J; Packer, Nicolle H

2018-03-14

Bio-imaging is a key technique in tracking and monitoring important biological processes and fundamental biomolecular interactions, however the interference of background autofluorescence with targeted fluorophores is problematic for many bio-imaging applications. This study reports on two novel methods for reducing interference with cellular autofluorescence for bio-imaging. The first method uses fluorescent nanodiamonds (FNDs), containing nitrogen vacancy centers. FNDs emit at near-infrared wavelengths typically higher than most cellular autofluorescence; and when appropriately functionalized, can be used for background-free imaging of targeted biomolecules. The second method uses europium-chelating tags with long fluorescence lifetimes. These europium-chelating tags enhance background-free imaging due to the short fluorescent lifetimes of cellular autofluorescence. In this study, we used both methods to target E-selectin, a transmembrane glycoprotein that is activated by inflammation, to demonstrate background-free fluorescent staining in fixed endothelial cells. Our findings indicate that both FND and Europium based staining can improve fluorescent bio-imaging capabilities by reducing competition with cellular autofluorescence. 30 nm nanodiamonds coated with the E-selectin antibody was found to enable the most sensitive detective of E-selectin in inflamed cells, with a 40-fold increase in intensity detected.

Assessment of swelling-activated Cl- channels using the halide-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl)quinolinium.

PubMed Central

Srinivas, S P; Bonanno, J A; Hughes, B A

1998-01-01

This study describes a quantitative analysis of the enhancement in anion permeability through swelling-activated Cl- channels, using the halide-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Cultured bovine corneal endothelial monolayers perfused with NO3- Ringer's were exposed to I- pulses under isosmotic and, subsequently, hyposmotic conditions. Changes in SPQ fluorescence due to I- influx were significantly faster under hyposmotic than under isosmotic conditions. Plasma membrane potential (Em) was -58 and -32 mV under isosmotic and hyposmotic conditions, respectively. An expression for the ratio of I- permeability under hyposmotic condition to that under isosmotic condition (termed enhancement ratio or ER) was derived by combining the Stern-Volmer equation (for modeling SPQ fluorescence quenching by I-) and the Goldman flux equation (for modeling the electrodiffusive unidirectional I- influx). The fluorescence values and slopes at the inflection points of the SPQ fluorescence profile during I- influx, together with Em under isosmotic and hyposmotic conditions, were used to calculate ER. Based on this approach, endothelial cells were shown to express swelling-activated Cl- channels with ER = 4.9 when the hyposmotic shock was 110 +/- 10 mosM. These results illustrate the application of the SPQ-based method for quantitative characterization of swelling-activated Cl- channels in monolayers. PMID:9649372

Visualization of MMP-2 Activity Using Dual-Probe Nanoparticles to Detect Potential Metastatic Cancer Cells

PubMed Central

Kim, Sung Hoon; Lee, Hyun; Kim, Bohee; Kim, Yoon Suk; Key, Jaehong

2018-01-01

Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes capable of degrading extracellular matrix components. Previous studies have shown that the upregulation of MMP-2 is closely related to metastatic cancers. While Western blotting, zymography, and Enzyme-Linked Immunosorbent Assays (ELISA) can be used to measure the amount of MMP-2 activity, it is not possible to visualize the dynamic MMP-2 activities of cancer cells using these techniques. In this study, MMP-2-activated poly(lactic-co-glycolic acid) with polyethylenimine (MMP-2-PLGA-PEI) nanoparticles were developed to visualize time-dependent MMP-2 activities. The MMP-2-PLGA-PEI nanoparticles contain MMP-2-activated probes that were detectable via fluorescence microscopy only in the presence of MMP-2 activity, while the Rhodamine-based probes in the nanoparticles were used to continuously visualize the location of the nanoparticles. This approach allowed us to visualize MMP-2 activities in cancer cells and their microenvironment. Our results showed that the MMP-2-PLGA-PEI nanoparticles were able to distinguish between MMP-2-positive (HaCat) and MMP-2-negative (MCF-7) cells. While the MMP-2-PLGA-PEI nanoparticles gave fluorescent signals recovered by active recombinant MMP-2, there was no signal recovery in the presence of an MMP-2 inhibitor. In conclusion, MMP-2-PLGA-PEI nanoparticles are an effective tool to visualize dynamic MMP-2 activities of potential metastatic cancer cells. PMID:29466303

Study of endothelial cell apoptosis using fluorescence resonance energy transfer (FRET) biosensor cell line with hemodynamic microfluidic chip system.

PubMed

Yu, J Q; Liu, X F; Chin, L K; Liu, A Q; Luo, K Q

2013-07-21

To better understand how hyperglycemia induces endothelial cell dysfunction under the diabetic conditions, a hemodynamic microfluidic chip system was developed. The system combines a caspase-3-based fluorescence resonance energy transfer (FRET) biosensor cell line which can detect endothelial cell apoptosis in real-time, post-treatment effect and with a limited cell sample, by using a microfluidic chip which can mimic the physiological pulsatile flow profile in the blood vessel. The caspase-3-based FRET biosensor endothelial cell line (HUVEC-C3) can produce a FRET-based sensor protein capable of probing caspase-3 activation. When the endothelial cells undergo apoptosis, the color of the sensor cells changes from green to blue, thus sensing apoptosis. A double-labeling fluorescent technique (yo pro-1 and propidium iodide) was used to validate the findings revealed by the FRET-based caspase sensor. The results show high rates of apoptosis and necrosis of endothelial cells when high glucose concentration was applied in our hemodynamic microfluidic chip combined with an exhaustive pulsatile flow profile. The two apoptosis detection techniques (fluorescent method and FRET biosensor) are comparable; but FRET biosensor offers more advantages such as real-time observation and a convenient operating process to generate more accurate and reliable data. Furthermore, the activation of the FRET biosensor also confirms the endothelial cell apoptosis induced by the abnormal pulsatile shear stress and high glucose concentration is through caspase-3 pathway. A 12% apoptotic rate (nearly a 4-fold increase compared to the static condition) was observed when the endothelial cells were exposed to a high glucose concentration of 20 mM under 2 h exhaustive pulsatile shear stress of 30 dyne cm(-2) and followed with another 10 h normal pulsatile shear stress of 15 dyne cm(-2). Therefore, the most important finding of this study is to develop a novel endothelial cell apoptosis detection method, which combines the microfluidic chip system and FRET biosensor. This finding may provide new insight into how glucose causes endothelial cell dysfunction, which is the major cause of diabetes-derived complications.

Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives.

PubMed

Stockert, Juan C; Horobin, Richard W; Colombo, Lucas L; Blázquez-Castro, Alfonso

2018-04-01

For many years various tetrazolium salts and their formazan products have been employed in histochemistry and for assessing cell viability. For the latter application, the most widely used are 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and 5-cyano-2,3-di-(p-tolyl)-tetrazolium chloride (CTC) for viability assays of eukaryotic cells and bacteria, respectively. In these cases, the nicotinamide-adenine-dinucleotide (NAD(P)H) coenzyme and dehydrogenases from metabolically active cells reduce tetrazolium salts to strongly colored and lipophilic formazan products, which are then quantified by absorbance (MTT) or fluorescence (CTC). More recently, certain sulfonated tetrazolium, which give rise to water-soluble formazans, have also proved useful for cytotoxicity assays. We describe several aspects of the application of tetrazolium salts and formazans in biomedical cell biology research, mainly regarding formazan-based colorimetric assays, cellular reduction of MTT, and localization and fluorescence of the MTT formazan in lipidic cell structures. In addition, some pharmacological and labeling perspectives of these compounds are also described. Copyright © 2018 Elsevier GmbH. All rights reserved.

The Role of IQGAP1 in Breast Carcinoma

DTIC Science & Technology

2011-10-01

study! of! the! pathogenesis! of! breast! cancer.! These! include! analysis ! of! intracellular! signaling!by!Western!blotting,! determination!of! cell...proliferation!by! sulforhodamine!B! staining,! fluorescence: activated!cell!sorting!(FACS)! analysis ,!stable!cell!line!generation,!production!of!and...transduction!using!retroviral! and!lentiviral!supernatants,! immunocytochemistry!and!confocal! laser!microscopy,! immunohistochemistry,!and! analysis

Toward a Droplet-Based Single-Cell Radiometric Assay.

PubMed

Gallina, Maria Elena; Kim, Tae Jin; Shelor, Mark; Vasquez, Jaime; Mongersun, Amy; Kim, Minkyu; Tang, Sindy K Y; Abbyad, Paul; Pratx, Guillem

2017-06-20

Radiotracers are widely used to track molecular processes, both in vitro and in vivo, with high sensitivity and specificity. However, most radionuclide detection methods have spatial resolution inadequate for single-cell analysis. A few existing methods can extract single-cell information from radioactive decays, but the stochastic nature of the process precludes high-throughput measurement (and sorting) of single cells. In this work, we introduce a new concept for translating radioactive decays occurring stochastically within radiolabeled single-cells into an integrated, long-lasting fluorescence signal. Single cells are encapsulated in radiofluorogenic droplets containing molecular probes sensitive to byproducts of ionizing radiation (primarily reactive oxygen species, or ROS). Different probes were examined in bulk solutions, and dihydrorhodamine 123 (DHRh 123) was selected as the lead candidate due to its sensitivity and reproducibility. Fluorescence intensity of DHRh 123 in bulk increased at a rate of 54% per Gy of X-ray radiation and 15% per MBq/ml of 2-deoxy-2-[ 18 F]-fluoro-d-glucose ([ 18 F]FDG). Fluorescence imaging of microfluidic droplets showed the same linear response, but droplets were less sensitive overall than the bulk ROS sensor (detection limit of 3 Gy per droplet). Finally, droplets encapsulating radiolabeled cancer cells allowed, for the first time, the detection of [ 18 F]FDG radiotracer uptake in single cells through fluorescence activation. With further improvements, we expect this technology to enable quantitative measurement and selective sorting of single cells based on the uptake of radiolabeled small molecules.

Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis.

PubMed

Weber, K; Mock, U; Petrowitz, B; Bartsch, U; Fehse, B

2010-04-01

Vector-encoded fluorescent proteins (FPs) facilitate unambiguous identification or sorting of gene-modified cells by fluorescence-activated cell sorting (FACS). Exploiting this feature, we have recently developed lentiviral gene ontology (LeGO) vectors (www.LentiGO-Vectors.de) for multi-gene analysis in different target cells. In this study, we extend the LeGO principle by introducing 10 different drug-selectable FPs created by fusing one of the five selection marker (protecting against blasticidin, hygromycin, neomycin, puromycin and zeocin) and one of the five FP genes (Cerulean, eGFP, Venus, dTomato and mCherry). All tested fusion proteins allowed both fluorescence-mediated detection and drug-mediated selection of LeGO-transduced cells. Newly generated codon-optimized hygromycin- and neomycin-resistance genes showed improved expression as compared with their ancestors. New LeGO constructs were produced at titers >10(6) per ml (for non-concentrated supernatants). We show efficient combinatorial marking and selection of various cells, including mesenchymal stem cells, simultaneously transduced with different LeGO constructs. Inclusion of the cytomegalovirus early enhancer/chicken beta-actin promoter into LeGO vectors facilitated robust transgene expression in and selection of neural stem cells and their differentiated progeny. We suppose that the new drug-selectable markers combining advantages of FACS and drug selection are well suited for numerous applications and vector systems. Their inclusion into LeGO vectors opens new possibilities for (stem) cell tracking and functional multi-gene analysis.

Lucky Imaging: Improved Localization Accuracy for Single Molecule Imaging

PubMed Central

Cronin, Bríd; de Wet, Ben; Wallace, Mark I.

2009-01-01

We apply the astronomical data-analysis technique, Lucky imaging, to improve resolution in single molecule fluorescence microscopy. We show that by selectively discarding data points from individual single-molecule trajectories, imaging resolution can be improved by a factor of 1.6 for individual fluorophores and up to 5.6 for more complex images. The method is illustrated using images of fluorescent dye molecules and quantum dots, and the in vivo imaging of fluorescently labeled linker for activation of T cells. PMID:19348772

In vitro studies with renal proximal tubule cells show direct cytotoxicity of Androctonus australis hector scorpion venom triggered by oxidative stress, caspase activation and apoptosis

PubMed Central

Saidani, Chanez; Hammoudi-Triki, Djelila; Laraba-Djebari, Fatima; Taub, Mary

2016-01-01

Scorpion envenomation injures a number of organs, including the kidney. Mechanisms proposed to explain the renal tubule injury include direct effects of venom on tubule epithelial cells, as well as indirect effects of the autonomic nervous system, and inflammation. Here, we report direct effects of Androctonus australis hector (Aah) scorpion venom on the viability of Renal Proximal Tubule (RPT) cells in vitro, unlike distal tubule and collecting duct cells. Extensive NucGreen nuclear staining was observed in immortalized rabbit RPT cells following treatment with Aah venom, consistent with cytotoxicity. The involvement of oxidative stress is supported by the observations that 1) anti-oxidants mitigated the Aah venom-induced decrease in the number of viable RPT cells, and 2) Aah venom-treated RPT cells were intensively stained with the CellROX® Deep Red reagent, an indicator of Reactive Oxygen Species (ROS). Relevance to normal RPT cells is supported by the red fluorescence observed in Aah venom treated primary rabbit RPT cell cultures following their incubation with the Flica reagent (indicative of caspase activation and apoptosis), and the green fluorescence of Sytox Green (indicative of dead cells). PMID:27470530

In vivo imaging of membrane type-1 matrix metalloproteinase with a novel activatable near-infrared fluorescence probe.

PubMed

Shimizu, Yoichi; Temma, Takashi; Hara, Isao; Makino, Akira; Kondo, Naoya; Ozeki, Ei-Ichi; Ono, Masahiro; Saji, Hideo

2014-08-01

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a protease activating MMP-2 that mediates cleavage of extracellular matrix components and plays pivotal roles in tumor migration, invasion and metastasis. Because in vivo noninvasive imaging of MT1-MMP would be useful for tumor diagnosis, we developed a novel near-infrared (NIR) fluorescence probe that can be activated following interaction with MT1-MMP in vivo. MT1-hIC7L is an activatable fluorescence probe comprised of anti-MT1-MMP monoclonal antibodies conjugated to self-assembling polymer micelles that encapsulate NIR dyes (IC7-1, λem : 858 nm) at concentrations sufficient to cause fluorescence self-quenching. In aqueous buffer, MT1-hIC7L fluorescence was suppressed to background levels and increased approximately 35.5-fold in the presence of detergent. Cellular uptake experiments revealed that in MT1-MMP positive C6 glioma cells, MT1-hIC7L showed significantly higher fluorescence that increased with time as compared to hIC7L, a negative control probe lacking the anti-MT1-MMP monoclonal antibody. In MT1-MMP negative MCF-7 breast adenocarcinoma cells, both MT1-hIC7L and hIC7L showed no obvious fluorescence. In addition, the fluorescence intensity of C6 cells treated with MT1-hIC7L was suppressed by pre-treatment with an MT1-MMP endocytosis inhibitor (P < 0.05). In vivo optical imaging using probes intravenously administered to tumor-bearing mice showed that MT1-hIC7L specifically visualized C6 tumors (tumor-to-background ratios: 3.8 ± 0.3 [MT1-hIC7L] vs 3.1 ± 0.2 [hIC7L] 48 h after administration, P < 0.05), while the probes showed similarly low fluorescence in MCF-7 tumors. Together, these results show that MT1-hIC7L would be a potential activatable NIR probe for specifically detecting MT1-MMP-expressing tumors. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Small-Molecule Photostabilizing Agents are Modifiers of Lipid Bilayer Properties

PubMed Central

Alejo, Jose L.; Blanchard, Scott C.; Andersen, Olaf S.

2013-01-01

Small-molecule photostabilizing or protective agents (PAs) provide essential support for the stability demands on fluorescent dyes in single-molecule spectroscopy and fluorescence microscopy. These agents are employed also in studies of cell membranes and model systems mimicking lipid bilayer environments, but there is little information about their possible effects on membrane structure and physical properties. Given the impact of amphipathic small molecules on bilayer properties such as elasticity and intrinsic curvature, we investigated the effects of six commonly used PAs—cyclooctatetraene (COT), para-nitrobenzyl alcohol (NBA), Trolox (TX), 1,4-diazabicyclo[2.2.2]octane (DABCO), para-nitrobenzoic acid (pNBA), and n-propyl gallate (nPG)—on bilayer properties using a gramicidin A (gA)-based fluorescence quench assay to probe for PA-induced changes in the gramicidin monomer↔dimer equilibrium. The experiments were done using fluorophore-loaded large unilamellar vesicles that had been doped with gA, and changes in the gA monomer↔dimer equilibrium were assayed using a gA channel-permeable fluorescence quencher (Tl+). Changes in bilayer properties caused by, e.g., PA adsorption at the bilayer/solution interface that alter the equilibrium constant for gA channel formation, and thus the number of conducting gA channels in the large unilamellar vesicle membrane, will be detectable as changes in the rate of Tl+ influx—the fluorescence quench rate. Over the experimentally relevant millimolar concentration range, TX, NBA, and pNBA, caused comparable increases in gA channel activity. COT, also in the millimolar range, caused a slight decrease in gA channel activity. nPG increased channel activity at submillimolar concentrations. DABCO did not alter gA activity. Five of the six tested PAs thus alter lipid bilayer properties at experimentally relevant concentrations, which becomes important for the design and analysis of fluorescence studies in cells and model membrane systems. We therefore tested combinations of COT, NBA, and TX; the combinations altered the fluorescence quench rate less than would be predicted assuming their effects on bilayer properties were additive. The combination of equimolar concentrations of COT and NBA caused minimal changes in the fluorescence quench rate. PMID:23746513

“Optical communication with brain cells by means of an implanted duplex micro-device with optogenetics and Ca2+ fluoroimaging”

PubMed Central

Kobayashi, Takuma; Haruta, Makito; Sasagawa, Kiyotaka; Matsumata, Miho; Eizumi, Kawori; Kitsumoto, Chikara; Motoyama, Mayumi; Maezawa, Yasuyo; Ohta, Yasumi; Noda, Toshihiko; Tokuda, Takashi; Ishikawa, Yasuyuki; Ohta, Jun

2016-01-01

To better understand the brain function based on neural activity, a minimally invasive analysis technology in a freely moving animal is necessary. Such technology would provide new knowledge in neuroscience and contribute to regenerative medical techniques and prosthetics care. An application that combines optogenetics for voluntarily stimulating nerves, imaging to visualize neural activity, and a wearable micro-instrument for implantation into the brain could meet the abovementioned demand. To this end, a micro-device that can be applied to the brain less invasively and a system for controlling the device has been newly developed in this study. Since the novel implantable device has dual LEDs and a CMOS image sensor, photostimulation and fluorescence imaging can be performed simultaneously. The device enables bidirectional communication with the brain by means of light. In the present study, the device was evaluated in an in vitro experiment using a new on-chip 3D neuroculture with an extracellular matrix gel and an in vivo experiment involving regenerative medical transplantation and gene delivery to the brain by using both photosensitive channel and fluorescent Ca2+ indicator. The device succeeded in activating cells locally by selective photostimulation, and the physiological Ca2+ dynamics of neural cells were visualized simultaneously by fluorescence imaging. PMID:26878910

“Optical communication with brain cells by means of an implanted duplex micro-device with optogenetics and Ca2+ fluoroimaging”

NASA Astrophysics Data System (ADS)

Kobayashi, Takuma; Haruta, Makito; Sasagawa, Kiyotaka; Matsumata, Miho; Eizumi, Kawori; Kitsumoto, Chikara; Motoyama, Mayumi; Maezawa, Yasuyo; Ohta, Yasumi; Noda, Toshihiko; Tokuda, Takashi; Ishikawa, Yasuyuki; Ohta, Jun

2016-02-01

To better understand the brain function based on neural activity, a minimally invasive analysis technology in a freely moving animal is necessary. Such technology would provide new knowledge in neuroscience and contribute to regenerative medical techniques and prosthetics care. An application that combines optogenetics for voluntarily stimulating nerves, imaging to visualize neural activity, and a wearable micro-instrument for implantation into the brain could meet the abovementioned demand. To this end, a micro-device that can be applied to the brain less invasively and a system for controlling the device has been newly developed in this study. Since the novel implantable device has dual LEDs and a CMOS image sensor, photostimulation and fluorescence imaging can be performed simultaneously. The device enables bidirectional communication with the brain by means of light. In the present study, the device was evaluated in an in vitro experiment using a new on-chip 3D neuroculture with an extracellular matrix gel and an in vivo experiment involving regenerative medical transplantation and gene delivery to the brain by using both photosensitive channel and fluorescent Ca2+ indicator. The device succeeded in activating cells locally by selective photostimulation, and the physiological Ca2+ dynamics of neural cells were visualized simultaneously by fluorescence imaging.

An innovative pre-targeting strategy for tumor cell specific imaging and therapy.

PubMed

Qin, Si-Yong; Peng, Meng-Yun; Rong, Lei; Jia, Hui-Zhen; Chen, Si; Cheng, Si-Xue; Feng, Jun; Zhang, Xian-Zheng

2015-09-21

A programmed pre-targeting system for tumor cell imaging and targeting therapy was established based on the "biotin-avidin" interaction. In this programmed functional system, transferrin-biotin can be actively captured by tumor cells with the overexpression of transferrin receptors, thus achieving the pre-targeting modality. Depending upon avidin-biotin recognition, the attachment of multivalent FITC-avidin to biotinylated tumor cells not only offered the rapid fluorescence labelling, but also endowed the pre-targeted cells with targeting sites for the specifically designed biotinylated peptide nano-drug. Owing to the successful pre-targeting, tumorous HepG2 and HeLa cells were effectively distinguished from the normal 3T3 cells via fluorescence imaging. In addition, the self-assembled peptide nano-drug resulted in enhanced cell apoptosis in the observed HepG2 cells. The tumor cell specific pre-targeting strategy is applicable for a variety of different imaging and therapeutic agents for tumor treatments.

Fluorescence imaging analysis of taxol-induced ASTC-a-1 cell death with cell swelling and cytoplasmic vacuolization

NASA Astrophysics Data System (ADS)

Chen, Tong-sheng; Sun, Lei; Wang, Longxiang; Wang, Huiying

2008-02-01

Taxol (Paclitaxel), an isolated component from the bark of the Pacific yew Taxus brevifolia, exhibits a broad spectrum of clinical activity against human cancers. Taxol can promote microtubule (MT) assembly, inhibit depolymerization, and change MT dynamics, resulting in disruption of the normal reorganization of the microtubule network required for mitosis and cell proliferation. However, the molecular mechanism of taxol-induced cell death is still unclear. In this report, CCK-8 was used to assay the inhibition of taxol on the human lung adenocarcinoma (ASTC-a-1) cells viability, confocal fluorescence microscope was used to monitor the morphology changes of cells with taxol treatment. We for the first time describe the characteristics of taxol-induced cells swelling, cytoplasmic vacuolization and cell death. Taxol induced swelling, cytoplasmatic vacuolization and cell death without cell shrinkage and membrane rupture. These features differ from those of apoptosis and resemble the paraptosis, a novel nonapoptotic PCD.

Methods for the Detection of Autophagy in Mammalian Cells

PubMed Central

Zhang, Ziyan; Singh, Rajat; Aschner, Michael

2016-01-01

Macroautophagy (hereafter referred to as autophagy) is a degradation pathway that delivers cytoplasmic materials to lysosomes via double-membraned vesicles designated autophagosomes. Cytoplasmic constituents are sequestered into autophagosomes, which subsequently fuse with lysosomes, where the cargo is degraded. Autophagy is a crucial mechanism involved in many aspects of cell function, including cellular metabolism and energy balance; and alterations in autophagy have been linked to various human pathological processes. Thus, methods that accurately measure autophagic activity are necessary. In this unit, we introduce several approaches to analyze autophagy in mammalian cells, including immunoblotting analysis of LC3 and p62, detection of autophagosome formation by fluorescence microscopy, and monitoring autophagosome maturation by tandem mRFP-GFP fluorescence microscopy. Overall, we recommend a combined use of multiple methods to accurately assess the autophagic activity in any given biological setting. PMID:27479363

Characterization of Nonjunctional Hemichannels in Caterpillar Cells

PubMed Central

Luo, Kaijun; Turnbull, Matthew W.

2011-01-01

Recent studies have demonstrated that hemichannels, which form gap junctions when paired from apposing cells, may serve additional roles when unpaired including cell adhesion and paracrine communication. Hemichannels in mammals are formed by connexins or pannexins, while in insects they are formed by pannexin homologues termed innexins. The formation of functional gap junctions by insect innexins has been established, although their ability to form functional nonjunctional hemichannels has not been reported. Here the characteristics of nonjunctional hemichannels were examined in three lepidopteran cell types, two cell lines (High Five and Sf9) and explanted hemocytes from Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae). Selective fluorescent dye uptake by hemichannels was observed in a significant minority of cells, using fluorescence microscopy and flow cytometry. Carbenoxelone, an inhibitor of mammalian junctions, disrupted dye uptake, while flufenamic acid and mefloquine did not. The presence of Ca2+ and Mg2+ in the media increased hemichannel activity. Additionally, lipopolysaccharide, a stimulator of immune activity in lepidopterans, decreased dye uptake. These results demonstrate for the first time the activity of nonjunctional hemichannels in insect cells, as well as pharmacological tools to manipulate them. These results will facilitate the further examination of the role of innexins and nonjunctional hemichannels in insect cell biology, including paracrine signaling, and comparative studies of mammalian pannexins and insect innexins. PMID:21521140

Autophagy induction by silibinin positively contributes to its anti-metastatic capacity via AMPK/mTOR pathway in renal cell carcinoma.

PubMed

Li, Feng; Ma, Zhenkun; Guan, Zhenfeng; Chen, Yule; Wu, Kaijie; Guo, Peng; Wang, Xinyang; He, Dalin; Zeng, Jin

2015-04-15

Silibinin, a dietary cancer chemopreventive flavonoid from the seeds of milk thistle, has been reported to exhibit anti-metastatic effects on renal cell carcinoma (RCC), but the mechanism underlying this phenomenon is not fully understood. The present study aimed at examining the potential role of autophagy in regulating silibinin-induced anti-metastatic effects on RCC cells. Using RCC ACHN and 786-O cells as a model system in vitro, we found that silibinin treatment increased the expression of LC3-II, resulted in the formation of autophagolysosome vacuoles, and caused a punctate fluorescence pattern with the monomeric red fluorescence protein-enhanced green fluorescence protein-LC3 (mRFP-EGFP-LC3) protein, which all are markers for cellular autophagy. Autophagy flux was induced by silibinin in RCC cells, as determined by LC3 turnover assay. Mechanically, the adenosine 5'-monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway was identified as involved in regulation of silibinin-induced autophagy. Furthermore, autophagy induction was demonstrated to positively contribute to silibinin-induced anti-metastatic effects on RCC cells in vitro. Activation of autophagy enhanced silibinin-induced inhibition of migration and invasion of RCC cells, while inhibition of autophagy attenuated it. These findings thus provide new information about the potential link between autophagy and metastasis inhibition induced by silibinin, and the induction of autophagy may shed some light into future treatment strategies for metastatic RCC.

Autophagy Induction by Silibinin Positively Contributes to Its Anti-Metastatic Capacity via AMPK/mTOR Pathway in Renal Cell Carcinoma

PubMed Central

Li, Feng; Ma, Zhenkun; Guan, Zhenfeng; Chen, Yule; Wu, Kaijie; Guo, Peng; Wang, Xinyang; He, Dalin; Zeng, Jin

2015-01-01

Silibinin, a dietary cancer chemopreventive flavonoid from the seeds of milk thistle, has been reported to exhibit anti-metastatic effects on renal cell carcinoma (RCC), but the mechanism underlying this phenomenon is not fully understood. The present study aimed at examining the potential role of autophagy in regulating silibinin-induced anti-metastatic effects on RCC cells. Using RCC ACHN and 786-O cells as a model system in vitro, we found that silibinin treatment increased the expression of LC3-II, resulted in the formation of autophagolysosome vacuoles, and caused a punctate fluorescence pattern with the monomeric red fluorescence protein-enhanced green fluorescence protein-LC3 (mRFP-EGFP-LC3) protein, which all are markers for cellular autophagy. Autophagy flux was induced by silibinin in RCC cells, as determined by LC3 turnover assay. Mechanically, the adenosine 5'-monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway was identified as involved in regulation of silibinin-induced autophagy. Furthermore, autophagy induction was demonstrated to positively contribute to silibinin-induced anti-metastatic effects on RCC cells in vitro. Activation of autophagy enhanced silibinin-induced inhibition of migration and invasion of RCC cells, while inhibition of autophagy attenuated it. These findings thus provide new information about the potential link between autophagy and metastasis inhibition induced by silibinin, and the induction of autophagy may shed some light into future treatment strategies for metastatic RCC. PMID:25884331

In Vivo Visualization of Endoplasmic Reticulum Stress in the Retina Using the ERAI Reporter Mouse.

PubMed

Alavi, Marcel V; Chiang, Wei-Chieh; Kroeger, Heike; Yasumura, Douglas; Matthes, Michael T; Iwawaki, Takao; LaVail, Matthew M; Gould, Douglas B; Lin, Jonathan H

2015-10-01

Endoplasmic reticulum (ER) stress activates inositol requiring enzyme 1 (IRE1), a key regulator of the unfolded protein response. The ER stress activated indicator (ERAI) transgenic mouse expresses a yellow fluorescent GFP variant (Venus) when IRE1 is activated by ER stress. We tested whether ERAI mice would allow for real-time longitudinal studies of ER stress in living mouse eyes. We chemically and genetically induced ER stress, and qualitatively and quantitatively studied the Venus signal by fluorescence ophthalmoscopy. We determined retinal cell types that contribute to the signal by immunohistology, and we performed molecular and biochemical assays using whole retinal lysates to assess activity of the IRE1 pathway. We found qualitative increase in vivo in fluorescence signal at sites of intravitreal tunicamycin injection in ERAI eyes, and quantitative increase in ERAI mice mated to RhoP23H mice expressing ER stress-inducing misfolded rhodopsin protein. As expected, we found that increased Venus signal arose primarily from photoreceptors in RhoP23H/+;ERAI mice. We found increased Xbp1S and XBP1s transcriptional target mRNA levels in RhoP23H/+;ERAI retinas compared to Rho+/+;ERAI retinas, and that Venus signal increased in ERAI retinas as a function of age. Fluorescence ophthalmoscopy of ERAI mice enables in vivo visualization of retinas undergoing ER stress. ER stress activated indicator mice enable identification of individual retinal cells undergoing ER stress by immunohistochemistry. ER stress activated indicator mice show higher Venus signal at older ages, likely arising from amplification of basal retinal ER stress levels by GFP's inherent stability.

Spatio-temporal kinetics of growth hormone receptor signaling in single cells using FRET microscopy.

PubMed

Biener-Ramanujan, Eva; Ramanujan, V Krishnan; Herman, Brian; Gertler, Arieh

2006-08-01

The growth hormone (GH) receptor (R)-mediated JAK2 (Janus kinase-2)-STAT5 (signaling transducer and activator of transcription-5) pathway involves a cascade of protein-protein interactions and tyrosine phosphorylations that occur in a spatially and temporally sensitive manner in cells. To study GHR dimerization or GH-induced conformational change of predimerized GHRs and STAT5 activation kinetics in intact cells, fluorescence resonance energy transfer (FRET) and live-cell imaging methods were employed. FRET measurements at the membrane of HEK-293T cells co-expressing GHRs tagged at the C-terminus with cyan (C) and yellow (Y) fluorescent proteins (FPs) revealed transient GHR dimerization lasting 2-3 min, with a maximum at 3 min after GH stimulation, which was sufficient to induce STAT5 activation. The transient nature of the dimerization or GH-induced conformational change of predimerized GHRs kinetics was not a result of GHR internalization, as neither potassium- nor cholesterol-depletion treatments prolonged the FRET signal. YFP-tagged STAT5 recruitment to the membrane, binding to GHR-CFP, and phosphorylation, occurred within minutes of GH stimulation. Activated STAT5a-YFP did not show nuclear accumulation, despite nuclear pSTAT5 increase, suggesting high turnover of STAT5 nuclear shuttling. Although GHR dimerization and STAT5 activation have been reported previously, this is the first spatially resolved demonstration of GHR-signaling kinetics in intact cells.

A biotemplated nickel nanostructure: Synthesis, characterization and antibacterial activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ashtari, Khadijeh; Fasihi, Javad; Mollania, Nasrin

Highlights: • Nickel nanostructure-encapsulated bacteria were prepared using electroless deposition. • Bacterium surface was activated by red-ox reaction of its surface amino acids. • Interfacial changes at cell surfaces were investigated using fluorescence spectroscopy. • TEM and AFM depicted morphological changes. • Antibacterial activity of nanostructure was examined against different bacteria strains. - Abstract: Nickel nanostructure-encapsulated bacteria were prepared using the electroless deposition procedure and activation of bacterium cell surface by red-ox reaction of surface amino acids. The electroless deposition step occurred in the presence of Ni(II) and dimethyl amine boran (DMAB). Interfacial changes at bacteria cell surfaces during themore » coating process were investigated using fluorescence spectroscopy. Fluorescence of tryptophan residues was completely quenched after the deposition of nickel onto bacteria surfaces. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) depicted morphological changes on the surface of the bacterium. It was found that the Ni coated nanostructure was mechanically stable after ultrasonication for 20 min. Significant increase in surface roughness of bacteria was also observed after deposition of Ni clusters. The amount of coated Ni on the bacteria surface was calculated as 36% w/w. The antibacterial activity of fabricated nanostructure in culture media was examined against three different bacteria strains; Escherichia coli, Bacillus subtilis and Xantomonas campestris. The minimum inhibitory concentrations (MIC) were determined as 500 mg/L, 350 mg/L and 200 mg/L against bacteria, respectively.« less

Properties of a fluorescent bezafibrate derivative (DNS-X). A new tool to study peroxisome proliferation and fatty acid beta-oxidation.

PubMed

Berlot, J P; Lutz, T; Cherkaoui Malki, M; Nicolas-Frances, V; Jannin, B; Latruffe, N

2000-12-01

The first peroxisome proliferator-activated receptor (PPAR) was cloned in 1990 by Issemann and Green. Many studies have reported the importance of this receptor in the control of gene expression of enzymes involved in lipid metabolic pathways including mitochondrial and peroxisomal fatty acid beta-oxidation, lipoprotein structure [apolipoprotein (apo) A2, apo CIII], and fatty acid synthase. By using radiolabeled molecules, it was shown that peroxisome proliferators bind and activate PPAR. As an alternative method, we developed a fluorescent dansyl (1-dimethylaminonaphthalene-5-sulfonyl) derivative peroxisome proliferator from bezafibrate (DNS-X), a hypolipidemic agent that exhibits an in vitro peroxisome proliferative activity on rat Fao-hepatic derived cultured cells. However, until now, the effect of this new compound on the liver of animals and subcellular localization was unknown. In addition to in vivo rat studies, we present a more efficient large-scale technique of DNS-X purification. Treating rats (DNS-X in the diet at 0.3% w/w) for 6 d leads to a hepatomegaly and a marked increase in liver peroxisomal palmitoyl-CoA oxidase activity. We also developed a method to localize and quantify DNS-X in tissues or cell compartment organelles. The primarily cytosolic distribution of DNS-X was confirmed by direct visualization using fluorescence microscopy of cultured Fao cells. Finally, transfection assay demonstrated that DNS-X enhanced the PPAR alpha activity as well as other peroxisome proliferators do.

Combination of Fluorescence In Situ Hybridization with Staining Techniques for Cell Viability and Accumulation of PHA and polyP in Microorganisms in Complex Microbial Systems

NASA Astrophysics Data System (ADS)

Nielsen, Jeppe Lund; Kragelund, Caroline; Nielsen, Per Halkjær

Fluorescence in situ hybridization (FISH) can be combined with a number of staining techniques to reveal the relationships between the microorganisms and their function in complex microbial systems with a single-cell resolution. In this chapter, we have focused on staining methods for intracellular storage compounds (polyhydroxyalkanoates, polyphosphate) and a measure for cell viability, reduction of the tetrazolium-based redox stain CTC. These protocols are optimized for the study of microorganisms in waste-water treatment (activated sludge and biofilms), but they may also be used with minor modifications in many other ecosystems.

Mechanotransduction in Endothelial Cells Studied with Fluorescence Imaging

NASA Astrophysics Data System (ADS)

Chien, Shu

2011-01-01

Mechanotransduction involves the conversion of mechanical stimuli to intracellular signaling to modulate gene and protein expressions and hence cellular functions in endothelial cells, thus playing importance roles in the regulation of homeostasis in health and disease. The aim of this paper is to investigate the dynamics of mechanotransduction in endothelial cells by the use of fluorescent resonance energy transfer (FRET) to study the temporal and spatial activation of Src kinase and focal adhesion kinase, both of which play critical roles in many cellular processes. The results have contributed to the elucidation of the roles of these two important signaling molecules and their interactions in mediating mechanotransduction.

Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression.

PubMed

Zager, Valerija; Cemazar, Maja; Hreljac, Irena; Lah, Tamara T; Sersa, Gregor; Filipic, Metka

2010-03-01

Human exposure to genotoxic agents in the environment and everyday life represents a serious health threat. Fast and reliable assessment of genotoxicity of chemicals is of main importance in the fields of new chemicals and drug development as well as in environmental monitoring. The tumor suppressor gene p21, the major downstream target gene of activated p53 which is responsible for cell cycle arrest following DNA damage, has been shown to be specifically up-regulated by genotoxic carcinogens. The aim of our study was to develop a human cell-based biosensor system for simple and fast detection of genotoxic agents. Metabolically active HepG2 human hepatoma cells were transfected with plasmid encoding Enhanced Green Fluorescent Protein (EGFP) under the control of the p21 promoter (p21HepG2GFP). DNA damage was induced by genotoxic agents with known mechanisms of action. The increase in fluorescence intensity, due to p21 mediated EGFP expression, was measured with a fluorescence microplate reader. The viability of treated cells was determined by the colorimetric MTS assay. The directly acting alkylating agent methylmethane sulphonate (MMS) showed significant increase in EGFP production after 48 h at 20 μg/mL. The indirectly acting carcinogen benzo(a)pyren (BaP) and the cross-linking agent cisplatin (CisPt) induced a dose- dependent increase in EGFP fluorescence, which was already significant at concentrations 0.13 μg/mL and 0.41 μg/mL, respectively. Vinblastine (VLB), a spindle poison that does not induce direct DNA damage, induced only a small increase in EGFP fluorescence intensity after 24 h at the lowest concentration (0.1 μg/mL), while exposure to higher concentrations was associated with significantly reduced cell viability. The results of our study demonstrated that this novel assay based on the stably transformed cell line p21HepG2GFP can be used as a fast and simple biosensor system for detection of genetic damage caused by chemical agents.

Large-Scale Femtoliter Droplet Array for Single Cell Efflux Assay of Bacteria.

PubMed

Iino, Ryota; Sakakihara, Shouichi; Matsumoto, Yoshimi; Nishino, Kunihiko

2018-01-01

Large-scale femtoliter droplet array as a platform for single cell efflux assay of bacteria is described. Device microfabrication, femtoliter droplet array formation and concomitant enclosure of single bacterial cells, fluorescence-based detection of efflux activity at the single cell level, and collection of single cells from droplet and subsequent gene analysis are described in detail.

A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways*

PubMed Central

Lecat, Sandra; Matthes, Hans W.D.; Pepperkok, Rainer; Simpson, Jeremy C.; Galzi, Jean-Luc

2015-01-01

Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening. PMID:25759509

A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways.

PubMed

Lecat, Sandra; Matthes, Hans W D; Pepperkok, Rainer; Simpson, Jeremy C; Galzi, Jean-Luc

2015-05-01

Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Entry of a Six-Residue Antimicrobial Peptide Derived from Lactoferricin B into Single Vesicles and Escherichia coli Cells without Damaging their Membranes.

PubMed

Moniruzzaman, Md; Islam, Md Zahidul; Sharmin, Sabrina; Dohra, Hideo; Yamazaki, Masahito

2017-08-22

Lactoferricin B (LfcinB) and shorter versions of this peptide have antimicrobial activity. However, the elementary processes of interactions of these peptides with lipid membranes and bacteria are still not well understood. To elucidate the mechanism of their antimicrobial activity, we investigated the interactions of LfcinB (4-9) (its sequence of RRWQWR) with Escherichia coli cells and giant unilamellar vesicles (GUVs). LfcinB (4-9) and lissamine rhodamine B red-labeled LfcinB (4-9) (Rh-LfcinB (4-9)) did not induce an influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli cells into their cytoplasm, indicating that no damage occurred in their plasma membrane. To examine the activity of LfcinB (4-9) to enter E. coli cytoplasm, we investigated the interaction of Rh-LfcinB (4-9) with single cells of E. coli containing calcein using confocal microscopy. We found that Rh-LfcinB (4-9) entered the cytoplasm without leakage of calcein. Next, we investigated the interactions of Rh-LfcinB (4-9) with single GUVs of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) mixtures containing a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using the single GUV method. The results indicate that Rh-LfcinB (4-9) outside the GUV translocated through the GUV membrane and entered its lumen without leakage of AF647. Interaction of Rh-LfcinB (4-9) with DNA increased its fluorescence intensity greatly. Therefore, we can conclude that Rh-LfcinB (4-9) can translocate across lipid membrane regions of the plasma membrane of E. coli cells to enter their cytoplasm without leakage of calcein and its antimicrobial activity is not due to damage of their plasma membranes.

High-throughput living cell-based optical biosensor for detection of bacterial lipopolysaccharide (LPS) using a red fluorescent protein reporter system.

PubMed

Jiang, Hui; Jiang, Donglei; Shao, Jingdong; Sun, Xiulan; Wang, Jiasheng

2016-11-14

Due to the high toxicity of bacterial lipopolysaccharide (LPS), resulting in sepsis and septic shock, two major causes of death worldwide, significant effort is directed toward the development of specific trace-level LPS detection systems. Here, we report sensitive, user-friendly, high-throughput LPS detection in a 96-well microplate using a transcriptional biosensor system, based on 293/hTLR4A-MD2-CD14 cells that are transformed by a red fluorescent protein (mCherry) gene under the transcriptional control of an NF-κB response element. The recognition of LPS activates the biosensor cell, TLR4, and the co-receptor-induced NF-κB signaling pathway, which results in the expression of mCherry fluorescent protein. The novel cell-based biosensor detects LPS with specificity at low concentration. The cell-based biosensor was evaluated by testing LPS isolated from 14 bacteria. Of the tested bacteria, 13 isolated Enterobacteraceous LPSs with hexa-acylated structures were found to increase red fluorescence and one penta-acylated LPS from Pseudomonadaceae appeared less potent. The proposed biosensor has potential for use in the LPS detection in foodstuff and biological products, as well as bacteria identification, assisting the control of foodborne diseases.

Light-patterning of synthetic tissues with single droplet resolution.

PubMed

Booth, Michael J; Restrepo Schild, Vanessa; Box, Stuart J; Bayley, Hagan

2017-08-24

Synthetic tissues can be generated by forming networks of aqueous droplets in lipid-containing oil. Each droplet contains a cell-free expression system and is connected to its neighbor through a lipid bilayer. In the present work, we have demonstrated precise external control of such networks by activating protein expression within single droplets, by using light-activated DNA to encode either a fluorescent or a pore-forming protein. By controlling the extent of activation, synthetic tissues were generated with graded levels of protein expression in patterns of single droplets. Further, we have demonstrated reversible activation within individual compartments in synthetic tissues by turning a fluorescent protein on-and-off. This is the first example of the high-resolution patterning of droplet networks, following their formation. Single-droplet control will be essential to power subsets of compartments within synthetic tissues or to stimulate subsets of cells when synthetic tissues are interfaced with living tissues.

Membrane recycling at the infranuclear pole of the outer hair cell

NASA Astrophysics Data System (ADS)

Harasztosi, Csaba; Harasztosi, Emese; Gummer, Anthony W.

2015-12-01

Rapid endocytic activity of outer hair cells (OHCs) in the guinea-pig cochlea has been already studied using the fluorescent membrane marker FM1-43. It was demonstrated that vesicles were endocytosed at the apical pole of OHCs and transcytosed to the basolateral membrane and through a central strand towards the nucleus. The significance of endocytic activity in the infranuclear region is still not clear. Therefore, in this study endocytic activity at the synaptic pole of OHCs was investigated. Confocal laser scanning microscopy was used to visualize dye uptake of OHCs isolated from the guinea-pig cochlea. Signal intensity changes were quantified in the apical and basal poles relative to the signal at the membrane. Data showed no significant difference in fluorescent signal intensity changes between the opposite poles of the OHC. These results suggest that endocytic activities in both the basal and the apical poles contribute equally to the membrane recycling of OHCs.

Quantitative in vivo fluorescence cross-correlation analyses highlight the importance of competitive effects in the regulation of protein-protein interactions.

PubMed

Sadaie, Wakako; Harada, Yoshie; Matsuda, Michiyuki; Aoki, Kazuhiro

2014-09-01

Computer-assisted simulation is a promising approach for clarifying complicated signaling networks. However, this approach is currently limited by a deficiency of kinetic parameters determined in living cells. To overcome this problem, we applied fluorescence cross-correlation spectrometry (FCCS) to measure dissociation constant (Kd) values of signaling molecule complexes in living cells (in vivo Kd). Among the pairs of fluorescent molecules tested, that of monomerized enhanced green fluorescent protein (mEGFP) and HaloTag-tetramethylrhodamine was most suitable for the measurement of in vivo Kd by FCCS. Using this pair, we determined 22 in vivo Kd values of signaling molecule complexes comprising the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway. With these parameters, we developed a kinetic simulation model of the EGFR-Ras-ERK MAP kinase pathway and uncovered a potential role played by stoichiometry in Shc binding to EGFR during the peak activations of Ras, MEK, and ERK. Intriguingly, most of the in vivo Kd values determined in this study were higher than the in vitro Kd values reported previously, suggesting the significance of competitive bindings inside cells. These in vivo Kd values will provide a sound basis for the quantitative understanding of signal transduction. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

A novel intrinsically fluorescent probe for study of uptake and trafficking of 25-hydroxycholesterol[S

PubMed Central

Iaea, David B.; Gale, Sarah E.; Bielska, Agata A.; Krishnan, Kathiresan; Fujiwara, Hideji; Jiang, Hui; Maxfield, Frederick R.; Schlesinger, Paul H.; Covey, Douglas F.; Schaffer, Jean E.; Ory, Daniel S.

2015-01-01

Cholesterol homeostasis is regulated not only by cholesterol, but also by oxygenated cholesterol species, referred to as oxysterols. Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), regulate cholesterol homeostasis through feedback inhibition and feed-forward activation of transcriptional pathways that govern cholesterol synthesis, uptake, and elimination, as well as through direct nongenomic actions that modulate cholesterol accessibility in membranes. Elucidating the cellular distribution of 25-HC is required to understand its biological activity at the molecular level. However, studying oxysterol distribution and behavior within cells has proven difficult due to the lack of fluorescent analogs of 25-HC that retain its chemical and physical properties. To address this, we synthesized a novel intrinsically fluorescent 25-HC mimetic, 25-hydroxycholestatrienol (25-HCTL). We show that 25-HCTL modulates sterol homeostatic responses in a similar manner as 25-HC. 25-HCTL associates with lipoproteins in media and is taken up by cells through LDL-mediated endocytosis. In cultured cells, 25-HCTL redistributes among cellular membranes and, at steady state, has a similar distribution as cholesterol, being enriched in both the endocytic recycling compartment as well as the plasma membrane. Our findings indicate that 25-HCTL is a faithful fluorescent 25-HC mimetic that can be used to investigate the mechanisms through which 25-HC regulates sterol homeostatic pathways. PMID:26497473

Cell surface antigens in renal tumour cells: detection by immunoluminescence and enzymatic analysis

PubMed Central

Laube, F; Göhring, B; Sann, H; Willhardt, I

2001-01-01

Two renal cell carcinoma cell lines (49RC 43STR and 75RC 2STR) were characterized by detection of the cell surface proteins: CD44(var), intercellular adhesion molecule-1 (ICAM-1), urokinase-type plasminogen activator (uPA) and its receptor and aminopeptidase N (APN). To detect their localization the immunoluminescent technique was used. In addition, the enzyme activity of uPA and APN was investigated in cell suspensions as well as in monolayers. The latter procedure was more advantageous since the additional use of HPLC permits a single registration of the fluorescent hydrolysis-product AMC (7-amino-4-methylcoumarin) without interference by cellular autofluorescence or non-reacted fluorescent substrate. Unlike 75RC 2STR, the cell line 49RC 43STR expressed high levels of uPA and APN. Contrary to that the cell line 75RC 2STR expressed high levels of ICAM-1 and CD44(v6), whereas 49RC 43STR showed a low level of ICAM-1 and no distinct light signal with anti-CD44(v6). The uPA activity was measured directly as well as indirectly (via plasmin) with the substrate Z-Gly-Gly-Arg-AMC. Both activator and plasmin activity were inhibited by D-Val-Phe-Lys-CMK and phenylmethylsulfonyl fluoride. The anti-catalytic antibody to uPA and that to uPA receptor were found to be inhibiting the uPA activity in a concentration-dependent manner. APN activity was assayed using alanine-p-nitroanilide. Peptidase activity was effectively inhibited by 1,10-phenanthroline and partly inhibited by ethylenediamine-tetraacetic acid. © 2001 Cancer Research Campaignhttp://www.bjcancer.com PMID:11556847

Fluorescence contrast-enhanced proliferative lesion imaging by enema administration of indocyanine green in a rat model of colon carcinogenesis

PubMed Central

Onda, Nobuhiko; Mizutani-Morita, Reiko; Yamashita, Susumu; Nagahara, Rei; Matsumoto, Shinya; Yoshida, Toshinori; Shibutani, Makoto

2017-01-01

The fluorescent contrast agent indocyanine green (ICG) is approved by the Food and Drug Administration for clinical applications. We previously reported that cultured human colon tumor cells preferentially take up ICG by endocytic activity in association with disruption of their tight junctions. The present study explored ICG availability in fluorescence imaging of the colon to identify proliferative lesions during colonoscopy. The cellular uptake of ICG in cultured rat colon tumor cells was examined using live-cell imaging. Colon lesions in rats administered an ICG-containing enema were further assessed in rats with azoxymethane-induced colon carcinogenesis, using in vivo endoscopy, ex vivo microscopy, and immunofluorescence microscopy. The uptake of ICG by the cultured cells was temperature-dependent. The intracellular retention of the dye in the membrane trafficking system suggested endocytosis as the uptake mechanism. ICG administered via enema accumulated in colon proliferative lesions ranging from tiny aberrant crypt foci to adenomas and localized in proliferating cells. Fluorescence endoscopy detected these ICG-positive colonic proliferative lesions in vivo. The immunoreactivity of the tight-junction molecule occludin was altered in the proliferative lesions, suggesting the disruption of the integrity of tight junctions. These results suggest that fluorescence contrast-enhanced imaging following the administration of an ICG-containing enema can enhance the detection of mucosal proliferative lesions of the colon during colonoscopy. The tissue preference of ICG in the rat model evaluated in this study can be attributed to the disruption of tight junctions, which in turn promotes endocytosis by proliferative cells and the cellular uptake of ICG. PMID:29163827

Fluorescence Live Cell Imaging

PubMed Central

Ettinger, Andreas

2014-01-01

Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio, and to provide a suitable environment for cells or tissues to replicate physiological cell dynamics. This chapter aims to give a general overview on microscope design choices critical for fluorescence live cell imaging that apply to most fluorescence microscopy modalities, and on environmental control with a focus on mammalian tissue culture cells. In addition, we provide guidance on how to design and evaluate fluorescent protein constructs by spinning disk confocal microscopy. PMID:24974023

Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin

PubMed Central

Saha, Suvrajit; Lee, Il-Hyung; Polley, Anirban; Groves, Jay T.; Rao, Madan; Mayor, Satyajit

2015-01-01

Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24–37°C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an “active actin-membrane composite” cell surface. PMID:26378258

Effects of Cot expression on the nuclear translocation of NF-kappaB in RBL-2H3 cells.

PubMed

Chikamatsu, Satomi; Furuno, Tadahide; Kinoshita, Yosuke; Inoh, Yoshikazu; Hirashima, Naohide; Teshima, Reiko; Nakanishi, Mamoru

2007-03-01

Cot is a serine/threonine protein kinase and is classified as a mitogen-activated protein (MAP) kinase kinase kinase. Overexpression of this protein has been shown to activate the extracellular signal-regulated kinase, the c-Jun N-terminal kinase, and the p38 MAP kinase pathways and to stimulate NF-AT and NF-kappaB-dependent transcription. Here we have shown that Cot kinase activity is intimately involved in the high affinity receptor for IgE (FcvarepsilonRI)-mediated nuclear translocation of NF-kappaB1 independent of NF-kappaB-inducing kinase (NIK) in rat basophilic leukemia (RBL-2H3) cells. A transfected green fluorescent protein-tagged NF-kappaB1 (GFP-NF-kappaB1) resided in the cytoplasm in RBL-2H3 cells and it remained in the cytoplasm even when Cot tagged with red fluorescent protein (Cot-RFP) was co-expressed. Western blotting analysis showed that IkappaB kinases (IKKs) were expressed in RBL-2H3 cells but NIK was not. GFP-NF-kappaB1 translocated from the cytoplasm to the nucleus after the aggregation of FcvarepsilonRI in Cot-transfected cells but not in kinase-deficient Cot-transfected cells. This finding gives a new insight into the role of Cot in the FcvarepsilonRI-mediated NF-kappaB activation in mast cells.

Use of DEAD-box polypeptide-4 (Ddx4) gene promoter-driven fluorescent reporter mice to identify mitotically active germ cells in post-natal mouse ovaries

PubMed Central

Park, Eun-Sil; Tilly, Jonathan L.

2015-01-01

Several laboratories have independently isolated mitotically active germ cells, termed female germline stem cells or oogonial stem cells (OSCs), from adult mouse ovaries. However, a recent study using Ddx4-Cre;Rosa26 reporter mice concluded that such germ cells do not exist. Given the disparity in conclusions drawn in this study compared with others, we felt it was important to re-assess the utility of Ddx4-Cre;Rosa26 reporter mice for identification of OSCs in adult mouse ovaries. Transgenic Ddx4-Cre mice were crossed with Rosa26tdTm/tdTm mice to drive restricted tomato red (tdTm) gene expression in cells in which the Ddx4 gene promoter has been activated. Crude dispersion of ovaries from recombined offspring generated cell fractions containing tdTm-positive immature oocytes, which are incapable of proliferation and thus probably represent the uncharacterized reporter-positive ovarian cells identified in the paper Zhang et al. (2012) as being mitotically inactive. Dispersed ovaries further subjected to fluorescence-activated cell sorting yielded a large population of non-germline tdTm-positive cells, indicative of promoter ‘leakiness’ in the Ddx4-Cre mouse line. Nonetheless, a small percentage of these tdTm-positive cells exhibited externalized (extracellular, ec) expression of Ddx4 protein (ecDdx4-positive), expressed markers of primitive germ cells but not of oocytes, and actively proliferated in culture, all of which are characteristic features of OSCs. Thus, crude dispersion of ovaries collected from Ddx4 gene promoter-driven reporter mice is not, by itself, a reliable approach to identify OSCs, whereas the same ovarian dispersates further subjected to cell sorting strategies yield purified OSCs that can be expanded in culture. PMID:25147160

Identification, visualization, and sorting of translationally active microbial consortia from deep-sea methane seeps

NASA Astrophysics Data System (ADS)

Hatzenpichler, R.; Connon, S. A.; Goudeau, D.; Malmstrom, R.; Woyke, T.; Orphan, V. J.

2015-12-01

Within the past few years, great progress has been made in tapping the genomes of individual cells separated from environmental samples. Unfortunately, however, most often these efforts have been target blind, as they did not pre-select for taxa of interest or focus on metabolically active cells that could be considered key species of the system at the time. This problem is particularly pronounced in low-turnover systems such as deep sea sediments. In an effort to tap the genetic potential hidden within functionally active cells, we have recently developed an approach for the in situ fluorescent tracking of protein synthesis in uncultured cells via bioorthogonal non-canonical amino acid-tagging (BONCAT). This technique depends on the incorporation of synthetic amino acids that carry chemically modifiable tags into newly made proteins, which later can be visualized via click chemistry-mediated fluorescence-labeling. BONCAT is thus able to specifically target proteins that have been expressed in reaction to an experimental condition. We are particularly interested in using BONCAT to understand the functional potential of slow-growing syntrophic consortia of anaerobic methanotrophic archaea and sulfate-reducing bacteria which together catalyze the anaerobic oxidation of methane (AOM) in marine methane seeps. In order to specifically target consortia that are active under varying environmental regimes, we are studying different subpopulations of these inter-domain consortia via a combination of BONCAT with rRNA-targeted FISH. We then couple the BONCAT-enabled staining of active consortia with their separation from inactive members of the community via fluorescence-activated cell-sorting (FACS) and metagenomic sequencing of individual consortia. Using this approach, we were able to identify previously unrecognized AOM-partnerships. By comparing the mini-metagenomes obtained from individual consortia with each other we are starting to gain a more hollistic understanding of the genetic similarities and niche-determining characteristics of a range of functional and taxonomic clades of AOM-consortia.

Human transport protein carrier for controlled photoactivation of antitumor prodrug and real-time intracellular tumor imaging.

PubMed

Li, Xi; Mu, Jing; Liu, Fang; Tan, Eddy Wei Ping; Khezri, Bahareh; Webster, Richard D; Yeow, Edwin Kok Lee; Xing, Bengang

2015-05-20

Current anticancer chemotherapy often suffers from poor tumor selectivity and serious drug resistance. Proper vectors for targeted delivery and controlled drug release play crucial roles in improving the therapeutic selectivity to tumor areas and also overcoming the resistance of cancer cells. In this work, we developed a novel human serum albumin (HSA) protein-based nanocarrier system, which combines the photoactivatable Pt(IV) antitumor prodrug for realizing the controlled release and fluorescent light-up probe for evaluations of drug action and efficacy. The constructed Pt(IV)-probe@HSA platform can be locally activated by light irradiation to release the active Pt species, which results in enhanced cell death at both drug-sensitive A2780 and cisplatin-resistant A2780cis cell lines when compared to the free prodrug molecules. Simultaneously, the cytotoxicity caused by light controlled drug release would further lead to the cellular apoptosis and trigger the activation of caspases 3, one crucial protease enzyme in apoptotic process, which could cleave the recognition peptide moiety (DEVD) with a flanking fluorescent resonance energy transfer (FRET) pair containing near-infrared (NIR) fluorophore Cy5 and quencher Qsy21 on the HSA nanocarrier surface. The turn-on fluorescence in response to caspase-3 could be assessed by fluorescence microscopy and flow cytometry analysis. Our results supported the hypothesis that such a unique design may present a successful platform for multiple roles: (i) a biocompatible protein-based nanocarrier for drug delivery, (ii) the controlled drug release with strengthened therapeutic effects, (iii) real-time monitoring of antitumor drug efficacy at the earlier stage.

Synthesis and preliminary biological evaluations of fluorescent or 149Promethium labeled Trastuzumab-polyethylenimine

DOE PAGES

Fitzsimmons, Jonathan; Nayak, Tapan; Cutler, Cathy; ...

2015-12-30

Radioimmunotherapy utilize a targeting antibody coupled to a therapeutic isotope to target and treat a tumor or disease. In this study we examine the synthesis and cell binding of a polymer scaffold containing a radiotherapeutic isotope and a targeting antibody. Methods: The multistep synthesis of a fluorescent or 149Promethium-labeled Trastuzumab-polyethyleneimine (PEI), Trastuzumab, or PEI is described. In vitro uptake, internalization and/or the binding affinity to the Her2/neu expressing human breast adenocarcinoma SKBr3 cells was investigated with the labeled compounds. Fluorescent-labeled Trastuzumab-PEI was internalized more into cells at 2 and 18 h than fluorescent-labeled Trastuzumab or PEI. The fluorescent-labeled Trastuzumab wasmore » concentrated on the cell surface at 2 and 18 h and the labeled PEI had minimal uptake. DOTA-PEI was prepared and contained an average of 16 chelates per PEI; the compound was radio-labeled with 149Promethium and conjugated to Trastuzumab. The purified 149Pm-DOTA-PEI-Trastuzumab had a radiochemical purity of 96.7% and a specific activity of 0.118 TBq/g. The compound demonstrated a dissociation constant for the Her2/neu receptor of 20.30 ± 6.91 nM. In conclusion, the results indicate the DOTA-PEI-Trastuzumab compound has potential as a targeted therapeutic carrier, and future in vivo studies should be performed.« less

Review on near-infrared heptamethine cyanine dyes as theranostic agents for tumor imaging, targeting, and photodynamic therapy

NASA Astrophysics Data System (ADS)

Shi, Changhong; Wu, Jason Boyang; Pan, Dongfeng

2016-05-01

A class of near-infrared fluorescence (NIRF) heptamethine cyanine dyes that are taken up and accumulated specifically in cancer cells without chemical conjugation have recently emerged as promising tools for tumor imaging and targeting. In addition to their fluorescence and nuclear imaging-based tumor-imaging properties, these dyes can be developed as drug carriers to safely deliver chemotherapy drugs to tumors. They can also be used as effective agents for photodynamic therapy with remarkable tumoricidal activity via photodependent cytotoxic activity. The preferential uptake of dyes into cancer but not normal cells is co-operatively mediated by the prevailing activation of a group of organic anion-transporting polypeptides on cancer cell membranes, as well as tumor hypoxia and increased mitochondrial membrane potential in cancer cells. Such mechanistic explorations have greatly advanced the current application and future development of NIRF dyes and their derivatives as anticancer theranostic agents. This review summarizes current knowledge and emerging advances in NIRF dyes, including molecular characterization, photophysical properties, multimodal development and uptake mechanisms, and their growing potential for preclinical and clinical use.

Non-invasive imaging and cellular tracking of pulmonary emboli by near-infrared fluorescence and positron-emission tomography

PubMed Central

Page, Michael J.; Lourenço, André L.; David, Tovo; LeBeau, Aaron M.; Cattaruzza, Fiore; Castro, Helena C.; VanBrocklin, Henry F.; Coughlin, Shaun R.; Craik, Charles S.

2015-01-01

Functional imaging of proteolytic activity is an emerging strategy to quantify disease and response to therapy at the molecular level. We present a new peptide-based imaging probe technology that advances these goals by exploiting enzymatic activity to deposit probes labelled with near-infrared (NIR) fluorophores or radioisotopes in cell membranes of disease-associated proteolysis. This strategy allows for non-invasive detection of protease activity in vivo and ex vivo by tracking deposited probes in tissues. We demonstrate non-invasive detection of thrombin generation in a murine model of pulmonary embolism using our protease-activated peptide probes in microscopic clots within the lungs with NIR fluorescence optical imaging and positron-emission tomography. Thrombin activity is imaged deep in tissue and tracked predominantly to platelets within the lumen of blood vessels. The modular design of our probes allows for facile investigation of other proteases, and their contributions to disease by tailoring the protease activation and cell-binding elements. PMID:26423607

Fast-Response Turn-on Fluorescent Probes Based on Thiolysis of NBD Amine for H2 S Bioimaging.

PubMed

Wang, Runyu; Li, Zhifei; Zhang, Changyu; Li, Yanyan; Xu, Guoce; Zhang, Qiang-Zhe; Li, Lu-Yuan; Yi, Long; Xi, Zhen

2016-05-17

Hydrogen sulfide (H2 S) is an important endogenous signaling molecule with multiple biological functions. New selective fluorescent turn-on probes based on fast thiolyling of NBD (7-nitro-1,2,3-benzoxadiazole) amine were explored for sensing H2 S in aqueous buffer and in living cells. The syntheses of both probes are simple and quite straightforward. The probes are highly sensitive and selective toward H2 S over other biologically relevant species. The fluorescein-NBD-based probe showed 65-fold green fluorescent increase upon H2 S activation. The rhodamine-NBD-based probe reacted rapidly with H2 S (t1/2 ≈1 min) to give a 4.5-fold increase in red fluorescence. Moreover, both probes were successfully used for monitoring H2 S in living cells and in mice. Based on such probe-based tools, we could observe H2 O2 -induced H2 S biogenesis in a concentration-dependent and time-dependent fashion in living cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In situ fluorescence spectroscopy correlates ionomer degradation to reactive oxygen species generation in an operating fuel cell.

PubMed

Prabhakaran, Venkateshkumar; Arges, Christopher G; Ramani, Vijay

2013-11-21

The rate of generation of reactive oxygen species (ROS) within the polymer electrolyte membrane (PEM) of an operating proton exchange member fuel cell (PEMFC) was monitored using in situ fluorescence spectroscopy. A modified barrier layer was introduced between the PEM and the electrocatalyst layer to eliminate metal-dye interactions and fluorescence resonance energy transfer (FRET) effects during measurements. Standard fuel cell operating parameters (temperature, relative humidity, and electrode potential) were systematically varied to evaluate their influence on the rate of ROS generation during PEMFC operation. Independently, the macroscopic rate of PEM degradation was measured by monitoring the fluoride ion emission rate (FER) in the effluent stream at each operating condition. The ROS generation reaction rate constant (estimated from the in situ fluorescence experiments) correlated perfectly with the measured FER across all conditions, demonstrating unequivocally for the first time that a direct correlation exists between in situ ROS generation and PEM macroscopic degradation. The activation energy for ROS generation within the PEM was estimated to be 12.5 kJ mol(-1).

A microprobe for parallel optical and electrical recordings from single neurons in vivo.

PubMed

LeChasseur, Yoan; Dufour, Suzie; Lavertu, Guillaume; Bories, Cyril; Deschênes, Martin; Vallée, Réal; De Koninck, Yves

2011-04-01

Recording electrical activity from identified neurons in intact tissue is key to understanding their role in information processing. Recent fluorescence labeling techniques have opened new possibilities to combine electrophysiological recording with optical detection of individual neurons deep in brain tissue. For this purpose we developed dual-core fiberoptics-based microprobes, with an optical core to locally excite and collect fluorescence, and an electrolyte-filled hollow core for extracellular single unit electrophysiology. This design provides microprobes with tips < 10 μm, enabling analyses with single-cell optical resolution. We demonstrate combined electrical and optical detection of single fluorescent neurons in rats and mice. We combined electrical recordings and optical Ca²(+) measurements from single thalamic relay neurons in rats, and achieved detection and activation of single channelrhodopsin-expressing neurons in Thy1::ChR2-YFP transgenic mice. The microprobe expands possibilities for in vivo electrophysiological recording, providing parallel access to single-cell optical monitoring and control.

[Visualization and Functional Regulation of Live Cell Proteins Based on Labeling Probe Design].

PubMed

Mizukami, Shin; Kikuchi, Kazuya

2016-01-01

  There are several approaches to understanding the physiological roles of biomolecules: (1) by observing the localization or activities of biomolecules (based on microscopic imaging experiments with fluorescent proteins or fluorescent probes) and (2) by investigating the cellular response via activation or suppression of functions of the target molecule (by using inhibitors, antagonists, siRNAs, etc.). In this context, protein-labeling technology serves as a powerful tool that can be used in various experiments, such as for fluorescence imaging of target proteins. Recently, we developed a protein-labeling technology that uses a mutant β-lactamase (a bacterial hydrolase) as the tag protein. In this protein-labeling technology, also referred to as the BL-tag technology, various β-lactam compounds were used as specific ligands that were covalently labeled to the tag. One major advantage of this labeling technology is that various functions can be carried out by suitably designing both the functional moieties such as the fluorophore and the β-lactam ligand structure. In this review, we briefly introduce the BL-tag technology and describe our future outlook for this technology, such as in fluorescence imaging of biomolecules and functional regulation of cellular proteins in living cells.

Introducing inducible fluorescent split cholesterol oxidase to mammalian cells.

PubMed

Chernov, Konstantin G; Neuvonen, Maarit; Brock, Ivonne; Ikonen, Elina; Verkhusha, Vladislav V

2017-05-26

Cholesterol oxidase (COase) is a bacterial enzyme catalyzing the first step in the biodegradation of cholesterol. COase is an important biotechnological tool for clinical diagnostics and production of steroid drugs and insecticides. It is also used for tracking intracellular cholesterol; however, its utility is limited by the lack of an efficient temporal control of its activity. To overcome this we have developed a regulatable fragment complementation system for COase cloned from Chromobacterium sp. The enzyme was split into two moieties that were fused to FKBP (FK506-binding protein) and FRB (rapamycin-binding domain) pair and split GFP fragments. The addition of rapamycin reconstituted a fluorescent enzyme, termed split GFP-COase, the fluorescence level of which correlated with its oxidation activity. A rapid decrease of cellular cholesterol induced by intracellular expression of the split GFP-COase promoted the dissociation of a cholesterol biosensor D4H from the plasma membrane. The process was reversible as upon rapamycin removal, the split GFP-COase fluorescence was lost, and cellular cholesterol levels returned to normal. These data demonstrate that the split GFP-COase provides a novel tool to manipulate cholesterol in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Delivery of Small Interfering RNA to Inhibit Vascular Endothelial Growth Factor in Zebrafish Using Natural Brain Endothelia Cell-Secreted Exosome Nanovesicles for the Treatment of Brain Cancer.

PubMed

Yang, Tianzhi; Fogarty, Brittany; LaForge, Bret; Aziz, Salma; Pham, Thuy; Lai, Leanne; Bai, Shuhua

2017-03-01

Although small interfering RNA (siRNA) holds great therapeutic promise, its delivery to the disease site remains a paramount obstacle. In this study, we tested whether brain endothelial cell-derived exosomes could deliver siRNA across the blood-brain barrier (BBB) in zebrafish. Natural exosomes were isolated from brain endothelial bEND.3 cell culture media and vascular endothelial growth factor (VEGF) siRNA was loaded in exosomes with the assistance of a transfection reagent. While fluorescence-activated cell flow cytometry and immunocytochemistry staining studies indicated that wild-type exosomes significantly increased the uptake of fluorescence-labeled siRNA in the autologous brain endothelial cells, decreased fluorescence intensity was observed in the cells treated with the tetraspanin CD63 antibody-blocked exosome-delivered formulation (p < 0.05). In the transport study, exosomes also enhanced the permeability of rhodamine 123 in a co-cultured monolayer of brain endothelial bEND.3 cell and astrocyte. Inhibition at the expression of VEGF RNA and protein levels was observed in glioblastoma-astrocytoma U-87 MG cells treated with exosome-delivered siRNAs. Imaging results showed that exosome delivered more siRNAs across the BBB in Tg(fli1:GFP) zebrafish. In a xenotransplanted brain tumor model, exosome-delivered VEGF siRNAs decreased the fluorescence intensity of labeled cancer cells in the brain of zebrafish. Brain endothelial cell-derived exosomes could be potentially used as a natural carrier for the brain delivery of exogenous siRNA.

Tracking transcriptional activities with high-content epifluorescent imaging

NASA Astrophysics Data System (ADS)

Hua, Jianping; Sima, Chao; Cypert, Milana; Gooden, Gerald C.; Shack, Sonsoles; Alla, Lalitamba; Smith, Edward A.; Trent, Jeffrey M.; Dougherty, Edward R.; Bittner, Michael L.

2012-04-01

High-content cell imaging based on fluorescent protein reporters has recently been used to track the transcriptional activities of multiple genes under different external stimuli for extended periods. This technology enhances our ability to discover treatment-induced regulatory mechanisms, temporally order their onsets and recognize their relationships. To fully realize these possibilities and explore their potential in biological and pharmaceutical applications, we introduce a new data processing procedure to extract information about the dynamics of cell processes based on this technology. The proposed procedure contains two parts: (1) image processing, where the fluorescent images are processed to identify individual cells and allow their transcriptional activity levels to be quantified; and (2) data representation, where the extracted time course data are summarized and represented in a way that facilitates efficient evaluation. Experiments show that the proposed procedure achieves fast and robust image segmentation with sufficient accuracy. The extracted cellular dynamics are highly reproducible and sensitive enough to detect subtle activity differences and identify mechanisms responding to selected perturbations. This method should be able to help biologists identify the alterations of cellular mechanisms that allow drug candidates to change cell behavior and thereby improve the efficiency of drug discovery and treatment design.

Real-time detecting gelatinases activity in living cells by FRET imaging

NASA Astrophysics Data System (ADS)

Yang, Jie; Zhang, Zhihong; Liu, Bifeng; Luo, Qingming

2006-01-01

Degradation of the extracellular matrix by Matrix metalloproteinases (MMPs) not only enhances tumor invasion, but also affects tumor cell behaviour and leads to cancer progression. To monitor gelatinases (contain MMP2 and MMP9) activity in living cells, we constructed a vector that encoded a gelatinases recognition site (GRS) between citrine (mutation of EYFP Q69M) in N terminal and ECFP in C terminal. Because Gelatinases are secretory proteins and act outside of cell, an expressing vector displayed the fusion protein on cellular surface was used for this FRET gene probe. On expression of YFP-GRS-ECFP in MCF-7 cells that expressed no gelatinases, we were able to observe the efficient transfer of energy from excited ECFP to YFP within the YFP-GRS-ECFP molecule. However, the fusion protein YFP-GRS-ECFP was expressed in MDA-MB 453s cell line with high secretory gelatinases, so YFP-GRS-ECFP was cleaved by gelatinases, no such transfer of energy was detected and fluorescence signal disappeared in YFP channel since YFP protein was cut down. Moreover, Doxycycline, a MMP inhibitor, could make FRET signal increase and fluorescence signal appeared in YFP channel. Thus, the FRET probe YFP-GRS-ECFP can sensitively and reliably monitor gelatinases activation in living cells and can be used for screening MMP inhibitors.

Dual turn-on fluorescence signal-based controlled release system for real-time monitoring of drug release dynamics in living cells and tumor tissues.

PubMed

Kong, Xiuqi; Dong, Baoli; Song, Xuezhen; Wang, Chao; Zhang, Nan; Lin, Weiying

2018-01-01

Controlled release systems with capabilities for direct and real-time monitoring of the release and dynamics of drugs in living systems are of great value for cancer chemotherapy. Herein, we describe a novel dual turn-on fluorescence signal-based controlled release system ( CDox ), in which the chemotherapy drug doxorubicin ( Dox ) and the fluorescent dye ( CH ) are conjugated by a hydrazone moiety, a pH-responsive cleavable linker. CDox itself shows nearly no fluorescence as the fluorescence of CH and Dox is essentially quenched by the C=N isomerization and N-N free rotation. However, when activated under acidic conditions, CDox could be hydrolyzed to afford Dox and CH , resulting in dual turn-on signals with emission peaks at 595 nm and 488 nm, respectively. Notably, CDox exhibits a desirable controlled release feature as the hydrolysis rate is limited by the steric hindrance effect from both the Dox and CH moieties. Cytotoxicity assays indicate that CDox shows much lower cytotoxicity relative to Dox , and displays higher cell inhibition rate to cancer than normal cells. With the aid of the dual turn-on fluorescence at different wavelengths, the drug release dynamics of CDox in living HepG2 and 4T-1 cells was monitored in double channels in a real-time fashion. Importantly, two-photon fluorescence imaging of CDox in living tumor tissues was also successfully performed by high-definition 3D imaging. We expect that the unique controlled release system illustrated herein could provide a powerful means to investigate modes of action of drugs, which is critical for development of much more robust and effective chemotherapy drugs.

Expression and testing in plants of ArcLight, a genetically-encoded voltage indicator used in neuroscience research.

PubMed

Matzke, Antonius J M; Matzke, Marjori

2015-10-12

It is increasingly appreciated that electrical controls acting at the cellular and supra-cellular levels influence development and initiate rapid responses to environmental cues. An emerging method for non-invasive optical imaging of electrical activity at cell membranes uses genetically-encoded voltage indicators (GEVIs). Developed by neuroscientists to chart neuronal circuits in animals, GEVIs comprise a fluorescent protein that is fused to a voltage-sensing domain. One well-known GEVI, ArcLight, undergoes strong shifts in fluorescence intensity in response to voltage changes in mammalian cells. ArcLight consists of super-ecliptic (SE) pHluorin (pH-sensitive fluorescent protein) with an A227D substitution, which confers voltage sensitivity in neurons, fused to the voltage-sensing domain of the voltage-sensing phosphatase of C iona i ntestinalis (Ci-VSD). In an ongoing effort to adapt tools of optical electrophysiology for plants, we describe here the expression and testing of ArcLight and various derivatives in different membranes of root cells in Arabidopsis thaliana. Transgenic constructs were designed to express ArcLight and various derivatives targeted to the plasma membrane and nuclear membranes of Arabidopsis root cells. In transgenic seedlings, changes in fluorescence intensity of these reporter proteins following extracellular ATP (eATP) application were monitored using a fluorescence microscope equipped with a high speed camera. Coordinate reductions in fluorescence intensity of ArcLight and Ci-VSD-containing derivatives were observed at both the plasma membrane and nuclear membranes following eATP treatments. However, similar responses were observed for derivatives lacking the Ci-VSD. The dispensability of the Ci-VSD suggests that in plants, where H(+) ions contribute substantially to electrical activities, the voltage-sensing ability of ArcLight is subordinate to the pH sensitivity of its SEpHluorin base. The transient reduction of ArcLight fluorescence triggered by eATP most likely reflects changes in pH and not membrane voltage. The pH sensitivity of ArcLight precludes its use as a direct sensor of membrane voltage in plants. Nevertheless, ArcLight and derivatives situated in the plasma membrane and nuclear membranes may offer robust, fluorescence intensity-based pH indicators for monitoring concurrent changes in pH at these discrete membrane systems. Such tools will assist analyses of pH as a signal and/or messenger at the cell surface and the nuclear periphery in living plants.

Small-molecule control of protein function through Staudinger reduction

NASA Astrophysics Data System (ADS)

Luo, Ji; Liu, Qingyang; Morihiro, Kunihiko; Deiters, Alexander

2016-11-01

Using small molecules to control the function of proteins in live cells with complete specificity is highly desirable, but challenging. Here we report a small-molecule switch that can be used to control protein activity. The approach uses a phosphine-mediated Staudinger reduction to activate protein function. Genetic encoding of an ortho-azidobenzyloxycarbonyl amino acid using a pyrrolysyl transfer RNA synthetase/tRNACUA pair in mammalian cells enables the site-specific introduction of a small-molecule-removable protecting group into the protein of interest. Strategic placement of this group renders the protein inactive until deprotection through a bioorthogonal Staudinger reduction delivers the active wild-type protein. This developed methodology was applied to the conditional control of several cellular processes, including bioluminescence (luciferase), fluorescence (enhanced green fluorescent protein), protein translocation (nuclear localization sequence), DNA recombination (Cre) and gene editing (Cas9).

Carbon "Quantum" Dots for Fluorescence Labeling of Cells.

PubMed

Liu, Jia-Hui; Cao, Li; LeCroy, Gregory E; Wang, Ping; Meziani, Mohammed J; Dong, Yiyang; Liu, Yuanfang; Luo, Pengju G; Sun, Ya-Ping

2015-09-02

The specifically synthesized and selected carbon dots of relatively high fluorescence quantum yields were evaluated in their fluorescence labeling of cells. For the cancer cell lines, the cellular uptake of the carbon dots was generally efficient, resulting in the labeling of the cells with bright fluorescence emissions for both one- and two-photon excitations from predominantly the cell membrane and cytoplasm. In the exploration on labeling the live stem cells, the cellular uptake of the carbon dots was relatively less efficient, though fluorescence emissions could still be adequately detected in the labeled cells, with the emissions again predominantly from the cell membrane and cytoplasm. This combined with the observed more efficient internalization of the same carbon dots by the fixed stem cells might suggest some significant selectivity of the stem cells toward surface functionalities of the carbon dots. The needs and possible strategies for more systematic and comparative studies on the fluorescence labeling of different cells, including especially live stem cells, by carbon dots as a new class of brightly fluorescent probes are discussed.

Computer-aided design of peptide near infrared fluorescent probe for tumor diagnosis

NASA Astrophysics Data System (ADS)

Zhang, Congying; Gu, Yueqing

2014-09-01

Integrin αvβ3 receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth, so they become hot research tagets in cancer diagnosis. Peptides possess several attractive features when compared to protein and small molecule, such as small size and high structural compatibility with target proteins. Efficient design of high-affinity peptide ligands to Integrin αvβ3 receptors has been an important problem. Designed peptides in silico provide a valuable and high-selectivity peptide, meanwhile decrease the time of drug screening. In this study, we design peptide which can bind with integrin αvβ3 via computer, and then synthesis near infrared fluorescent probe. The characterization of this near infrared fluorescent probe was detected by UV. To investigate the tumor cell targeting of this probe, it was labeled with visible fluorescent dye Rhodamine B (RhB) for microscopy. To evaluate the targeting capability of this near infrared fluorescent probe, mice bearing integrin αvβ3 positive tumor xenografts were used. In vitro cellular experiments indicated that this probe have a clear binding affinity to αvβ3-positive tumor cells. In vivo experiments confirmed the receptor binding specificity of this probe. The peptide of computational design can bind with integrin αvβ3. Combined peptide near-infrared fluorescent probe with imaging technology use for clinical and tumor diagnosis have a greater development in future.

Non-Cytotoxic Quantum Dot–Chitosan Nanogel Biosensing Probe for Potential Cancer Targeting Agent

PubMed Central

Maxwell, Tyler; Banu, Tahmina; Price, Edward; Tharkur, Jeremy; Campos, Maria Gabriela Nogueira; Gesquiere, Andre; Santra, Swadeshmukul

2015-01-01

Quantum dot (Qdot) biosensors have consistently provided valuable information to researchers about cellular activity due to their unique fluorescent properties. Many of the most popularly used Qdots contain cadmium, posing the risk of toxicity that could negate their attractive optical properties. The design of a non-cytotoxic probe usually involves multiple components and a complex synthesis process. In this paper, the design and synthesis of a non-cytotoxic Qdot-chitosan nanogel composite using straight-forward cyanogen bromide (CNBr) coupling is reported. The probe was characterized by spectroscopy (UV-Vis, fluorescence), microscopy (Fluorescence, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Dynamic Light Scattering. This activatable (“OFF”/“ON”) probe contains a core–shell Qdot (CdS:Mn/ZnS) capped with dopamine, which acts as a fluorescence quencher and a model drug. Dopamine capped “OFF” Qdots can undergo ligand exchange with intercellular glutathione, which turns the Qdots “ON” to restore fluorescence. These Qdots were then coated with chitosan (natural biocompatible polymer) functionalized with folic acid (targeting motif) and Fluorescein Isothiocyanate (FITC; fluorescent dye). To demonstrate cancer cell targetability, the interaction of the probe with cells that express different folate receptor levels was analyzed, and the cytotoxicity of the probe was evaluated on these cells and was shown to be nontoxic even at concentrations as high as 100 mg/L. PMID:28347126

Fluorescent Nanocrystals Reveal Regulated Portals of Entry into and Between the Cells of Hydra

PubMed Central

Tortiglione, Claudia; Quarta, Alessandra; Malvindi, Maria Ada; Tino, Angela; Pellegrino, Teresa

2009-01-01

Initially viewed as innovative carriers for biomedical applications, with unique photophysical properties and great versatility to be decorated at their surface with suitable molecules, nanoparticles can also play active roles in mediating biological effects, suggesting the need to deeply investigate the mechanisms underlying cell-nanoparticle interaction and to identify the molecular players. Here we show that the cell uptake of fluorescent CdSe/CdS quantum rods (QRs) by Hydra vulgaris, a simple model organism at the base of metazoan evolution, can be tuned by modifying nanoparticle surface charge. At acidic pH, amino-PEG coated QRs, showing positive surface charge, are actively internalized by tentacle and body ectodermal cells, while negatively charged nanoparticles are not uptaken. In order to identify the molecular factors underlying QR uptake at acidic pH, we provide functional evidence of annexins involvement and explain the QR uptake as the combined result of QR positive charge and annexin membrane insertion. Moreover, tracking QR labelled cells during development and regeneration allowed us to uncover novel intercellular trafficking and cell dynamics underlying the remarkable plasticity of this ancient organism. PMID:19888325

Effect of Starvation and the Viable-but-Nonculturable State on Green Fluorescent Protein (GFP) Fluorescence in GFP-Tagged Pseudomonas fluorescens A506

PubMed Central

Lowder, M.; Unge, A.; Maraha, N.; Jansson, J. K.; Swiggett, J.; Oliver, J. D.

2000-01-01

The green fluorescent protein (GFP) gene, gfp, of the jellyfish Aequorea victoria is being used as a reporter system for gene expression and as a marker for tracking prokaryotes and eukaryotes. Cells that have been genetically altered with the gfp gene produce a protein that fluoresces when it is excited by UV light. This unique phenotype allows gfp-tagged cells to be specifically monitored by nondestructive means. In this study we determined whether a gfp-tagged strain of Pseudomonas fluorescens continued to fluoresce under conditions under which the cells were starved, viable but nonculturable (VBNC), or dead. Epifluorescent microscopy, flow cytometry, and spectrofluorometry were used to measure fluorescence intensity in starved, VBNC, and dead or dying cells. Results obtained by using flow cytometry indicated that microcosms containing VBNC cells, which were obtained by incubation under stress conditions (starvation at 37.5°C), fluoresced at an intensity that was at least 80% of the intensity of nonstressed cultures. Similarly, microcosms containing starved cells incubated at 5 and 30°C had fluorescence intensities that were 90 to 110% of the intensity of nonstressed cells. VBNC cells remained fluorescent during the entire 6-month incubation period. In addition, cells starved at 5 or 30°C remained fluorescent for at least 11 months. Treatment of the cells with UV light or incubation at 39 or 50°C resulted in a loss of GFP from the cells. There was a strong correlation between cell death and leakage of GFP from the cells, although the extent of leakage varied depending on the treatment. Most dead cells were not GFP fluorescent, but a small proportion of the dead cells retained some GFP at a lower concentration than the concentration in live cells. Our results suggest that gfp-tagged cells remain fluorescent following starvation and entry into the VBNC state but that fluorescence is lost when the cells die, presumably because membrane integrity is lost. PMID:10919764

The effect of RU 486 and related compounds on cultured macrophage differentiation and function.

PubMed

Roberts, C P; Murphy, A A; Santanam, N; Parthasarathy, S

1996-08-01

Our purpose was to examine RU 486 and related compounds on macrophage scavenger receptors and cellular adhesion. THP-1 cells were activated with phorbol myristate acetate and treated with dexamethasone, levonorgestrel, and RU 486 alone or in combination. Scavenger receptor activity was determined by counting adhered cells. In addition, fluorescently labeled acetyl low density lipoprotein uptake was determined. Both dexamethasone and RU 486 significantly decreased activated macrophages (81% and 26% of control). Levonorgestrel stimulated adherent cells in activated monocytes (130% of control). RU 486 and dexamethasone were antagonistic when combined (p < 0.001). In contrast, dexamethasone could not overcome the stimulatory effect of levonorgestrel (p < 0.001). Fluorescent studies yielded similar results. RU 486 is a known antiglucocorticoid with novel antioxidant properties. Levonorgestrel has antiglucocorticoid but no antioxidant activity. Glucocorticoids decrease scavenger receptors and antioxidants regulate inflammatory cytokines. RU 486 antagonized the inhibitory effect of dexamethasone on scavenger receptors, whereas levonorgestrel was stimulatory. It is therapeutically important to up-regulate scavenger receptor activity by antiglucocorticoids in the peritoneal cavity of women with endometriosis. However, because these mechanisms also induce inflammatory cytokines, a balance of antioxidants and antiglucocorticoids may prove beneficial.

Novel KCNQ2 channel activators discovered using fluorescence-based and automated patch-clamp-based high-throughput screening techniques

PubMed Central

Yue, Jin-feng; Qiao, Guan-hua; Liu, Ni; Nan, Fa-jun; Gao, Zhao-bing

2016-01-01

Aim: To establish an improved, high-throughput screening techniques for identifying novel KCNQ2 channel activators. Methods: KCNQ2 channels were stably expressed in CHO cells (KCNQ2 cells). Thallium flux assay was used for primary screening, and 384-well automated patch-clamp IonWorks Barracuda was used for hit validation. Two validated activators were characterized using a conventional patch-clamp recording technique. Results: From a collection of 80 000 compounds, the primary screening revealed a total of 565 compounds that potentiated the fluorescence signals in thallium flux assay by more than 150%. When the 565 hits were examined in IonWorks Barracuda, 38 compounds significantly enhanced the outward currents recorded in KCNQ2 cells, and were confirmed as KCNQ2 activators. In the conventional patch-clamp recordings, two validated activators ZG1732 and ZG2083 enhanced KCNQ2 currents with EC50 values of 1.04±0.18 μmol/L and 1.37±0.06 μmol/L, respectively. Conclusion: The combination of thallium flux assay and IonWorks Barracuda assay is an efficient high-throughput screening (HTS) route for discovering KCNQ2 activators. PMID:26725738

Synthesis and characterization of N-parinaroyl analogs of ganglioside GM3 and de-N-acetyl GM3. Interactions with the EGF receptor kinase

NASA Technical Reports Server (NTRS)

Song, W.; Welti, R.; Hafner-Strauss, S.; Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

1993-01-01

A specific plasma membrane glycosphingolipid, known as ganglioside GM3, can regulate the intrinsic tyrosyl kinase activity of the epidermal growth factor (EGF) receptor; this modulation is not associated with alterations in hormone binding to the receptor. GM3 inhibits EGF receptor tyrosyl kinase activity in detergent micelles, in plasma membrane vesicles, and in whole cells. In addition, immunoaffinity-purified EGF receptor preparations contain ganglioside GM3 (Hanai et al. (1988) J. Biol. Chem. 263, 10915-10921), implying that the glycosphingolipid is intimately associated with the receptor kinase in cell membranes. Both the nature of this association and the molecular mechanism of kinase inhibition remain to be elucidated. In this report, we describe the synthesis of a fluorescent analog of ganglioside GM3, in which the native fatty acid was replaced with trans-parinaric acid. This glycosphingolipid inhibited the receptor kinase activity in a manner similar to that of the native ganglioside. A modified fluorescent glycosphingolipid, N-trans-parinaroyl de-N-acetyl ganglioside GM3, was also prepared. This analog, like the nonfluorescent de-N-acetyl ganglioside GM3, had no effect on receptor kinase activity. Results from tryptophan fluorescence quenching and steady-state anisotropy measurements in membranes containing these fluorescent probes and the human EGF receptor were consistent with the notion that GM3, but not de-N-acetyl GM3, interacts specifically with the receptor in intact membranes.

A pink mouse reports the switch from red to green fluorescence upon Cre-mediated recombination.

PubMed

Hartwich, Heiner; Satheesh, Somisetty V; Nothwang, Hans Gerd

2012-06-14

Targeted genetic modification in the mouse becomes increasingly important in biomedical and basic science. This goal is most often achieved by use of the Cre/loxP system and numerous Cre-driver mouse lines are currently generated. Their initial characterization requires reporter mouse lines to study the in vivo spatiotemporal activity of Cre. Here, we report a dual fluorescence reporter mouse line, which switches expression from the red fluorescent protein mCherry to eGFP after Cre-mediated recombination. Both fluorescent proteins are expressed from the ubiquitously active and strong CAGGS promoter. Among the founders, we noticed a pink mouse line, expressing high levels of the red fluorescent protein mCherry throughout the entire body. Presence of mCherry in the living animal as well as in almost all organs was clearly visible without optical equipment. Upon Cre-activity, mCherry expression was switched to eGFP, demonstrating functionality of this reporter mouse line. The pink mouse presented here is an attractive novel reporter line for fluorescence-based monitoring of Cre-activity. The high expression of mCherry, which is visible to the naked eye, facilitates breeding and crossing, as no genotyping is required to identify mice carrying the reporter allele. The presence of two fluorescent proteins allows in vivo monitoring of recombined and non-recombined cells. Finally, the pink mouse is an eye-catching animal model to demonstrate the power of transgenic techniques in teaching courses.

Fluorometric method of quantitative cell mutagenesis

DOEpatents

Dolbeare, Frank A.

1982-01-01

A method for assaying a cell culture for mutagenesis is described. A cell culture is stained first with a histochemical stain, and then a fluorescent stain. Normal cells in the culture are stained by both the histochemical and fluorescent stains, while abnormal cells are stained only by the fluorescent stain. The two stains are chosen so that the histochemical stain absorbs the wavelengths that the fluorescent stain emits. After the counterstained culture is subjected to exciting light, the fluorescence from the abnormal cells is detected.

Fluorometric method of quantitative cell mutagenesis

DOEpatents

Dolbeare, F.A.

1980-12-12

A method for assaying a cell culture for mutagenesis is described. A cell culture is stained first with a histochemical stain, and then a fluorescent stain. Normal cells in the culture are stained by both the histochemical and fluorescent stains, while abnormal cells are stained only by the fluorescent stain. The two stains are chosen so that the histochemical stain absorbs the wavelengths that the fluorescent stain emits. After the counterstained culture is subjected to exciting light, the fluorescence from the abnormal cells is detected.

Albumin Enhances Caspofungin Activity against Aspergillus Species by Facilitating Drug Delivery to Germinating Hyphae

PubMed Central

Ioannou, Petros; Andrianaki, Aggeliki; Akoumianaki, Tonia; Kyrmizi, Irene; Albert, Nathaniel; Perlin, David; Samonis, George

2015-01-01

The modest in vitro activity of echinocandins against Aspergillus implies that host-related factors augment the action of these antifungal agents in vivo. We found that, in contrast to the other antifungal agents (voriconazole, amphotericin B) tested, caspofungin exhibited a profound increase in activity against various Aspergillus species under conditions of cell culture growth, as evidenced by a ≥4-fold decrease in minimum effective concentrations (MECs) (P = 0. 0005). Importantly, the enhanced activity of caspofungin against Aspergillus spp. under cell culture conditions was strictly dependent on serum albumin and was not observed with the other two echinocandins, micafungin and anidulafungin. Of interest, fluorescently labeled albumin bound preferentially on the surface of germinating Aspergillus hyphae, and this interaction was further enhanced upon treatment with caspofungin. In addition, supplementation of cell culture medium with albumin resulted in a significant, 5-fold increase in association of fluorescently labeled caspofungin with Aspergillus hyphae (P < 0.0001). Collectively, we found a novel synergistic interaction between albumin and caspofungin, with albumin acting as a potential carrier molecule to facilitate antifungal drug delivery to Aspergillus hyphae. PMID:26643329

Single-cell Raman and fluorescence microscopy reveal the association of lipid bodies with phagosomes in leukocytes

PubMed Central

van Manen, Henk-Jan; Kraan, Yvonne M.; Roos, Dirk; Otto, Cees

2005-01-01

Cellular imaging techniques based on vibrational spectroscopy have become powerful tools in cell biology because the molecular composition of subcellular compartments can be visualized without the need for labeling. Using high-resolution, nonresonant confocal Raman microscopy on individual cells, we demonstrate here that lipid bodies (LBs) rich in arachidonate as revealed by their Raman spectra associate with latex bead-containing phagosomes in neutrophilic granulocytes. This finding was corroborated in macrophages and in PLB-985 cells, which can be induced to differentiate into neutrophil-like cells, by selective staining of LBs and visualization by confocal fluorescence microscopy. We further show that the accumulation of LBs near phagosomes is mediated at least in part by the flavohemoprotein gp91phox (in which “phox” is phagocyte oxidase), because different LB distributions around phagocytosed latex beads were observed in WT and gp91phox-deficient PLB-985 cells. gp91phox, which accumulates in the phagosomal membrane, is the catalytic subunit of the leukocyte NADPH oxidase, a critical enzyme in the innate immune response. Finally, time-lapse fluorescence microscopy experiments on neutrophils revealed that the LB-phagosome association is transient, similar to the “kiss-and-run” behavior displayed by endosomes involved in phagosome maturation. Because arachidonic acid (AA) has been shown to be involved in NADPH oxidase activation and phagosome maturation in neutrophils and macrophages, respectively, the findings reported here suggest that LBs may provide a reservoir of AA for local activation of these essential leukocyte functions. PMID:16002471

The Z-cad dual fluorescent sensor detects dynamic changes between the epithelial and mesenchymal cellular states.

PubMed

Toneff, M J; Sreekumar, A; Tinnirello, A; Hollander, P Den; Habib, S; Li, S; Ellis, M J; Xin, L; Mani, S A; Rosen, J M

2016-06-17

The epithelial to mesenchymal transition (EMT) has been implicated in metastasis and therapy resistance of carcinomas and can endow cancer cells with cancer stem cell (CSC) properties. The ability to detect cancer cells that are undergoing or have completed EMT has typically relied on the expression of cell surface antigens that correlate with an EMT/CSC phenotype. Alternatively these cells may be permanently marked through Cre-mediated recombination or through immunostaining of fixed cells. The EMT process is dynamic, and these existing methods cannot reveal such changes within live cells. The development of fluorescent sensors that mirror the dynamic EMT state by following the expression of bona fide EMT regulators in live cells would provide a valuable new tool for characterizing EMT. In addition, these sensors will allow direct observation of cellular plasticity with respect to the epithelial/mesenchymal state to enable more effective studies of EMT in cancer and development. We generated a lentiviral-based, dual fluorescent reporter system, designated as the Z-cad dual sensor, comprising destabilized green fluorescent protein containing the ZEB1 3' UTR and red fluorescent protein driven by the E-cadherin (CDH1) promoter. Using this sensor, we robustly detected EMT and mesenchymal to epithelial transition (MET) in breast cancer cells by flow cytometry and fluorescence microscopy. Importantly, we observed dynamic changes in cellular populations undergoing MET. Additionally, we used the Z-cad sensor to identify and isolate minor subpopulations of cells displaying mesenchymal properties within a population comprising predominately epithelial-like cells. The Z-cad dual sensor identified cells with CSC-like properties more effectively than either the ZEB1 3' UTR or E-cadherin sensor alone. The Z-cad dual sensor effectively reports the activities of two factors critical in determining the epithelial/mesenchymal state of carcinoma cells. The ability of this stably integrating dual sensor system to detect dynamic fluctuations between these two states through live cell imaging offers a significant improvement over existing methods and helps facilitate the study of EMT/MET plasticity in response to different stimuli and in cancer pathogenesis. Finally, the versatile Z-cad sensor can be adapted to a variety of in vitro or in vivo systems to elucidate whether EMT/MET contributes to normal and disease phenotypes.

Curcumin induced autophagy anticancer effects on human lung adenocarcinoma cell line A549

PubMed Central

Liu, Furong; Gao, Song; Yang, Yuxuan; Zhao, Xiaodan; Fan, Yameng; Ma, Wenxia; Yang, Danrong; Yang, Aimin; Yu, Yan

2017-01-01

To investigate the anticancer effects of curcumin-induced autophagy and its effects on the human lung adenocarcinoma A549 cell line, inverted phase contrast microscopy was used to observe alterations to the cytomorphology of cells. An MTT assay was used to measure cell viability. Autophagy was detected using acridine orange (AO) staining and 3-methyladenine (3-MA) was used as an autophagy-specific inhibitor. Dose- and time-dependent A549 cell viability inhibition was observed following curcumin treatment. A dose-dependent increase in the red fluorescent structures in A549 cells was identified following curcumin treatment for 48 h through AO staining. In addition, the activation of autophagy was determined through changes in the number of autophagic vesicles (AVs; fluorescent particles) infected with monodansylcadaverine (MDC). The fluorescence intensity and density of AVs in the curcumin-treated groups were higher at 48 h compared with the control group. Finally, the MTT assay demonstrated that the survival rates of the curcumin-treated cells were increased when pretreated with 3-MA for 3 h, indicating that the inhibitory effect of curcumin on A549 cells is reduced following the inhibition of autophagy. Furthermore, AO and MDC staining confirmed that 3-MA does inhibit the induction of autophagy. Thus, it was hypothesized that the induction of autophagy is partially involved in the reduction of cell viability observed following curcumin treatment. The anticancer effects of curcumin on A549 cells can be reduced using autophagy inhibitors. This suggests a possible cancer therapeutic application of curcumin through the activation of autophagy. These findings have improved the understanding of the mechanism underlying the anticancer property of curcumin. PMID:28928819

Curcumin induced autophagy anticancer effects on human lung adenocarcinoma cell line A549.

PubMed

Liu, Furong; Gao, Song; Yang, Yuxuan; Zhao, Xiaodan; Fan, Yameng; Ma, Wenxia; Yang, Danrong; Yang, Aimin; Yu, Yan

2017-09-01

To investigate the anticancer effects of curcumin-induced autophagy and its effects on the human lung adenocarcinoma A549 cell line, inverted phase contrast microscopy was used to observe alterations to the cytomorphology of cells. An MTT assay was used to measure cell viability. Autophagy was detected using acridine orange (AO) staining and 3-methyladenine (3-MA) was used as an autophagy-specific inhibitor. Dose- and time-dependent A549 cell viability inhibition was observed following curcumin treatment. A dose-dependent increase in the red fluorescent structures in A549 cells was identified following curcumin treatment for 48 h through AO staining. In addition, the activation of autophagy was determined through changes in the number of autophagic vesicles (AVs; fluorescent particles) infected with monodansylcadaverine (MDC). The fluorescence intensity and density of AVs in the curcumin-treated groups were higher at 48 h compared with the control group. Finally, the MTT assay demonstrated that the survival rates of the curcumin-treated cells were increased when pretreated with 3-MA for 3 h, indicating that the inhibitory effect of curcumin on A549 cells is reduced following the inhibition of autophagy. Furthermore, AO and MDC staining confirmed that 3-MA does inhibit the induction of autophagy. Thus, it was hypothesized that the induction of autophagy is partially involved in the reduction of cell viability observed following curcumin treatment. The anticancer effects of curcumin on A549 cells can be reduced using autophagy inhibitors. This suggests a possible cancer therapeutic application of curcumin through the activation of autophagy. These findings have improved the understanding of the mechanism underlying the anticancer property of curcumin.

Reassessing the Role of Type II Toxin-Antitoxin Systems in Formation of Escherichia coli Type II Persister Cells.

PubMed

Goormaghtigh, Frédéric; Fraikin, Nathan; Putrinš, Marta; Hallaert, Thibaut; Hauryliuk, Vasili; Garcia-Pino, Abel; Sjödin, Andreas; Kasvandik, Sergo; Udekwu, Klas; Tenson, Tanel; Kaldalu, Niilo; Van Melderen, Laurence

2018-06-12

Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bacterial population to survive antibiotic treatments. Upon removal of the antibiotic, persister cells resume growth and give rise to viable progeny. Type II toxin-antitoxin (TA) systems were assumed to play a key role in the formation of persister cells in Escherichia coli based on the observation that successive deletions of TA systems decreased persistence frequency. In addition, the model proposed that stochastic fluctuations of (p)ppGpp levels are the basis for triggering activation of TA systems. Cells in which TA systems are activated are thought to enter a dormancy state and therefore survive the antibiotic treatment. Using independently constructed strains and newly designed fluorescent reporters, we reassessed the roles of TA modules in persistence both at the population and single-cell levels. Our data confirm that the deletion of 10 TA systems does not affect persistence to ofloxacin or ampicillin. Moreover, microfluidic experiments performed with a strain reporting the induction of the yefM-yoeB TA system allowed the observation of a small number of type II persister cells that resume growth after removal of ampicillin. However, we were unable to establish a correlation between high fluorescence and persistence, since the fluorescence of persister cells was comparable to that of the bulk of the population and none of the cells showing high fluorescence were able to resume growth upon removal of the antibiotic. Altogether, these data show that there is no direct link between induction of TA systems and persistence to antibiotics. IMPORTANCE Within a growing bacterial population, a small subpopulation of cells is able to survive antibiotic treatment by entering a transient state of dormancy referred to as persistence. Persistence is thought to be the cause of relapsing bacterial infections and is a major public health concern. Type II toxin-antitoxin systems are small modules composed of a toxic protein and an antitoxin protein counteracting the toxin activity. These systems were thought to be pivotal players in persistence until recent developments in the field. Our results demonstrate that previous influential reports had technical flaws and that there is no direct link between induction of TA systems and persistence to antibiotics. Copyright © 2018 Goormaghtigh et al.

Stimulation of Suicidal Erythrocyte Death by Tafenoquine.

PubMed

Al Mamun Bhuyan, Abdulla; Bissinger, Rosi; Stockinger, Katja; Lang, Florian

2016-01-01

The 8-aminoquinoline tafenoquine has been shown to be effective against Plasmodia, Leishmania and Trypanosoma. The substance is at least in part effective by triggering apoptosis of the parasites. Similar to apoptosis, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the regulation of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, zVAD sensitive caspases, SB203580 sensitive p38 kinase, staurosporine sensitive protein kinase C as well as D4476 sensitive casein kinase. The present study explored, whether tafenoquine induces eryptosis and aimed to possibly identify cellular mechanisms involved. Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence, and ceramide abundance utilizing specific antibodies. A 48 hours exposure of human erythrocytes to tafenoquine (500 ng/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased Fluo3-fluorescence, and significantly increased DCFDA fluorescence. Tafenoquine did not significantly modify ceramide abundance. The effect of tafenoquine on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. The effect of tafenoquine on annexin-V-binding was not significantly blunted by zVAD (10 µM), SB203580 (2 µM) or staurosporine (1 µM). The effect of tafenoquine on annexin-V-binding was significantly blunted but not abolished by D4476 (10 µM). Tafenoquine triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to stimulation of Ca2+ entry, oxidative stress and possibly activation of casein kinase. © 2016 The Author(s) Published by S. Karger AG, Basel.

Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

PubMed Central

Choi, Hoseok; Choi, Bomi; Seo, Ju Tae; Lee, Kyung Jin; Gye, Myung Chan; Kim, Young-Pil

2016-01-01

Assaying the glycogen synthase kinase-3 (GSK3) activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed) peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts. PMID:27092510

Autofluorescence of human cells in vitro as a biomarker of their metabolic activity

NASA Astrophysics Data System (ADS)

Dobrzyńska, Monika; Stepińska, Małgorzata; Lewandowski, Rafał; Gietka, Andrzej; Łapiński, Mariusz P.; Trafny, ElŻbieta A.

2016-12-01

Autofluorescence (AF) is the natural emission of light by intrinsic fluorophores. Oxidized mitochondrial flavins, lipofuscin and reduced nicotinamideadenine dinucleotide phosphate (NAD(P)H) are the main sources of the autofluorescence in cells upon excitation with visible light. The aim of the study was to investigate changes in the metabolism of four cell lines by monitoring their autofluorescence with a microplate reader. Autofluorescence intensities of cells were collected at two wavelengths for the excitation and fluorescence emission: for endogenous NAD(P)H at 366/450 nm, for the oxidized flavoproteins and lipofuscin at 460/540 nm. Human mesenchymal stem cells (hMSC), epithelial cells from mammary gland (MCF 10A), breast ductal carcinoma (T-47D) prostate carcinoma (DU-145) were observed daily for 16 days. The level of NAD(P)H autofluorescence did not differ among the cell lines investigated. The significant increase in oxidized flavoproteins fluorescence intensity was recorded for hMSC and ranged from 140 to 175% of control. During 28 days differentiation process, the NAD(P)H, FAD and lipofuscin fluorescence intensities were recorded every day, and the redox ratio was then calculated. The redox ratio gradually decreased during the last eight days of osteogenesis and adipogenesis. Therefore, in our opinion the NAD(P)H, FAD, and lipofuscin fluorescence emission at the wavelengths selected are the optimal parameters to be collected during the differentiation process in order to monitor the metabolism of hMSC undergoing structural and morphological changes.

Application of green fluorescent protein to measure antimicrobial efficacy and the kinetics of cell death against Escherichia coli.

PubMed

Greenhalgh, Richard; Greenhalgh, Malcolm; Alshareef, Fadwa; Robson, Geoffrey D

2017-10-01

Industrial antimicrobials have been extensively used to control unwanted microbial growth by incorporation into a variety of products such as plastics and paints, reducing biodeterioration and biofouling and extending the lifespan of the product. Industrial antimicrobials generally have broad sites of action affecting core cellular functions such as central metabolism, enzyme function, cell wall or DNA synthesis and can either be biocidal or biostatic. In addition, susceptibility can be affected by the metabolic state of the microbe, with metabolically inactive cells generally more resistant than metabolically active cells. Previously it was demonstrated that cytosolically expressed green fluorescent protein could be used as a real-time viability indicator in the yeast Aureobasidium pullulans based on the pH dependent fluorescence of GFP and the collapse of the proton gradient across the cell membrane during cell death. In this study we report on the development and validation of an equivalent GFP fluorescence viability assay in Escherichia coli and used this assay to study the effect of five antimicrobials commonly used in plastics; 4,5-dichloro-2-octyl-isothiazol-3-one (DCOIT), sodium pyrithione, 1,2-benzisothiazol-3-one (BIT), 2-octyl-isothiazol-3-one (OIT) and n-butyl-1,2-benzisothiazol-3-one (BBIT). The results demonstrate broad differences amongst the antimicrobials in both relative efficacy, rate of effect and for some antimicrobials, marked differences in sensitivity toward growing and non-growing cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Do Binucleate Cardiomyocytes Have A Role in Myocardial Repair? Insights Using Isolated Rodent Myocytes and Cell Culture

PubMed Central

Stephen, Michael J; Poindexter, Brian J; Moolman, Johan A; Sheikh-Hamad, David; Bick, Roger J

2009-01-01

Neonatal and adult cardiomyocytes were isolated from rat hearts. Some of the adult myocytes were cultured to allow for cell dedifferentiation, a phenomenon thought to mimic cell changes that occur in stressed myocardium, with myocytes regressing to a fetal pattern of metabolism and stellate neonatal shape. Using fluorescence deconvolution microscopy, cells were probed with fluorescent markers and scanned for a number of proteins associated with ion control, calcium movements and cardiac function. Image analysis of deconvoluted image stacks and sequential real-time image recordings of calcium transients of cells were made. All three myocyte groups were predominantly comprised of binucleate cells. Clustering of proteins to a single nucleus was a common observation, suggesting that one nucleus is active in protein synthesis pathways, while the other nucleus assumes a ‘dormant’ or different role and that cardiomyocytes might be mitotically active even in late development, or specific protein syntheses could be targeted and regulated for reintroduction into the cell cycle. Such possibilities would extend cardiac disease associated stem cell research and therapy options, while producing valuable insights into developmental and death pathways of binucleate cardiomyocytes (word count 183). PMID:19430572

Fluorescent sensors of protein kinases: from basics to biomedical applications.

PubMed

Nhu Ngoc Van, Thi; Morris, May C

2013-01-01

Protein kinases constitute a major class of enzymes underlying essentially all biological processes. These enzymes present similar structural folds, yet their mechanism of action and of regulation vary largely, as well as their substrate specificity and their subcellular localization. Classical approaches to study the function/activity of protein kinases rely on radioactive endpoint assays, which do not allow for characterization of their dynamic activity in their native environment. The development of fluorescent biosensors has provided a whole new avenue for studying protein kinase behavior and regulation in living cells in real time with high spatial and temporal resolution. Two major classes of biosensors have been developed: genetically encoded single-chain fluorescence resonance energy transfer biosensors and peptide/protein biosensors coupled to small synthetic fluorophores which are sensitive to changes in their environment. In this review, we discuss the developments in fluorescent biosensor technology related to protein kinase sensing and the different strategies employed to monitor protein kinase activity, conformation, or relative abundance, as well as kinase regulation and subcellular dynamics in living cells. Moreover, we discuss their application in biomedical settings, for diagnostics and therapeutics, to image disease progression and monitor response to therapeutics, in drug discovery programs, for high-throughput screening assays, for postscreen characterization of drug candidates, and for clinical evaluation of novel drugs. Copyright © 2013 Elsevier Inc. All rights reserved.

[Standardized indirect immunofluorescence. Differentiation of mitochondrial, microsomal and ribosomal antibodies].

PubMed

Storch, W

1977-02-15

By an extensive standardisation of the indirect immunofluorescence for the demonstration espeially of mitochondrial antibodies we succeeded in recognizing atypical fluorescence patterns and in describing their exact localisation. On the basis of absorption studies with mitochondrias, microsomas and ribosomas by comparative observation of sections of liver, stomach and kidneys of rats the preferred sort of reaction and the intensity of fluorescence of antibodies against mitochondria, microsomas and ribosomas were empirically established. Antimitochondrial antibodies react above all with the parietal cells of the stomach and the distal epithelia of the tubulus of the kidney. Antibodies against microsomas of liver and kidney are characterized by a brilliant diffuse cytoplasmatic fluorescence of the hepatocytes and by a comparatively weaker fluorescence of exclusively proximal tubuli of the kidneys of rats. Antibodies against ribosomas lead to a fluorescence especially of the main cells of the stomach. The differentiation of several cytoplasmatic antibodies is among others of interest for the diagnosis of certain autoimmune diseases. Although there are numerous still unclear findings and "overlap" phenomena the existence of high titre antibodies against mitochondrias speaks for a primarily biliary cirrhosis or a pseudo-LE-syndrome, the existence of antibodies against microsomas of kidney and liver of rats for a special form of a chronically active hepatitis and the existence of the very rare antibodies against ribosomas for an active lupus erythematodes disseminatus.

Laser-Induced Fluorescence Detection in High-Throughput Screening of Heterogeneous Catalysts and Single Cells Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Hui

2001-01-01

Laser-induced fluorescence detection is one of the most sensitive detection techniques and it has found enormous applications in various areas. The purpose of this research was to develop detection approaches based on laser-induced fluorescence detection in two different areas, heterogeneous catalysts screening and single cell study. First, we introduced laser-induced imaging (LIFI) as a high-throughput screening technique for heterogeneous catalysts to explore the use of this high-throughput screening technique in discovery and study of various heterogeneous catalyst systems. This scheme is based on the fact that the creation or the destruction of chemical bonds alters the fluorescence properties of suitablymore » designed molecules. By irradiating the region immediately above the catalytic surface with a laser, the fluorescence intensity of a selected product or reactant can be imaged by a charge-coupled device (CCD) camera to follow the catalytic activity as a function of time and space. By screening the catalytic activity of vanadium pentoxide catalysts in oxidation of naphthalene, we demonstrated LIFI has good detection performance and the spatial and temporal resolution needed for high-throughput screening of heterogeneous catalysts. The sample packing density can reach up to 250 x 250 subunits/cm 2 for 40-μm wells. This experimental set-up also can screen solid catalysts via near infrared thermography detection.« less

Synthesis and bioimaging of biodegradable red fluorescent organic nanoparticles with aggregation-induced emission characteristics.

PubMed

Xu, Dazhuang; Zou, Hui; Liu, Meiying; Tian, Jianwen; Huang, Hongye; Wan, Qing; Dai, Yanfeng; Wen, Yuanqing; Zhang, Xiaoyong; Wei, Yen

2017-12-15

Fluorescent organic nanoparticles (FONs) with aggregation-induced emission (AIE) features have recently emerged as promising fluorescent probes for biomedical applications owing to their excellent optical properties, designability and biocompatibility. Significant progress has been made recently for synthesis and biomedical applications of these AIE-active FONs. However, only very limited reports have demonstrated the fabrication of biodegradable AIE-active FONs with red fluorescence emission. In this study, a novel strategy has been developed for the preparation of biodegradable AIE-active polyurethanes (PUs) through a two-step polymerization, in which the diisocyanate-terminated polyethylene glycol (NCO-PEG-NCO) was synthesized and subsequently conjugated with diamine-containing AIE dye (NH 2 -Phe-NH 2 ). The successful synthesis of AIE-active Phe-PEG 2000 PUs is evidenced by a series of characterization techniques. Because of the formation of AIE-active amphiphilic PUs, the final copolymers can self-assemble into spherical nanoparticles, which exhibit strong luminescence and high water dispersion. The biological evaluation results suggest that the AIE-active Phe-PEG 2000 FONs possess low toxicity and desirable cell permeability. Therefore, we anticipate that these AIE-active FONs with biodegradable potential will trigger much research enthusiasm and effort toward the creation of new AIE-active materials with improved properties for various biomedical applications. Copyright © 2017 Elsevier Inc. All rights reserved.

Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio

PubMed Central

Tantama, Mathew; Martínez-François, Juan Ramón; Mongeon, Rebecca; Yellen, Gary

2013-01-01

The ATP:ADP ratio is a critical parameter of cellular energy status that regulates many metabolic activities. Here we report an optimized genetically-encoded fluorescent biosensor, PercevalHR, that senses the ATP:ADP ratio. PercevalHR is tuned to the range of intracellular ATP:ADP expected in mammalian cells, and it can be used with one- or two-photon microscopy in live samples. We use PercevalHR to visualize activity-dependent changes in ATP:ADP when neurons are exposed to multiple stimuli, demonstrating that it is a sensitive reporter of physiological changes in energy consumption and production. We also use PercevalHR to visualize intracellular ATP:ADP while simultaneously recording currents from ATP-sensitive potassium (KATP) channels in single cells, showing that PercevalHR enables the study of coordinated variation in ATP:ADP and KATP channel open probability in intact cells. With its ability to monitor changes in cellular energetics within seconds, PercevalHR should be a versatile tool for metabolic research. PMID:24096541

Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins.

PubMed

Vasilev, Kalin V; Gallo, Eugenio; Shank, Nathaniel; Jarvik, Jonathan W

2016-01-01

We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay.

SipA Activation of Caspase-3 Is a Decisive Mediator of Host Cell Survival at Early Stages of Salmonella enterica Serovar Typhimurium Infection

PubMed Central

McIntosh, Anne; Meikle, Lynsey M.; Ormsby, Michael J.; McCormick, Beth A.; Christie, John M.; Brewer, James M.; Roberts, Mark

2017-01-01

ABSTRACT Salmonella invasion protein A (SipA) is a dual-function effector protein that plays roles in both actin polymerization and caspase-3 activation in intestinal epithelial cells. To date its function in other cell types has remained largely unknown despite its expression in multiple cell types and its extracellular secretion during infection. Here we show that in macrophages SipA induces increased caspase-3 activation early in infection. This activation required a threshold level of SipA linked to multiplicity of infection and may be a limiting factor controlling bacterial numbers in infected macrophages. In polymorphonuclear leukocytes, SipA or other Salmonella pathogenicity island 1 effectors had no effect on induction of caspase-3 activation either alone or in the presence of whole bacteria. Tagging of SipA with the small fluorescent phiLOV tag, which can pass through the type three secretion system, allowed visualization and quantification of caspase-3 activation by SipA-phiLOV in macrophages. Additionally, SipA-phiLOV activation of caspase-3 could be tracked in the intestine through multiphoton laser scanning microscopy in an ex vivo intestinal model. This allowed visualization of areas where the intestinal epithelium had been compromised and demonstrated the potential use of this fluorescent tag for in vivo tracking of individual effectors. PMID:28630067

Active substrates improving sensitivity in biomedical fluorescence microscopy

NASA Astrophysics Data System (ADS)

Le Moal, E.; Leveque-Fort, S.; Fort, E.; Lacharme, J.-P.; Fontaine-Aupart, M.-P.; Ricolleau, C.

2005-08-01

Fluorescence is widely used as a spectroscopic tool or for biomedical imaging, in particular for DNA chips. In some cases, detection of very low molecular concentrations and precise localization of biomarkers are limited by the weakness of the fluorescence signal. We present a new method based on sample substrates that improve fluorescence detection sensitivity. These active substrates consist in glass slides covered with metal (gold or silver) and dielectric (alumina) films and can directly be used with common microscope set-up. Fluorescence enhancement affects both excitation and decay rates and is strongly dependant on the distance to the metal surface. Furthermore, fluorescence collection is improved since fluorophore emission lobes are advantageously modified close to a reflective surface. Finally, additional improvements are achieved by structuring the metallic layer. Substrates morphology was mapped by Atomic Force Microscopy (AFM). Substrates optical properties were studied using mono- and bi-photonic fluorescence microscopy with time resolution. An original set-up was implemented for spatial radiation pattern's measurement. Detection improvement was then tested on commercial devices. Several biomedical applications are presented. Enhancement by two orders of magnitude are achieved for DNA chips and signal-to-noise ratio is greatly increased for cells imaging.

Self-phase modulation and two-photon absorption imaging of cells and active neurons

NASA Astrophysics Data System (ADS)

Fischer, Martin C.; Liu, Henry; Piletic, Ivan R.; Ye, Tong; Yasuda, Ryohei; Warren, Warren S.

2007-02-01

Even though multi-photon fluorescence microscopy offers higher resolution and better penetration depth than traditional fluorescence microscopy, its use is restricted to the detection of molecules that fluoresce. Two-photon absorption (TPA) imaging can provide contrast in non-fluorescent molecules while retaining the high resolution and sectioning capabilities of nonlinear imaging modalities. In the long-wavelength water window, tissue TPA is dominated by the endogenous molecules melanin and hemoglobin with an almost complete absence of endogenous two-photon fluorescence. A complementary nonlinear contrast mechanism is self-phase modulation (SPM), which can provide intrinsic signatures that can depend on local tissue anisotropy, chemical environment, or other structural properties. We have developed a spectral hole refilling measurement technique for TPA and SPM measurements using shaped ultrafast laser pulses. Here we report on a microscopy setup to simultaneously acquire 3D, high-resolution TPA and SPM images. We have acquired data in mounted B16 melanoma cells with very modest laser power levels. We will also discuss the possible application of this measurement technique to neuronal imaging. Since SPM is sensitive to material structure we can expect SPM properties of neurons to change during neuronal firing. Using our hole-refilling technique we have now demonstrated strong novel intrinsic nonlinear signatures of neuronal activation in a hippocampal brain slice. The observed changes in nonlinear signal upon collective activation were up to factors of two, unlike other intrinsic optical signal changes on the percent level. These results show that TPA and SPM imaging can provide important novel functional contrast in tissue using very modest power levels suitable for in vivo applications.

Aggregation induced emission enhancement (AIEE) characteristics of quinoline based compound - A versatile fluorescent probe for pH, Fe(III) ion, BSA binding and optical cell imaging

NASA Astrophysics Data System (ADS)

Manikandan, Irulappan; Chang, Chien-Huei; Chen, Chia-Ling; Sathish, Veerasamy; Li, Wen-Shan; Malathi, Mahalingam

2017-07-01

Novel benzimidazoquinoline derivative (AVT) was synthesized through a substitution reaction and characterized by various spectral techniques. Analyzing the optical properties of AVT under absorption and emission spectral studies in different environments exclusively with respect to solvents and pH, intriguing characteristics viz. aggregation induced emission enhancement (AIEE) in the THF solvent and 'On-Off' pH sensing were found at neutral pH. Sensing nature of AVT with diverse metal ions and bovine serum albumin (BSA) was also studied. Among the metal ions, Fe3 + ion alone tunes the fluorescence intensity of AVT probe in aqueous medium from ;turn-on; to ;turn-off; through ligand (probe) to metal charge transfer (LMCT) mechanism. The probe AVT in aqueous medium interacts strongly with BSA due to Fluorescence Resonance Energy Transfer (FRET) and the conformational change in BSA was further analyzed using synchronous fluorescence techniques. Docking study of AVT with BSA reveals that the active site of binding is tryptophan residue which is also supported by the experimental results. Interestingly, fluorescent AVT probe in cells was examined through cellular imaging studies using BT-549 and MDA-MB-231 cells. Thus, the single molecule probe based detection of multiple species and stimuli were described.

Droplet Microfluidic Platform for the Determination of Single-Cell Lactate Release.

PubMed

Mongersun, Amy; Smeenk, Ian; Pratx, Guillem; Asuri, Prashanth; Abbyad, Paul

2016-03-15

Cancer cells release high levels of lactate that has been correlated to increased metastasis and tumor recurrence. Single-cell measurements of lactate release can identify malignant cells and help decipher metabolic cancer pathways. We present here a novel droplet microfluidic method that allows the fast and quantitative determination of lactate release in many single cells. Using passive forces, droplets encapsulated cells are positioned in an array. The single-cell lactate release rate is determined from the increase in droplet fluorescence as the lactate is enzymatically converted to a fluorescent product. The method is used to measure the cell-to-cell variance of lactate release in K562 leukemia and U87 glioblastoma cancer cell lines and under the chemical inhibition of lactate efflux. The technique can be used in the study of cancer biology, but more broadly in cell biology, to capture the full range of stochastic variations in glycolysis activity in heterogeneous cell populations in a repeatable and high-throughput manner.

Photoelectrocyclization as an activation mechanism for organelle-specific live-cell imaging probes.

PubMed

Tran, Mai N; Chenoweth, David M

2015-05-26

Photoactivatable fluorophores are useful tools in live-cell imaging owing to their potential for precise spatial and temporal control. In this report, a new photoactivatable organelle-specific live-cell imaging probe based on a 6π electrocyclization/oxidation mechanism is described. It is shown that this new probe is water-soluble, non-cytotoxic, cell-permeable, and useful for mitochondrial imaging. The probe displays large Stokes shifts in both pre-activated and activated forms, allowing simultaneous use with common dyes and fluorescent proteins. Sequential single-cell activation experiments in dense cellular environments demonstrate high spatial precision and utility in single- or multi-cell labeling experiments. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preparation and characterization of dextran nanobubbles for oxygen delivery.

PubMed

Cavalli, R; Bisazza, A; Giustetto, P; Civra, A; Lembo, D; Trotta, G; Guiot, C; Trotta, M

2009-11-03

Dextran nanobubbles were prepared with a dextran shell and a perfluoropentan core in which oxygen was stored. To increase the stability polyvinylpirrolidone was also added to the formulation as stabilizing agent. Rhodamine B was used as fluorescent marker to obtain fluorescent nanobubbles. The nanobubble formulations showed sizes of about 500nm, a negative surface charge and a good capacity of loading oxygen, no hemolytic activity or toxic effect on cell lines. The fluorescent labelled nanobubbles could be internalized in Vero cells. Oxygen-filled nanobubbles were able to release oxygen in different hypoxic solutions at different time after their preparation in in vitro experiments. The oxygen release kinetics could be enhanced after nanobubble insonation with ultrasound at 2.5MHz. The oxygen-filled nanobubble formulations might be proposed for therapeutic applications in various diseases.

Application of fluorescence spectroscopy for on-line bioprocess monitoring and control

NASA Astrophysics Data System (ADS)

Boehl, Daniela; Solle, D.; Toussaint, Hans J.; Menge, M.; Renemann, G.; Lindemann, Carsten; Hitzmann, Bernd; Scheper, Thomas-Helmut

2001-02-01

12 Modern bioprocess control requires fast data acquisition and in-time evaluation of bioprocess variables. On-line fluorescence spectroscopy for data acquisition and the use of chemometric methods accomplish these requirements. The presented investigations were performed with fluorescence spectrometers with wide ranges of excitation and emission wavelength. By detection of several biogenic fluorophors (amino acids, coenzymes and vitamins) a large amount of information about the state of the bioprocess are obtained. For the evaluation of the process variables partial least squares regression is used. This technique was applied to several bioprocesses: the production of ergotamine by Claviceps purpurea, the production of t-PA (tissue plasminogen activator) by animal cells and brewing processes. The main point of monitoring the brewing processes was to determine the process variables cell count and extract concentration.

A Sensitive and Versatile Fluorescent Activity Assay for ABHD6.

PubMed

Savinainen, Juha R; Navia-Paldanius, Dina; Laitinen, Jarmo T

2016-01-01

The α/β-hydrolase domain-containing 6 (ABHD6) enzyme is a newly found serine hydrolase whose substrate profile resembles that of monoacylglycerol lipase (MAGL), the major 2-arachidonoyl glycerol (2-AG) hydrolase in the brain. Here, we describe a sensitive fluorescent assay of ABHD6 activity in a 96-well-plate format that allows parallel testing of inhibitor activities of up to 40 compounds in a single assay. The method utilizes lysates of HEK293 cells transiently overexpressing human ABHD6 as the enzymatic source, and kinetically monitors glycerol liberated in the hydrolysis of 1(3)-AG, the preferred arachidonoyl glycerol isomer. Glycerol output is coupled to an enzymatic cascade generating the fluorescent end-product resorufin. The approach has major benefits compared to laborious traditional mass spectrometric methods and liquid scintillation-based assays, or approaches using unnatural substrates.

Labeling the oily core of nanocapsules and lipid-core nanocapsules with a triglyceride conjugated to a fluorescent dye as a strategy to particle tracking in biological studies

NASA Astrophysics Data System (ADS)

Fiel, Luana Almeida; Contri, Renata Vidor; Bica, Juliane Freitas; Figueiró, Fabrício; Battastini, Ana Maria Oliveira; Guterres, Sílvia Stanisçuaski; Pohlmann, Adriana Raffin

2014-05-01

The synthesis of novel fluorescent materials represents a very important step to obtain labeled nanoformulations in order to evaluate their biological behavior. The strategy of conjugating a fluorescent dye with triacylglycerol allows that either particles differing regarding supramolecular structure, i.e., nanoemulsions, nanocapsules, lipid-core nanocapsules, or surface charge, i.e., cationic nanocapsules and anionic nanocapsules, can be tracked using the same labeled material. In this way, a rhodamine B-conjugated triglyceride was obtained to prepare fluorescent polymeric nanocapsules. Different formulations were obtained, nanocapsules (NC) or lipid-core nanocapsules (LNC), using the labeled oil and Eudragit RS100, Eudragit S100, or poly(caprolactone) (PCL), respectively. The rhodamine B was coupled with the ricinolein by activating the carboxylic function using a carbodiimide derivative. Thin layer chromatography, proton nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy (FTIR), UV-vis, and fluorescence spectroscopy were used to identify the new product. Fluorescent nanocapsule aqueous suspensions were prepared by the solvent displacement method. Their pH values were 4.6 (NC-RS100), 3.5 (NC-S100), and 5.0 (LNC-PCL). The volume-weighted mean diameter ( D 4.3) and polydispersity values were 150 nm and 1.05 (NC-RS100), 350 nm and 2.28 (NC-S100), and 270 nm and 1.67 (LNC-PCL). The mean diameters determined by photon correlation spectroscopy (PCS) ( z-average) were around 200 nm. The zeta potential values were +5.85 mV (NC-RS100), -21.12 mV (NC-S100), and -19.25 mV (LNC-PCL). The wavelengths of maximum fluorescence emission were 567 nm (NC-RS100 and LNC-PCL) and 574 nm (NC-S100). Fluorescence microscopy was used to evaluate the cell uptake (human macrophage cell line) of the fluorescent nanocapsules in order to show the applicability of the approach. When the cells were treated with the fluorescent nanocapsules, red emission was detected around the cell nucleus. We demonstrated that the rhodamine B-conjugated triglyceride is a promising new material to obtain versatile dye-labeled nanocarriers presenting different chemical nature in their surfaces.

Fluorescence technique for on-line monitoring of state of hydrogen-producing microorganisms

DOEpatents

Seibert, Michael [Lakewood, CO; Makarova, Valeriya [Golden, CO; Tsygankov, Anatoly A [Pushchino, RU; Rubin, Andrew B [Moscow, RU

2007-06-12

In situ fluorescence method to monitor state of sulfur-deprived algal culture's ability to produce H.sub.2 under sulfur depletion, comprising: a) providing sulfur-deprived algal culture; b) illuminating culture; c) measuring onset of H.sub.2 percentage in produced gas phase at multiple times to ascertain point immediately after anerobiosis to obtain H.sub.2 data as function of time; and d) determining any abrupt change in three in situ fluorescence parameters; i) increase in F.sub.t (steady-state level of chlorophyll fluorescence in light adapted cells); ii) decrease in F.sub.m', (maximal saturating light induced fluorescence level in light adapted cells); and iii) decrease in .DELTA.F/F.sub.m'=(F.sub.m'-F.sub.t)/F.sub.m' (calculated photochemical activity of photosystem II (PSII) signaling full reduction of plastoquinone pool between PSII and PSI, which indicates start of anaerobic conditions that induces synthesis of hydrogenase enzyme for subsequent H.sub.2 production that signal oxidation of plastoquinone pool asmain factor to regulate H.sub.2 under sulfur depletion.

In vitro studies with renal proximal tubule cells show direct cytotoxicity of Androctonus australis hector scorpion venom triggered by oxidative stress, caspase activation and apoptosis.

PubMed

Saidani, Chanez; Hammoudi-Triki, Djelila; Laraba-Djebari, Fatima; Taub, Mary

2016-09-15

Scorpion envenomation injures a number of organs, including the kidney. Mechanisms proposed to explain the renal tubule injury include direct effects of venom on tubule epithelial cells, as well as indirect effects of the autonomic nervous system, and inflammation. Here, we report direct effects of Androctonus australis hector (Aah) scorpion venom on the viability of Renal Proximal Tubule (RPT) cells in vitro, unlike distal tubule and collecting duct cells. Extensive NucGreen nuclear staining was observed in immortalized rabbit RPT cells following treatment with Aah venom, consistent with cytotoxicity. The involvement of oxidative stress is supported by the observations that 1) anti-oxidants mitigated the Aah venom-induced decrease in the number of viable RPT cells, and 2) Aah venom-treated RPT cells were intensively stained with the CellROX(®) Deep Red reagent, an indicator of Reactive Oxygen Species (ROS). Relevance to normal RPT cells is supported by the red fluorescence observed in Aah venom treated primary rabbit RPT cell cultures following their incubation with the Flica reagent (indicative of caspase activation and apoptosis), and the green fluorescence of Sytox Green (indicative of dead cells). Copyright © 2016 Elsevier Ltd. All rights reserved.

Using simulated fluorescence cell micrographs for the evaluation of cell image segmentation algorithms.

PubMed

Wiesmann, Veit; Bergler, Matthias; Palmisano, Ralf; Prinzen, Martin; Franz, Daniela; Wittenberg, Thomas

2017-03-18

Manual assessment and evaluation of fluorescent micrograph cell experiments is time-consuming and tedious. Automated segmentation pipelines can ensure efficient and reproducible evaluation and analysis with constant high quality for all images of an experiment. Such cell segmentation approaches are usually validated and rated in comparison to manually annotated micrographs. Nevertheless, manual annotations are prone to errors and display inter- and intra-observer variability which influence the validation results of automated cell segmentation pipelines. We present a new approach to simulate fluorescent cell micrographs that provides an objective ground truth for the validation of cell segmentation methods. The cell simulation was evaluated twofold: (1) An expert observer study shows that the proposed approach generates realistic fluorescent cell micrograph simulations. (2) An automated segmentation pipeline on the simulated fluorescent cell micrographs reproduces segmentation performances of that pipeline on real fluorescent cell micrographs. The proposed simulation approach produces realistic fluorescent cell micrographs with corresponding ground truth. The simulated data is suited to evaluate image segmentation pipelines more efficiently and reproducibly than it is possible on manually annotated real micrographs.

Photoswitching Near-Infrared Fluorescence from Polymer Nanoparticles Catapults Signals over the Region of Noises and Interferences for Enhanced Sensitivity.

PubMed

Wang, Jie; Lv, Yanlin; Wan, Wei; Wang, Xuefei; Li, Alexander D Q; Tian, Zhiyuan

2016-02-01

As a very sensitive technique, photoswitchable fluorescence not only gains ultrasensitivity but also imparts many novel and unexpected applications. Applications of near-infrared (NIR) fluorescence have demonstrated low background noises, high tissue-penetrating ability, and an ability to reduce photodamage to live cells. Because of these desired features, NIR-fluorescent dyes have been the premium among fluorescent dyes, and probes with photoswitchable NIR fluorescence are even more desirable for enhanced signal quality in the emerging optical imaging modalities but rarely used because they are extremely challenging to design and construct. Using a spiropyran derivative functioning as both a photoswitch and a fluorophore to launch its periodically modulated red fluorescence excitation energy into a NIR acceptor, we fabricated core-shell polymer nanoparticles exhibiting a photoswitchable fluorescence signal within the biological window (∼700-1000 nm) with a peak maximum of 776 nm. Live cells constantly synthesize new molecules, including fluorescent molecules, and also endocytose exogenous particles, including fluorescent particles. Upon excitation at different wavelengths, these fluorescent species bring about background noises and interferences covering nearly the whole visible region and therefore render many intracellular targets unaddressable. The oscillating NIR fluorescence signal with an on/off ratio of up to 67 that the polymer nanoparticles display is beyond the typical background noises and interferences, thus producing superior sharpness, reliability, and signal-to-noise ratios in cellular imaging. Taking these salient features, we anticipate that these types of nanoparticles will be useful for in vivo imaging of biological tissue and other complex specimens, where two-photon activation and excitation are used in combination with NIR-fluorescence photoswitching.

Saccharomyces cerevisiae samples stained with FUN-1 dye can be stored at -20 degrees C for later observation.

PubMed

Eggleston, M D; Marshall, P A

2007-01-01

FUN-1, a fluorescent vital dye, has been observed to form cylindrical intravacuolar structures within the vacuoles of metabolically active yeast cells. FUN-1 staining, which begins as a diffuse pool of fluorescent cytoplasmic stain, uses an unknown endogenous biochemical processing mechanism to compact and form orange-red cylindrical intravacuolar structures within the cell vacuole. In the clinical setting, FUN-1 is primarily used for identification of fungal infection. FUN-1 is utilized in the laboratory to distinguish between metabolically active and dead fungal cells. Although this stain is useful for distinguishing between live and dead fungal dead cells, few studies have utilized this chemical. This lack of use in the scientific community may be due to the requirement that cells are visualized directly after staining. Thus, it would be of interest to be able to stain cells and store them for later use. Our lab examined the longevity of cylindrical intravacuolar structures in two strains of Saccharomyces cerevisiae stained with FUN-1 and stored at -20 degrees C. We found that cylindrical intravacuolar structures could be reliably observed and imaged utilizing differential interference contrast microscopy and fluorescence microscopy for 21 days. We also observed that cells stained with FUN-1 would resume propagation on yeast extract, peptone, dextrose (YPD) plates after being frozen at -20 degrees C for 21 days. These modifications to the published procedure for FUN-1 dye staining should allow for a more prevalent and less time sensitive use of this important biological tool.

Force-activatable coating enables high-resolution cellular force imaging directly on regular cell culture surfaces.

PubMed

Sarkar, Anwesha; Zhao, Yuanchang; Wang, Yongliang; Wang, Xuefeng

2018-06-25

Integrin-transmitted cellular forces are crucial mechanical signals regulating a vast range of cell functions. Although various methods have been developed to visualize and quantify cellular forces at the cell-matrix interface, a method with high performance and low technical barrier is still in demand. Here we developed a force-activatable coating (FAC), which can be simply coated on regular cell culture apparatus' surfaces by physical adsorption, and turn these surfaces to force reporting platforms that enable cellular force mapping directly by fluorescence imaging. The FAC molecule consists of an adhesive domain for surface coating and a force-reporting domain which can be activated to fluoresce by integrin molecular tension. The tension threshold required for FAC activation is tunable in 10-60 piconewton (pN), allowing the selective imaging of cellular force contributed by integrin tension at different force levels. We tested the performance of two FACs with tension thresholds of 12 and 54 pN (nominal values), respectively, on both glass and polystyrene surfaces. Cellular forces were successfully mapped by fluorescence imaging on all the surfaces. FAC-coated surfaces also enable co-imaging of cellular forces and cell structures in both live cells and immunostained cells, therefore opening a new avenue for the study of the interplay of force and structure. We demonstrated the co-imaging of integrin tension and talin clustering in live cells, and concluded that talin clustering always occurs before the generation of integrin tension above 54 pN, reinforcing the notion that talin is an important adaptor protein for integrin tension transmission. Overall, FAC provides a highly convenient approach that is accessible to general biological laboratories for the study of cellular forces with high sensitivity and resolution, thus holding the potential to greatly boost the research of cell mechanobiology.

Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells

PubMed Central

Lu, Jinjin; Zheng, Xiufen; Li, Fan; Yu, Yang; Chen, Zhong; Liu, Zheng; Wang, Zhihua; Xu, Hua; Yang, Weimin

2017-01-01

Intercellular transfer of organelles via tunneling nanotubes (TNTs) is a novel means of cell-to-cell communication. Here we demonstrate the existence of TNTs between co-cultured RT4 and T24 bladder cancer cells using light microscopy, fluorescence imaging, and scanning electron microscopy (SEM). Spontaneous unidirectional transfer of mitochondria from T24 to RT4 cells was detected using fluorescence imaging and flow cytometry. The distribution of mitochondria migrated from T24 cells was in good agreement with the original mitochondria in RT4 cells, which may imply mitochondrial fusion. We detected cytoskeleton reconstruction in RT4-Mito-T24 cells by observing F-actin redistribution. Akt, mTOR, and their downstream mediators were activated and increased. The resultant increase in the invasiveness of bladder cancer cells was detected in vitro and in vivo. These data indicate that TNTs promote intercellular mitochondrial transfer between heterogeneous cells, followed by an increase in the invasiveness of bladder cancer cells. PMID:28107184

Myricetin and methyl eugenol combination enhances the anticancer activity, cell cycle arrest and apoptosis induction of cis-platin against HeLa cervical cancer cell lines

PubMed Central

Yi, Jin-Ling; Shi, Song; Shen, Yan-Li; Wang, Ling; Chen, Hai-Yan; Zhu, Jun; Ding, Yan

2015-01-01

Drug combination therapies are common practice in the treatment of cancer. In this study, we evaluated the anticancer effects of myricetin (MYR), methyl eugenol (MEG) and cisplatin (CP) both separately as well as in combination against cervical cancer (HeLa) cells. To demonstrate whether MYR and MEG enhance the anticancer activity of CP against cervical cancer cells, we treated HeLa cells with MYR and MEG alone or in combination with cisplatin and evaluated cell growth and apoptosis using MTT (3 (4, 5 dimethyl thiazol 2yl) 2, 5 diphenyltetrazolium bromide) assay, LDH release assay, flow cytometry and fluorescence microscopy. The results revealed that, as compared to single drug treatment, the combination of MYR or MEG with CP resulted in greater effect in inhibiting cancer cell growth and inducing apoptosis. Cell apoptosis induction, Caspase-3 activity, cell cycle arrest and mitochondrial membrane potential loss were systematically studied to reveal the mechanisms of synergy between MYR, MEG and CP. Combination of MYR or MEG with CP resulted in more potent apoptosis induction as revealed by fluorescence microscopy using Hoechst 33258 and AO-ETBR staining. The combination treatment also increased the number of cells in G0/G1 phase dramatically as compared to single drug treatment. Mitochondrial membrane potential loss (ΛΨm) as well as Caspase-3 activity was much higher in combination treatment as compared to single drug treatment. Findings of this investigation suggest that MYR and MEG combined with cisplatin is a potential clinical chemotherapeutic approach in human cervical cancer. PMID:25972998

The novel phloroglucinol derivative BFP induces apoptosis of glioma cancer through reactive oxygen species and endoplasmic reticulum stress pathways.

PubMed

Lu, Dah-Yuu; Chang, Chih-Shiang; Yeh, Wei-Lan; Tang, Chih-Hsin; Cheung, Chi-Wai; Leung, Yuk-Man; Liu, Ju-Fang; Wong, Kar-Lok

2012-09-15

Prenyl-phloroglucinol derivatives from hop plants have been shown to have anticancer activities. This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (2,4-bis(4-fluorophenylacetyl)phloroglucinol; BFP). BFP induced cell death and anti-proliferation in three glioma, U251, U87 and C6 cells, but not in primary human astrocytes. BFP-induced concentration-dependently cell death in glioma cells was determined by MTT and SRB assay. Moreover, BFP-induced apoptotic cell death in glioma cells was measured by Hochest 33258 staining and fluorescence-activated cell sorter (FACS) of propidine iodine (PI) analysis. Treatment of U251 human glioma cells with BFP was also found to induce reactive oxygen species (ROS) generation, which was detected by a fluorescence dye used FACS analysis. Treatment of BFP also increased a number of signature endoplasmic reticulum (ER) stress markers glucose-regulated protein (GRP)-78, GRP-94, IRE1, phosphorylation of eukaryotic initiation factor-2α (eIF-2α) and up-regulation of CAAT/enhancer-binding protein homologous protein (CHOP). Moreover, treatment of BFP also increased the down-stream caspase activation, such as pro-caspase-7 and pro-caspase-12 degradation, suggesting the induction of ER stress. Furthermore, BFP also induced caspase-9 and caspase-3 activation as well as up-regulation of cleaved PARP expression. Treatment of antioxidants, or pre-transfection of cells with GRP78 or CHOP siRNA reduced BFP-mediated apoptotic-related protein expression. Taken together, the present study provides evidences to support that ROS generation, GRP78 and CHOP activation are mediating the BFP-induced human glioma cell apoptosis. Copyright © 2012 Elsevier GmbH. All rights reserved.

Development of fluorescent methods for DNA methyltransferase assay

NASA Astrophysics Data System (ADS)

Li, Yueying; Zou, Xiaoran; Ma, Fei; Tang, Bo; Zhang, Chun-yang

2017-03-01

DNA methylation modified by DNA methyltransferase (MTase) plays an important role in regulating gene transcription, cell growth and proliferation. The aberrant DNA MTase activity may lead to a variety of human diseases including cancers. Therefore, accurate and sensitive detection of DNA MTase activity is crucial to biomedical research, clinical diagnostics and therapy. However, conventional DNA MTase assays often suffer from labor-intensive operations and time-consuming procedures. Alternatively, fluorescent methods have significant advantages of simplicity and high sensitivity, and have been widely applied for DNA MTase assay. In this review, we summarize the recent advances in the development of fluorescent methods for DNA MTase assay. These emerging methods include amplification-free and the amplification-assisted assays. Moreover, we discuss the challenges and future directions of this area.

Selective detection of Co2+ by fluorescent nano probe: Diagnostic approach for analysis of environmental samples and biological activities

NASA Astrophysics Data System (ADS)

Mahajan, Prasad G.; Dige, Nilam C.; Desai, Netaji K.; Patil, Shivajirao R.; Kondalkar, Vijay V.; Hong, Seong-Karp; Lee, Ki Hwan

2018-06-01

Nowadays scientist over the world are engaging to put forth improved methods to detect metal ion in an aqueous medium based on fluorescence studies. A simple, selective and sensitive method was proposed for detection of Co2+ ion using fluorescent organic nanoparticles. We synthesized a fluorescent small molecule viz. 4,4‧-{benzene-1,4-diylbis-[(Z)methylylidenenitrilo]}dibenzoic acid (BMBA) to explore its suitability as sensor for Co2+ ion and biocompatibility in form of nanoparticles. Fluorescence nanoparticles (BMBANPs) prepared by simple reprecipitation method. Aggregation induced enhanced emission of BMBANPs exhibits the narrower particle size of 68 nm and sphere shape morphology. The selective fluorescence quenching was observed by addition of Co2+ and does not affected by presence of other coexisting ion solutions. The photo-physical properties, viz. UV-absorption, fluorescence emission, and lifetime measurements are in support of ligand-metal interaction followed by static fluorescence quenching phenomenon in emission of BMBANPs. Finally, we develop a simple analytical method for selective and sensitive determination of Co2+ ion in environmental samples. The cell culture E. coli, Bacillus sps., and M. tuberculosis H37RV strain in the vicinity of BMBANPs indicates virtuous anti-bacterial and anti-tuberculosis activity which is of additional novel application shown by prepared nanoparticles.

Thermally activated delayed fluorescence of fluorescein derivative for time-resolved and confocal fluorescence imaging.

PubMed

Xiong, Xiaoqing; Song, Fengling; Wang, Jingyun; Zhang, Yukang; Xue, Yingying; Sun, Liangliang; Jiang, Na; Gao, Pan; Tian, Lu; Peng, Xiaojun

2014-07-09

Compared with fluorescence imaging utilizing fluorophores whose lifetimes are in the order of nanoseconds, time-resolved fluorescence microscopy has more advantages in monitoring target fluorescence. In this work, compound DCF-MPYM, which is based on a fluorescein derivative, showed long-lived luminescence (22.11 μs in deaerated ethanol) and was used in time-resolved fluorescence imaging in living cells. Both nanosecond time-resolved transient difference absorption spectra and time-correlated single-photon counting (TCSPC) were employed to explain the long lifetime of the compound, which is rare in pure organic fluorophores without rare earth metals and heavy atoms. A mechanism of thermally activated delayed fluorescence (TADF) that considers the long wavelength fluorescence, large Stokes shift, and long-lived triplet state of DCF-MPYM was proposed. The energy gap (ΔEST) of DCF-MPYM between the singlet and triplet state was determined to be 28.36 meV by the decay rate of DF as a function of temperature. The ΔE(ST) was small enough to allow efficient intersystem crossing (ISC) and reverse ISC, leading to efficient TADF at room temperature. The straightforward synthesis of DCF-MPYM and wide availability of its starting materials contribute to the excellent potential of the compound to replace luminescent lanthanide complexes in future time-resolved imaging technologies.

Quenching-induced deactivation of photosensitizer by nanoencapsulation to improve phototherapy of cancer.

PubMed

Zeisser-Labouèbe, Magali; Mattiuzzo, Marc; Lange, Norbert; Gurny, Robert; Delie, Florence

2009-09-01

Photodynamic therapy has emerged as a promising alternative to current cancer treatment. However, conventional photosensitizers have several limitations due to their unsuitable pharmaceutical formulations and lack of selectivity. Our strategy was to exploit the advantages of nanoparticles and the quenching-induced deactivation of the model photosensitizer hypericin to produce "activatable" drug delivery systems. Efficient fluorescence and activity quenching were achieved by increasing the drug-loading rate of nanoparticles. In vitro assays confirmed the reversibility of hypericin deactivation, as the hypericin fluorescence and photodynamic activity were recovered upon cell internalization.

Real-time assessment of inflammation and treatment response in a mouse model of allergic airway inflammation

PubMed Central

Cortez-Retamozo, Virna; Swirski, Filip K.; Waterman, Peter; Yuan, Hushan; Figueiredo, Jose Luiz; Newton, Andita P.; Upadhyay, Rabi; Vinegoni, Claudio; Kohler, Rainer; Blois, Joseph; Smith, Adam; Nahrendorf, Matthias; Josephson, Lee; Weissleder, Ralph; Pittet, Mikael J.

2008-01-01

Eosinophils are multifunctional leukocytes that degrade and remodel tissue extracellular matrix through production of proteolytic enzymes, release of proinflammatory factors to initiate and propagate inflammatory responses, and direct activation of mucus secretion and smooth muscle cell constriction. Thus, eosinophils are central effector cells during allergic airway inflammation and an important clinical therapeutic target. Here we describe the use of an injectable MMP-targeted optical sensor that specifically and quantitatively resolves eosinophil activity in the lungs of mice with experimental allergic airway inflammation. Through the use of real-time molecular imaging methods, we report the visualization of eosinophil responses in vivo and at different scales. Eosinophil responses were seen at single-cell resolution in conducting airways using near-infrared fluorescence fiberoptic bronchoscopy, in lung parenchyma using intravital microscopy, and in the whole body using fluorescence-mediated molecular tomography. Using these real-time imaging methods, we confirmed the immunosuppressive effects of the glucocorticoid drug dexamethasone in the mouse model of allergic airway inflammation and identified a viridin-derived prodrug that potently inhibited the accumulation and enzyme activity of eosinophils in the lungs. The combination of sensitive enzyme-targeted sensors with noninvasive molecular imaging approaches permitted evaluation of airway inflammation severity and was used as a model to rapidly screen for new drug effects. Both fluorescence-mediated tomography and fiberoptic bronchoscopy techniques have the potential to be translated into the clinic. PMID:19033674

Evaluation of the oxidative stress of psoriatic fibroblasts based on spectral two-photon fluorescence lifetime imaging

NASA Astrophysics Data System (ADS)

Kapsokalyvas, Dimitrios; Barygina, Victoria; Cicchi, Riccardo; Fiorillo, Claudia; Pavone, Francesco S.

2013-02-01

Psoriasis is an autoimmune disease of the skin characterized by hyperkeratosis, hyperproliferation of the epidermis, inflammatory cell accumulation and increased dilatation of dermal papillary blood vessels. Metabolic activity is increased in the epidermis and the dermis. Oxidative stress is high mainly due to reactive oxygen species (ROS) originating from the skin environment and cellular metabolism. We employed a custom multiphoton microscope coupled with a FLIM setup to image primary culture fibroblast cells from perilesional and lesional psoriatic skin in-vitro. Twophoton excited fluorescence images revealed the morphological differences between healthy and psoriatic fibroblasts. Based on the spectral analysis of the NADH and FAD components the oxidative stress was assessed and found to be higher in psoriatic cells. Furthermore the fluorescence lifetime properties were investigated with a TCSPC FLIM module. Mean fluorescence lifetime was found to be longer in psoriatic lesional cells. Analysis of the fast (τ1) and slow (τ2) decay lifetimes revealed a decrease of the ratio of the contribution of the fast (α1) parameter to the contribution of the slow (α2) parameter. The fluorescence in the examined part of the spectrum is attributed mainly to NADH. The decrease of the ratio (α1)/ (α2) is believed to correlate strongly with the anti-oxidant properties of NADH which can lead to the variation of its population in high ROS environment. This methodology could serve as an index of the oxidative status in cells and furthermore could be used to probe the oxidative stress of tissues in-vivo.

Isolation of Primary Human Skeletal Muscle Cells

PubMed Central

Spinazzola, Janelle M.; Gussoni, Emanuela

2017-01-01

Primary myoblast culture is a valuable tool in research of muscle disease, pathophysiology, and pharmacology. This protocol describes techniques for dissociation of cells from human skeletal muscle biopsies and enrichment for a highly myogenic population by fluorescence-activated cell sorting (FACS). We also describe methods for assessing myogenicity and population expansion for subsequent in vitro study. PMID:29152538

A Method to Identify and Isolate Pluripotent Human Stem Cells and Mouse Epiblast Stem Cells Using Lipid Body-Associated Retinyl Ester Fluorescence

PubMed Central

Muthusamy, Thangaselvam; Mukherjee, Odity; Menon, Radhika; Megha, P.B.; Panicker, Mitradas M.

2014-01-01

Summary We describe the use of a characteristic blue fluorescence to identify and isolate pluripotent human embryonic stem cells and human-induced pluripotent stem cells. The blue fluorescence emission (450–500 nm) is readily observed by fluorescence microscopy and correlates with the expression of pluripotency markers (OCT4, SOX2, and NANOG). It allows easy identification and isolation of undifferentiated human pluripotent stem cells, high-throughput fluorescence sorting and subsequent propagation. The fluorescence appears early during somatic reprogramming. We show that the blue fluorescence arises from the sequestration of retinyl esters in cytoplasmic lipid bodies. The retinoid-sequestering lipid bodies are specific to human and mouse pluripotent stem cells of the primed or epiblast-like state and absent in naive mouse embryonic stem cells. Retinol, present in widely used stem cell culture media, is sequestered as retinyl ester specifically by primed pluripotent cells and also can induce the formation of these lipid bodies. PMID:25068130

Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry.

PubMed

Smurthwaite, Cameron A; Hilton, Brett J; O'Hanlon, Ryan; Stolp, Zachary D; Hancock, Bryan M; Abbadessa, Darin; Stotland, Aleksandr; Sklar, Larry A; Wolkowicz, Roland

2014-01-01

The discovery of the green fluorescent protein from Aequorea victoria has revolutionized the field of cell and molecular biology. Since its discovery a growing panel of fluorescent proteins, fluorophores and fluorescent-coupled staining methodologies, have expanded the analytical capabilities of flow cytometry. Here, we exploit the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins. For genetic engineering, we utilize retroviral technology, which allows for the expression of ectopic genetic information in a stable manner in mammalian cells. We have genetically barcoded both adherent and nonadherent cells with different fluorescent proteins. Multiplexing power was increased by combining both the number of distinct fluorescent proteins, and the fluorescence intensity in each channel. Moreover, retroviral expression has proven to be stable for at least a 6-month period, which is critical for applications such as biological screens. We have shown the applicability of fluorescent barcoded multiplexing to cell-based assays that rely themselves on genetic barcoding, or on classical staining protocols. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. Once cell lines are developed, no further manipulation or staining is required, decreasing time, nonspecific background associated with staining protocols, and cost. The increasing number of discovered and/or engineered fluorescent proteins with unique absorbance/emission spectra, combined with the growing number of detection devices and lasers, increases multiplexing versatility, making fluorescent genetic barcoding a powerful tool for flow cytometry-based analysis. © 2013 International Society for Advancement of Cytometry.

Methods on observation of fluorescence micro-imaging for microalgae

NASA Astrophysics Data System (ADS)

Ou, Lin; Zhuang, Hui-ru; Chen, Rong; Lei, Jin-pin; Liao, Xiao-hua; Lin, Wen-suo

2007-11-01

Objective: Auto-fluorescence micro-imaging of microalgae are observed by using of laser scanning confocal microscopy (LSCM) and fluorescence microscopy, so as to investigate the effect of auto fluorescence alteration on growth of irradiated microalgae irradiated, meanwhile, the method of microalgae cells stained also to be studied. Methods: Platymonas subcordiformis, Phaeodactylum tricormutum and Isochyrsis zhanjiangensis cells are stained with acridine orange, and observed by fluorescence microscopy; the three types microalgae mentioned above are irradiated by Nd:YAP laser with 10w at 1341nm, irradiating time:12s, 30s, 35s and 55s, than to be cultured 6 days, and the auto fluorescence images and fluorescence spectra of algae cells are obtained by LSCM on lambda scan mode, at excitation 488nm (Ar + laser). Results: It is showed that the shapes and the structural features of microalgae cells stained can be seen clearly, and the cytoplasm and nucleus also can be observed. The chloroplasts in cell is bigger on promoting effects, conversely, it is to be mutilated, deformation and shrink. Contrast to the CK, the peak positions of fluorescence of algae cells irradiated is similar to the whole while the peak light intensity alters. On irradiation of promoting dose, however, the auto fluorescence intensity is enhanced more than control. Conclusions: The method of cell stained can be used to observed genetic material in microalgae. There are obvious effects for laser irradiating to chloroplasts in cells, the bigger chloroplasts the greater fluorescence intensity. Physiological incentive effects of microalgae irradiated can be given expression on fluorescence characteristics and fluorescence intensity alteration of cells.

NAD(P)H Oxidase Activity in the Small Intestine Is Predominantly Found in Enterocytes, Not Professional Phagocytes.

PubMed

Lindquist, Randall L; Bayat-Sarmadi, Jannike; Leben, Ruth; Niesner, Raluca; Hauser, Anja E

2018-05-04

The balance between various cellular subsets of the innate and adaptive immune system and microbiota in the gastrointestinal tract is carefully regulated to maintain tolerance to the normal flora and dietary antigens, while protecting against pathogens. The intestinal epithelial cells and the network of dendritic cells and macrophages in the lamina propria are crucial lines of defense that regulate this balance. The complex relationship between the myeloid compartment (dendritic cells and macrophages) and lymphocyte compartment (T cells and innate lymphoid cells), as well as the impact of the epithelial cell layer have been studied in depth in recent years, revealing that the regulatory and effector functions of both innate and adaptive immune compartments exhibit more plasticity than had been previously appreciated. However, little is known about the metabolic activity of these cellular compartments, which is the basic function underlying all other additional tasks the cells perform. Here we perform intravital NAD(P)H fluorescence lifetime imaging in the small intestine of fluorescent reporter mice to monitor the NAD(P)H-dependent metabolism of epithelial and myeloid cells. The majority of myeloid cells which comprise the surveilling network in the lamina propria have a low metabolic activity and remain resting even upon stimulation. Only a few myeloid cells, typically localized at the tip of the villi, are metabolically active and are able to activate NADPH oxidases upon stimulation, leading to an oxidative burst. In contrast, the epithelial cells are metabolically highly active and, although not considered professional phagocytes, are also able to activate NADPH oxidases, leading to massive production of reactive oxygen species. Whereas the oxidative burst in myeloid cells is mainly catalyzed by the NOX2 isotype, in epithelial cells other isotypes of the NADPH oxidases family are involved, especially NOX4. They are constitutively expressed by the epithelial cells, but activated only on demand to ensure rapid defense against pathogens. This minimizes the potential for inadvertent damage from resting NOX activation, while maintaining the capacity to respond quickly if needed.

Efficient genome editing by FACS enrichment of paired D10A Cas9 nickases coupled with fluorescent proteins.

PubMed

Gopalappa, Ramu; Song, Myungjae; Chandrasekaran, Arun Pandian; Das, Soumyadip; Haq, Saba; Koh, Hyun Chul; Ramakrishna, Suresh

2018-05-31

Targeted genome editing by clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) raised concerns over off-target effects. The use of double-nicking strategy using paired Cas9 nickase has been developed to minimize off-target effects. However, it was reported that the efficiency of paired nickases were comparable or lower than that of either corresponding nuclease alone. Recently, we conducted a systematic comparison of the efficiencies of several paired Cas9 with their corresponding Cas9 nucleases and showed that paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption. However, sometimes the designed paired Cas9 nickases exhibited significantly lower mutation frequencies than nucleases, hampering the generation of cells containing paired Cas9 nickase-induced mutations. Here we implemented IRES peptide-conjugation of fluorescent protein to Cas9 nickase and subjected for fluorescence-activated cell sorting. The sorted cell populations are highly enriched with cells containing paired Cas9 nickase-induced mutations, by a factor of up to 40-fold as compared with the unsorted population. Furthermore, gene-disrupted single cell clones using paired nickases followed by FACS sorting strategy were generated highly efficiently, without compromising with its low off-target effects. We envision that our fluorescent protein coupled paired nickase-mediated gene disruption, facilitating efficient and highly specific genome editing in medical research.

Fluorescence Microspectroscopy for Testing the Dimerization Hypothesis of BACE1 Protein in Cultured HEK293 Cells

NASA Astrophysics Data System (ADS)

Gardeen, Spencer; Johnson, Joseph L.; Heikal, Ahmed A.

2016-06-01

Alzheimer's Disease (AD) is a neurodegenerative disorder that results from the formation of beta-amyloid plaques in the brain that trigger the known symptoms of memory loss in AD patients. The beta-amyloid plaques are formed by the proteolytic cleavage of the amyloid precursor protein (APP) by the proteases BACE1 and gamma-secretase. These enzyme-facilitated cleavages lead to the production of beta-amyloid fragments that aggregate to form plaques, which ultimately lead to neuronal cell death. Recent detergent protein extraction studies suggest that BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. In this contribution, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using complementary fluorescence spectroscopy and microscopy methods. Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal, and differential interference contrast to monitor the localization and distribution of intracellular BACE1. Complementary fluorescence lifetime and anisotropy measurements enabled us to examine the conformational and environmental changes of BACE1 as a function of substrate binding. Using fluorescence correlation spectroscopy, we also quantified the diffusion coefficient of BACE1-EGFP on the plasma membrane as a means to test the dimerization hypothesis as a fucntion of substrate-analog inhibitition. Our results represent an important first towards examining the substrate-mediated dimerization hypothesis of BACE1 in live cells.

A versatile microsatellite instability reporter system in human cells.

PubMed

Koole, Wouter; Schäfer, Henning S; Agami, Reuven; van Haaften, Gijs; Tijsterman, Marcel

2013-09-01

Here, we report the investigation of microsatellite instability (MSI) in human cells with a newly developed reporter system based on fluorescence. We composed a vector into which microsatellites of different lengths and nucleotide composition can be introduced between a functional copy of the fluorescent protein mCherry and an out-of-frame copy of EGFP; in vivo frameshifting will lead to EGFP expression, which can be quantified by fluorescence activated cell sorting (FACS). Via targeted recombineering, single copy reporters were introduced in HEK293 and MCF-7 cells. We found predominantly -1 and +1 base pair frameshifts, the levels of which are kept in tune by mismatch repair. We show that tract length and composition greatly influences MSI. In contrast, a tracts' potential to form a G-quadruplex structure, its strand orientation or its transcriptional status is not affecting MSI. We further validated the functionality of the reporter system for screening microsatellite mutagenicity of compounds and for identifying modifiers of MSI: using a retroviral miRNA expression library, we identified miR-21, which targets MSH2, as a miRNA that induces MSI when overexpressed. Our data also provide proof of principle for the strategy of combining fluorescent reporters with next-generation sequencing technology to identify genetic factors in specific pathways.

Mechanical dynamics in live cells and fluorescence-based force/tension sensors

PubMed Central

Yang, Chao; Zhang, Xiaohan; Guo, Yichen; Meng, Fanjie; Sachs, Frederick; Guo, Jun

2016-01-01

Three signaling systems play the fundamental roles in modulating cell activities: chemical, electrical, and mechanical. While the former two are well studied, the mechanical signaling system is still elusive because of the lack of methods to measure structural forces in real time at cellular and subcellular levels. Indeed, almost all biological processes are responsive to modulation by mechanical forces that trigger dispersive downstream electrical and biochemical pathways. Communication among the three systems is essential to make cells and tissues receptive to environmental changes. Cells have evolved many sophisticated mechanisms for the generation, perception and transduction of mechanical forces, including motor proteins and mechanosensors. In this review, we introduce some background information about mechanical dynamics in live cells, including the ubiquitous mechanical activity, various types of mechanical stimuli exerted on cells and the different mechanosensors. We also summarize recent results obtained using genetically encoded FRET (fluorescence resonance energy transfer)-based force/tension sensors; a new technique used to measure mechanical forces in structural proteins. The sensors have been incorporated into many specific structural proteins and have measured the force gradients in real time within live cells, tissues, and animals. PMID:25958335

Establishment of nude mice with complete loss of lymphocytes and NK cells and application for in vivo bio-imaging.

PubMed

Kariya, Ryusho; Matsuda, Kouki; Gotoh, Kumiko; Vaeteewoottacharn, Kulthida; Hattori, Shinichiro; Okada, Seiji

2014-01-01

Nude mice are used in human xenograft research; however, only 25-35% of human tumors have been successfully transplanted into nude mice and their application is limited due to high natural killer (NK) cell activity. More severely immunodeficient mice with loss of NK activity are needed to overcome this limitation. Balb/c nude Rag-2(-/-)Jak3(-/-) (Nude-RJ) mice were established by crossing Rag-2(-/-)Jak3(-/-) mice and nude mice. The K562 cell line was implanted subcutaneously to compare tumorigenicity between Nude-RJ mice and Nude mice. The cholangiocarcinoma mCherry expressing cell line (KKU-M213) was implanted subcutaneously, and fluorescence intensity and tumor weight were measured. Nude R/J mice showed complete loss of lymphocytes and NK cells. Xeno-transplantation of K562 cells showed higher proliferation in Nude R/J mice than nude mice. Subcutaneously-transplanted mCherry-transduced KKU-M213 cells were successfully detected with a fluorescence imager. Nude-R/J mice are valuable tools for in vivo imaging studies in biomedical research. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Time- and spectrally resolved characteristics of flavin fluorescence in U87MG cancer cells in culture

NASA Astrophysics Data System (ADS)

Horilova, Julia; Cunderlikova, Beata; Marcek Chorvatova, Alzbeta

2015-05-01

Early detection of cancer is crucial for the successful diagnostics of its presence and its subsequent treatment. To improve cancer detection, we tested the progressive multimodal optical imaging of U87MG cells in culture. A combination of steady-state spectroscopic methods with the time-resolved approach provides a new insight into the native metabolism when focused on endogenous tissue fluorescence. In this contribution, we evaluated the metabolic state of living U87MG cancer cells in culture by means of endogenous flavin fluorescence. Confocal microscopy and time-resolved fluorescence imaging were employed to gather spectrally and time-resolved images of the flavin fluorescence. We observed that flavin fluorescence in U87MG cells was predominantly localized outside the cell nucleus in mitochondria, while exhibiting a spectral maximum under 500 nm and fluorescence lifetimes under 1.4 ns, suggesting the presence of bound flavins. In some cells, flavin fluorescence was also detected inside the cell nuclei in the nucleoli, exhibiting longer fluorescence lifetimes and a red-shifted spectral maximum, pointing to the presence of free flavin. Extra-nuclear flavin fluorescence was diminished by 2-deoxyglucose, but failed to increase with 2,4-dinitrophenol, the uncoupler of oxidative phosphorylation, indicating that the cells use glycolysis, rather than oxidative phosphorylation for functioning. These gathered data are the first step toward monitoring the metabolic state of U87MG cancer cells.

Endo-β-Glucosidase Tag Allows Dual Detection of Fusion Proteins by Fluorescent Mechanism-Based Probes and Activity Measurement.

PubMed

Kallemeijn, Wouter W; Scheij, Saskia; Voorn-Brouwer, Tineke M; Witte, Martin D; Verhoek, Marri; Overkleeft, Hermen S; Boot, Rolf G; Aerts, Johannes M F G

2016-09-15

β-Glucoside-configured cyclophellitols are activity-based probes (ABPs) that allow sensitive detection of β-glucosidases. Their applicability to detect proteins fused with β-glucosidase was investigated in the cellular context. The tag was Rhodococcus sp. M-777 endoglycoceramidase II (EGCaseII), based on its lack of glycans and ability to hydrolyze fluorogenic 4-methylumbelliferyl β-d-lactoside (an activity absent in mammalian cells). Specific dual detection of fusion proteins was possible in vitro and in situ by using fluorescent ABPs and a fluorogenic substrate. Pre-blocking with conduritol β-epoxide (a poor inhibitor of EGCaseII) eliminated ABP labeling of endogenous β-glucosidases. ABPs equipped with biotin allowed convenient purification of the fusion proteins. Diversification of ABPs (distinct fluorophores, fluorogenic high-resolution detection moieties) should assist further research in living cells and organisms. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of fluorescent glucose bioprobes and their application on real-time and quantitative monitoring of glucose uptake in living cells.

PubMed

Lee, Hyang Yeon; Lee, Jae Jeong; Park, Jongmin; Park, Seung Bum

2011-01-03

We developed a novel fluorescent glucose bioprobe, GB2-Cy3, for the real-time and quantitative monitoring of glucose uptake in living cells. We synthesized a series of fluorescent glucose analogues by adding Cy3 fluorophores to the α-anomeric position of D-glucose through various linkers. Systematic and quantitative analysis of these Cy3-labeled glucose analogues revealed that GB2-Cy3 was the ideal fluorescent glucose bioprobe. The cellular uptake of this probe competed with the cellular uptake of D-glucose in the media and was mediated by a glucose-specific transport system, and not by passive diffusion. Flow cytometry and fluorescence microscopy analyses revealed that GB2-Cy3 is ten times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. GB2-Cy3 can also be utilized for the quantitative flow cytometry monitoring of glucose uptake in metabolically active C2C12 myocytes under various treatment conditions. As opposed to a glucose uptake assay performed by using radioisotope-labeled deoxy-D-glucose and a scintillation counter, GB2-Cy3 allows the real-time monitoring of glucose uptake in living cells under various experimental conditions by using fluorescence microscopy or confocal laser scanning microscopy (CLSM). Therefore, we believe that GB2-Cy3 can be utilized in high-content screening (HCS) for the discovery of novel therapeutic agents and for making significant advances in biomedical studies and diagnosis of various diseases, especially metabolic diseases. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Changes in River Organic Matter Through Time.

NASA Astrophysics Data System (ADS)

Hudson, N.; Baker, A.; Ward, D.

2006-12-01

Samples of river water from central England were collected during the summer base-flow period. They were analysed for BOD and filtered at 1.2μm and 0.1μm increments to obtain i) the colloidal and dissolved, and ii) dissolved filter sterilized fractions. Each filtered fraction was plated up for microbiological cell counts and the agar plates and water samples were stored under a range of environmental conditions (4° C dark, 11° C light/ dark, 11° C dark, and 20° C dark) for 26 days. Absorbance, fluorescence, pH, conductivity and total organic carbon (TOC) were measured and colony forming units (CFU) counted on days 1, 2, 3, 4, 5, 12, 19 and 26. The fluorescence intensity was recorded for 5 commonly studied regions: protein like fluorescence, indicative of microbial activity, represented by the fluorescent amino acids tyrosine and tryptophan (which has two clear fluorescence regions) and humic and fulvic acids derived from the break down of terrestrial and aquatic plant material. Humic and fulvic-like fluorescence increased in all samples under all storage conditions suggesting that peaks A and C probably include a microbial element, either a product of the living community or as dead cell material in all fraction sizes including <0.1μm. Tryptophan and tyrosine-like fluorescence intensities demonstrated less clear trends which may be reflective of the intrinsic variation in natural samples. Tryptophan-like fluorescence generally decreased or showed minimal change, except in samples exposed to light in which an increase was observed in line with algal growth. A decrease in intensity may relate to the use of the tryptophan-like material as a microbial substrate. The increase in tryptophan-like fluorescence intensity suggests that this fluorescent material is being produced, either by algae, or bacterial activity associated with algal growth. It may also occur as a result of changing water chemistry causing a change in molecular conformation, and resulting fluorescence, as an increase in pH was also observed in these samples. This work illustrates the dynamic character of river organic matter within a timescale and under conditions that are representative of the natural system.

A passive two-band sensor of sunlight-excited plant fluorescence

NASA Astrophysics Data System (ADS)

Kebabian, Paul L.; Theisen, Arnold F.; Kallelis, Spiros; Freedman, Andrew

1999-11-01

We have designed and built a passive remote sensor of sunlight-excited chlorophyll fluorescence (U.S. Patent No. 5,567,947, Oct. 22, 1996) which provides for the real-time, in situ sensing of photosynthetic activity in plants. This sensor, which operates as a Fraunhofer line discriminator, detects light at the cores of the lines comprising the atmospheric oxygen A and B bands, centered at 762 and 688 nm, respectively. These bands also correspond to wavelengths in the far-red and red chlorophyll fluorescence bands. The sensor is based on an induced fluorescence approach; as light collected from fluorescing plants is passed through a low-pressure cell containing oxygen, the oxygen absorbs the energy and subsequently reemits photons which are then detected by a photomultiplier tube. Since the oxygen in the cell absorbs light at the same wavelengths that have been strongly absorbed by the oxygen in the atmosphere, the response to incident sunlight is minimal. This mode of measurement is limited to target plants sufficiently close in range that the plants' fluorescence is not itself appreciably absorbed by atmospheric oxygen (˜200 m). In vivo measurements of fluorescence in the 760 and 690 nm bands of vegetation in full sunlight are also presented. Measurements of plant fluorescence at the single-plant canopy level were obtained from greenhouse-grown bean plants subjected to a range of nitrogen treatments. The ratio of the fluorescence obtained from the two measurement bands showed statistically significant variation with respect to nitrogen treatments.

Hybrid ZnPc@TiO2 nanostructures for targeted photodynamic therapy, bioimaging and doxorubicin delivery.

PubMed

Flak, Dorota; Yate, Luis; Nowaczyk, Grzegorz; Jurga, Stefan

2017-09-01

In this study ZnPc@TiO 2 hybrid nanostructures, both nanoparticles and nanotubes, as potential photosensitizers for the photodynamic therapy, fluorescent bioimaging agents, as well as anti-cancer drug nanocarriers, were prepared via zinc phthalocyanine (ZnPc) deposition on TiO 2 . In order to provide the selectivity of prepared hybrid nanostructures towards cancer cells they were modified with folic acid molecules (FA). The efficient attachment of both ZnPc and FA molecules was confirmed with dynamic light scattering (DLS), zeta potential measurements and X-ray photoelectron spectroscopy (XPS). It was presented that ZnPc and FA attachment has a strong effect on fluorescence emission properties of TiO 2 nanostructures, which can be further used for their simultaneous visualization upon cellular uptake. ZnPc@TiO 2 and FA/ZnPc@TiO 2 hybrid nanotubes were then employed as doxorubicin nanocarriers. It was demonstrated that doxorubicin can be easily loaded on these hybrid nanostructures via an electrostatic interaction and then released. In vitro cytotoxicity and photo-cytotoxic activity studies showed that prepared hybrid nanostructures were selectively targeting to cancer cells. Doxorubicin loaded hybrid nanostructures were significantly more cytotoxic than un-loaded ones and their cytotoxic effect was even more severe upon irradiation. The cellular uptake of prepared hybrid nanostructures and their localization in cells was monitored in vitro in 2D cell culture and tumor-like 3D multicellular culture environment with fluorescent confocal microscopy. These hybrid nanostructures preferentially penetrated into human cervical cancer cells (HeLa) than into normal fibroblasts (MSU-1.1) and were mainly localized within the cell cytoplasm. HeLa cells spheroids were also efficiently labelled by prepared hybrid nanostructures. Fluorescent imaging of Hela cells treated with doxorubicin loaded hybrid nanostructures showed that doxorubicin was effectively delivered into cells, released and evenly distributed in the cytoplasm. In conclusion, prepared hybrid nanostructures exhibit high potential as selective bioimaging agents next to their photodynamic activity and drug delivery ability. Copyright © 2017 Elsevier B.V. All rights reserved.

Toxic Effects of Ethyl Cinnamate on the Photosynthesis and Physiological Characteristics of Chlorella vulgaris Based on Chlorophyll Fluorescence and Flow Cytometry Analysis

PubMed Central

Jiao, Yang; Ouyang, Hui-Ling; Jiang, Yu-Jiao; Kong, Xiang-Zhen; He, Wei; Liu, Wen-Xiu; Yang, Bin; Xu, Fu-Liu

2015-01-01

The toxic effects of ethyl cinnamate on the photosynthetic and physiological characteristics of Chlorella vulgaris were studied based on chlorophyll fluorescence and flow cytometry analysis. Parameters, including biomass, F v/F m (maximal photochemical efficiency of PSII), ФPSII (actual photochemical efficiency of PSII in the light), FDA, and PI staining fluorescence, were measured. The results showed the following: (1) The inhibition on biomass increased as the exposure concentration increased. 1 mg/L ethyl cinnamate was sufficient to reduce the total biomass of C. vulgaris. The 48-h and 72-h EC50 values were 2.07 mg/L (1.94–2.20) and 1.89 mg/L (1.82–1.97). (2) After 24 h of exposure to 2–4 mg/L ethyl cinnamate, the photosynthesis of C. vulgaris almost ceased, manifesting in ФPSII being close to zero. After 72 h of exposure to 4 mg/L ethyl cinnamate, the F v/F m of C. vulgaris dropped to zero. (3) Ethyl cinnamate also affected the cellular physiology of C. vulgaris, but these effects resulted in the inhibition of cell yield rather than cell death. Exposure to ethyl cinnamate resulted in decreased esterase activities in C. vulgaris, increased average cell size, and altered intensities of chlorophyll a fluorescence. Overall, esterase activity was the most sensitive variable. PMID:26101784

Mesenchymal Stem Cells in the Bone Marrow Provide a Supportive Niche for Early Disseminated Breast Tumor-Initiating Cells

DTIC Science & Technology

2011-04-01

Differentiation of mouse embryonic stem cells Immunology: - Flow cytometry - Proliferation Assays - Chromium Release Assays - B...of metastatic cells in close proximation to hepatocytes in the liver. Additionally, re-expression of E-cadherin was observed in the membrane of the...profile CD44+/CD24low/ESA+ using fluorescence- activated cell sorting (FACS) [4]. Subcutaneous injection of low numbers of the sorted cell

A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity.

PubMed

Bandhuvula, Padmavathi; Fyrst, Henrik; Saba, Julie D

2007-12-01

Sphingosine-1-phosphate (S1P) lyase (SPL) catalyzes the conversion of S1P to ethanolamine phosphate and hexadecenal. This enzyme plays diverse roles in physiology and disease and, thus, may be useful as a disease marker and/or drug target. Unfortunately, the radioisotope-based assay currently used to quantify SPL activity is suboptimal. We have devised an assay using a commercially available omega(7-nitro-2-1,3-benzoxadiazol-4-yl)-d-erythro (NBD)-labeled fluorescent substrate. Alternatively, we provide a method for synthesis of the substrate from NBD-sphingosine. Enzyme activity is determined by following the formation of NBD-aldehyde product, which is isolated from unreacted substrate by lipid extraction and quantified after separation by HPLC using a C18 column. A fluorescent NBD-C18-sphingosine internal standard is used to control for extraction efficiency. The reaction is linear over 20 min and total protein concentrations of 20-200 mg/l. The sensitivity of the fluorescence assay is comparable to or better than that of the radioactive assay, and SPL levels as low as 8 pmol/mg/min were readily detected. Semicarbazide, a nonspecific SPL inhibitor, reduced SPL activity in vitro by approximately 70% using both standard and fluorescence methods. Product inhibition was not observed using ethanolamine phosphate and a commercially available source of hexadecenal. This method is suitable for quantifying SPL activity in a variety of cell and tissue sources.

Permeabilization Activated Reduction in Fluorescence (PARF): a novel method to measure kinetics of protein interactions with intracellular structures

PubMed Central

Singh, Pali P.; Hawthorne, Jenci L.; Davis, Christie A.; Quintero, Omar A.

2016-01-01

Understanding kinetic information is fundamental in understanding biological function. Advanced imaging technologies have fostered the development of kinetic analyses in cells. We have developed Permeabilization Activated Reduction in Fluorescence (PARF) analysis for determination of apparent t1/2 and immobile fraction, describing the dissociation of a protein of interest from intracellular structures. To create conditions where dissociation events are observable, cells expressing a fluorescently-tagged protein are permeabilized with digitonin, diluting the unbound protein into the extracellular media. As the media volume is much larger than the cytosolic volume, the concentration of the unbound pool decreases drastically, shifting the system out of equilibrium--favoring dissociation events. Loss of bound protein is observed as loss of fluorescence from intracellular structures and can be fit to an exponential decay. We compared PARF dissociation kinetics with previously published equilibrium kinetics as determined by FRAP. PARF dissociation rates agreed with the equilibrium-based FRAP analysis predictions of the magnitude of those rates. When used to investigate binding kinetics of a panel of cytoskeletal proteins, PARF analysis revealed that filament stabilization resulted in slower fluorescence loss. Additionally, commonly used “general” F-actin labels display differences in kinetic properties, suggesting that not all fluorescently-tagged actin labels interact with the actin network in the same way. We also observed differential dissociation kinetics for GFP-VASP depending on which cellular structure was being labeled. These results demonstrate that PARF analysis of non-equilibrium systems reveals kinetic information without the infrastructure investment required for other quantitative approaches such as FRAP, photoactivation, or in vitro reconstitution assays. PMID:27126922

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

PubMed Central

Zhu, Hongying; Ozcan, Aydogan

2013-01-01

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 μm over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water. PMID:23603893

Wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone.

PubMed

Zhu, Hongying; Ozcan, Aydogan

2013-04-11

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. - 10 μm over a very large field-of-view of - 81 mm(2). This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

FRET detection of lymphocyte function–associated antigen-1 conformational extension

PubMed Central

Chigaev, Alexandre; Smagley, Yelena; Haynes, Mark K.; Ursu, Oleg; Bologa, Cristian G.; Halip, Liliana; Oprea, Tudor; Waller, Anna; Carter, Mark B.; Zhang, Yinan; Wang, Wei; Buranda, Tione; Sklar, Larry A.

2015-01-01

Lymphocyte function–associated antigen 1 (LFA-1, CD11a/CD18, αLβ2-integrin) and its ligands are essential for adhesion between T-cells and antigen-presenting cells, formation of the immunological synapse, and other immune cell interactions. LFA-1 function is regulated through conformational changes that include the modulation of ligand binding affinity and molecular extension. However, the relationship between molecular conformation and function is unclear. Here fluorescence resonance energy transfer (FRET) with new LFA-1–specific fluorescent probes showed that triggering of the pathway used for T-cell activation induced rapid unquenching of the FRET signal consistent with extension of the molecule. Analysis of the FRET quenching at rest revealed an unexpected result that can be interpreted as a previously unknown LFA-1 conformation. PMID:25378583

Self-assembled Targeting of Cancer Cells by Iron(III)-doped, Silica Nanoparticles.

PubMed

Mitchell, K K Pohaku; Sandoval, S; Cortes-Mateos, M J; Alfaro, J G; Kummel, A C; Trogler, W C

2014-12-07

Iron(III)-doped silica nanoshells are shown to possess an in vitro cell-receptor mediated targeting functionality for endocytosis. Compared to plain silica nanoparticles, iron enriched ones are shown to be target-specific, a property that makes them potentially better vehicles for applications, such as drug delivery and tumor imaging, by making them more selective and thereby reducing the nanoparticle dose. Iron(III) in the nanoshells can interact with endogenous transferrin, a serum protein found in mammalian cell culture media, which subsequently promotes transport of the nanoshells into cells by the transferrin receptor-mediated endocytosis pathway. The enhanced uptake of the iron(III)-doped nanoshells relative to undoped silica nanoshells by a transferrin receptor-mediated pathway was established using fluorescence and confocal microscopy in an epithelial breast cancer cell line. This process was also confirmed using fluorescence activated cell sorting (FACS) measurements that show competitive blocking of nanoparticle uptake by added holo-transferrin.

Characterization of the Fluorescence Properties of 4-Dialkylaminochalcones and Investigation of the Cytotoxic Mechanism of Chalcones.

PubMed

Zhou, Bo; Jiang, Peixin; Lu, Junxuan; Xing, Chengguo

2016-07-01

Understanding the mechanisms responsible for the various biological activities of chalcones, particularly the direct cellular targets, presents an unmet challenge. Here, we prepared a series of fluorescent chalcone derivatives as chemical probes for their mechanistic investigation. Upon systematic physicochemical characterization, we explored their potential to elucidate the mode of action of chalcones' cytotoxicity. The fluorescence of the chalcones was found to be highly sensitive to structural and environmental factors. Structurally, a 4-dialkylamino group on the B ring, suitable electronic properties of the A ring substituents, and the planar conformation of the chalcone's core structure were essential for optimal fluorescence. Environmental factors influencing fluorescence included solvent polarity, pH, and the interactions of the chalcones with proteins and detergents. It was found that 18 chalcones showed a fluorescent brightness greater than 6000 M(-1)  cm(-1) in DMSO. However, water dramatically quenched the fluorescence, although it could be partially recovered in the presence of BSA or detergents. As expected, these fluorescent chalcones showed a sharp structure-activity relationship in their cellular cytotoxicity, leading to the identification of structurally similar cytotoxic and non-cytotoxic fluorescent chalcones as chemical probes. Confocal microscopy results revealed the co-localization of the cytotoxic probe C8 and tubulin in cells, supporting tubulin as the direct cellular target responsible for the cytotoxicity of chalcones. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous detection of Escherichia coli O157:H7 and Salmonella Typhimurium using quantum dots as fluorescence labels.

PubMed

Yang, Liju; Li, Yanbin

2006-03-01

In this study, we explored the use of semiconductor quantum dots (QDs) as fluorescence labels in immunoassays for simultaneous detection of two species of foodborne pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium. QDs with different sizes can be excited with a single wavelength of light, resulting in different emission peaks that can be measured simultaneously. Highly fluorescent semiconductor quantum dots with different emission wavelengths (525 nm and 705 nm) were conjugated to anti-E. coli O157 and anti-Salmonella antibodies, respectively. Target bacteria were separated from samples by using specific antibody coated magnetic beads. The bead-cell complexes reacted with QD-antibody conjugates to form bead-cell-QD complexes. Fluorescent microscopic images of QD labeled E. coli and Salmonella cells demonstrated that QD-antibody conjugates could evenly and completely attach to the surface of bacterial cells, indicating that the conjugated QD molecules still retain their effective fluorescence, while the conjugated antibody molecules remain active and are able to recognize their specific target bacteria in a complex mixture. The intensities of fluorescence emission peaks at 525 nm and 705 nm of the final complexes were measured for quantitative detection of E. coli O157:H7 and S. Typhimurium simultaneously. The fluorescence intensity (FI) as a function of cell number (N) was found for Salmonella and E. coli, respectively. The regression models can be expressed as: FI = 60.6 log N- 250.9 with R(2) = 0.97 for S. Typhimurium, and FI = 77.8 log N- 245.2 with R(2) = 0.91 for E. coli O157:H7 in the range of cell numbers from 10(4) to 10(7) cfu ml(-1). The detection limit of this method was 10(4) cfu ml(-1). The detection could be completed within 2 hours. The principle of this method could be extended to detect multiple species of bacteria (3-4 species) simultaneously, depending on the availability of each type of QD-antibody conjugates with a unique emission peak and the antibody coated magnetic beads specific to each species of bacteria.

Small-molecule photostabilizing agents are modifiers of lipid bilayer properties.

PubMed

Alejo, Jose L; Blanchard, Scott C; Andersen, Olaf S

2013-06-04

Small-molecule photostabilizing or protective agents (PAs) provide essential support for the stability demands on fluorescent dyes in single-molecule spectroscopy and fluorescence microscopy. These agents are employed also in studies of cell membranes and model systems mimicking lipid bilayer environments, but there is little information about their possible effects on membrane structure and physical properties. Given the impact of amphipathic small molecules on bilayer properties such as elasticity and intrinsic curvature, we investigated the effects of six commonly used PAs--cyclooctatetraene (COT), para-nitrobenzyl alcohol (NBA), Trolox (TX), 1,4-diazabicyclo[2.2.2]octane (DABCO), para-nitrobenzoic acid (pNBA), and n-propyl gallate (nPG)--on bilayer properties using a gramicidin A (gA)-based fluorescence quench assay to probe for PA-induced changes in the gramicidin monomer↔dimer equilibrium. The experiments were done using fluorophore-loaded large unilamellar vesicles that had been doped with gA, and changes in the gA monomer↔dimer equilibrium were assayed using a gA channel-permeable fluorescence quencher (Tl⁺). Changes in bilayer properties caused by, e.g., PA adsorption at the bilayer/solution interface that alter the equilibrium constant for gA channel formation, and thus the number of conducting gA channels in the large unilamellar vesicle membrane, will be detectable as changes in the rate of Tl⁺ influx-the fluorescence quench rate. Over the experimentally relevant millimolar concentration range, TX, NBA, and pNBA, caused comparable increases in gA channel activity. COT, also in the millimolar range, caused a slight decrease in gA channel activity. nPG increased channel activity at submillimolar concentrations. DABCO did not alter gA activity. Five of the six tested PAs thus alter lipid bilayer properties at experimentally relevant concentrations, which becomes important for the design and analysis of fluorescence studies in cells and model membrane systems. We therefore tested combinations of COT, NBA, and TX; the combinations altered the fluorescence quench rate less than would be predicted assuming their effects on bilayer properties were additive. The combination of equimolar concentrations of COT and NBA caused minimal changes in the fluorescence quench rate. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Imaging mycobacterial growth and division with a fluorogenic probe.

PubMed

Hodges, Heather L; Brown, Robert A; Crooks, John A; Weibel, Douglas B; Kiessling, Laura L

2018-05-15

Control and manipulation of bacterial populations requires an understanding of the factors that govern growth, division, and antibiotic action. Fluorescent and chemically reactive small molecule probes of cell envelope components can visualize these processes and advance our knowledge of cell envelope biosynthesis (e.g., peptidoglycan production). Still, fundamental gaps remain in our understanding of the spatial and temporal dynamics of cell envelope assembly. Previously described reporters require steps that limit their use to static imaging. Probes that can be used for real-time imaging would advance our understanding of cell envelope construction. To this end, we synthesized a fluorogenic probe that enables continuous live cell imaging in mycobacteria and related genera. This probe reports on the mycolyltransferases that assemble the mycolic acid membrane. This peptidoglycan-anchored bilayer-like assembly functions to protect these cells from antibiotics and host defenses. Our probe, quencher-trehalose-fluorophore (QTF), is an analog of the natural mycolyltransferase substrate. Mycolyltransferases process QTF by diverting their normal transesterification activity to hydrolysis, a process that unleashes fluorescence. QTF enables high contrast continuous imaging and the visualization of mycolyltransferase activity in cells. QTF revealed that mycolyltransferase activity is augmented before cell division and localized to the septa and cell poles, especially at the old pole. This observed localization suggests that mycolyltransferases are components of extracellular cell envelope assemblies, in analogy to the intracellular divisomes and polar elongation complexes. We anticipate QTF can be exploited to detect and monitor mycobacteria in physiologically relevant environments.

In vivo imaging of the dynamics of different variants of EGFR in glioblastomas.

PubMed

Shah, Khalid

2011-01-01

A number of altered pathways in cancer cells depend on growth factor receptors. The amplification/alteration of the epidermal growth factor receptor (EGFR) has been shown to play a significant role in enhancing tumor burden in a number of tumors, including malignant glioblastomas (GBM). To dissect the role of EGFR expression in tumor progression in mouse models of cancer and ultimately evaluate targeted therapies, it is necessary to visualize the dynamics of EGFR in real time in vivo. Non-invasive imaging based on quantitative and qualitative changes in light emission by fluorescent and bioluminescent markers offers a huge potential to facilitate drug development. Multiple approaches could be used to follow a molecular target or pathway with the fusion of a bioluminescent-fluorescent marker. This unit describes a protocol for simultaneously imaging EGFR activity and progression of GBM in a mouse model. Human glioma cells transduced with lentiviral vectors bearing different combinations of fluorescent and bioluminescent proteins either fused to EGFR or expressed alone can be grown as monolayers and maintained over several passages. The unit begins with a method for transducing glioma cells with lentiviral vectors for stable expression of these fluorescent and bioluminescent markers in vitro, followed by transplantation of engineered glioma cells in mice, and, finally, sequential bioluminescent imaging of EGFR expression and GBM progression in mice. The protocol details characterization of engineered glioma cells in culture, surgical preparation, craniotomy, cell implantation, animal recovery, and imaging procedures to study kinetics of EGFR expression and GBM progression.

NADPH oxidase 2 (NOX2) enzyme activation in patients with chronic inflammatory demyelinating polyneuropathy.

PubMed

Marrali, G; Salamone, P; Casale, F; Fuda, G; Cugnasco, P; Caorsi, C; Amoroso, A; Calvo, A; Lopiano, L; Cocito, D; Chiò, A

2016-05-01

Chronic inflammatory demyelinating polyneuropathy (CIDP) is an acquired immunomediated condition affecting the peripheral nervous system where probably macrophages are the primary effector cells for demyelination. Reactive oxygen species (ROS), catalyzed by the NOX family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes, can induce peroxidation and are potentially injurious to myelin. Our aim was to assess the activity of NOX2, an isoform of NOX, in a series of CIDP patients and to analyze the effect of intravenous immunoglobulin (IVIg) on NOX2. Thirty CIDP patients treated with IVIg and 30 control subjects were enrolled. To evaluate NOX2 activity, neutrophil and monocyte oxidative burst was measured directly in fresh whole blood using the Phagoburst™ assay, a fluorescence-activated cell sorting method. The mean fluorescence intensity, emitted in response to different stimuli, leads to the production of ROS and corresponds to the percentage of oxidizing cells and their enzymatic activity. Mean fluorescence intensity values for granulocyte and monocyte burst in patients (mean 633.3, SD 191; mean 111.8, SD 28.5) were different from those measured in healthy controls (granulocytes, mean 436.6, SD 137.0, P = 0.0003; monocytes, mean 78.2, SD 17.3, P = 0.000001). Moreover, IVIg administration increased both granulocyte (P = 0.005) and monocyte (P = 0.0009) burst. Our findings demonstrate that oxidative burst is significantly increased in CIDP patients and that treatment with IVIg enhances oxidative values, thus representing a possible IVIg therapeutic effect linked to a regulatory effect of ROS. Based on this, the development of treatments targeting the specific activation of NOX may be beneficial in autoimmune disorders. © 2016 EAN.

[First attempts of detecting fetal cells in the maternal circulation].

PubMed

Nagy, Gyula Richárd; Bán, Zoltán; Sipos, Ferenc; Fent, János; Oroszné Nagy, Judit; Beke, Artúr; Furész, József; Papp, Zoltán

2004-10-31

In prenatal diagnosis there is great interest for noninvasive diagnostic methods. Authors report their first results in detecting fetal cells in the maternal circulation during pregnancy. The aim of the study was to detect fetal gender from maternal peripheral blood samples during pregnancy. Authors have analysed fetal nucleated red blood cells. In 12 cases after a double density Percoll gradient separation they labelled the surface antigens of the cells with anti-glycophorin-A and anti-CD45 fluorescent antibodies, did an intracellular staining of the epsilon haemoglobin chain, and analysed the cells with flow cytometry. The CD45 negative/glycophorin-A positive/epsilon-haemoglobin chain positive cells were considered as fetal cells. Having the results, in another 13 cases magnetic activated cell sorting with CD71 antibody were used as an enrichment step. Authors made an intracellular staining of the epsilon haemoglobin chain, the positive cells were isolated by micromanipulation, and analysed by single cell fluorescent polymerase chain reaction. Primers for the amelogenin gene were used to detect fetal gender. Only the Percoll enrichment step itself is not enough for using the samples for diagnostic molecular-biologic examinations, a following enrichment step is needed. For this the authors used magnetic activated cell sorting with CD71 antibody. With the help of this enrichment step, after the intracellular staining of the epsilon haemoglobin chain the direct micromanipulator isolation of the epsilon haemoglobin chain positive cells could be done. After analysing single cells by fluorescent polymerase chain reaction, in 8 out of the 11 comparable cases the results were similar to those, what was found during the genetic amniocentesis. In 2 cases from this 8, genetic amniocentesis proved Klinefelter syndrome, which they could also confirm with the examination of fetal cells in the maternal circulation. The results of the study suggest that the method described above can be useful in prenatal genetic diagnosis, and improving it could be useful to detect other genetic abnormalities (chromosomal abnormalities, single gene disorders) as well.

Cryo-imaging of fluorescently labeled single cells in a mouse

NASA Astrophysics Data System (ADS)

Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

2009-02-01

We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron-scale, fluorescence, and bright field image data. Here we describe our image preprocessing, analysis, and visualization techniques. Processing improves axial resolution, reduces subsurface fluorescence by 97%, and enables single cell detection and counting. High quality 3D volume renderings enable us to evaluate cell distribution patterns. Applications include the myriad of biomedical experiments using fluorescent reporter gene and exogenous fluorophore labeling of cells in applications such as stem cell regenerative medicine, cancer, tissue engineering, etc.