Comparison of the Second-Generation Digene Hybrid Capture Assay with the Branched-DNA Assay for Measurement of Hepatitis B Virus DNA in Serum

PubMed Central

Ho, Stephen K. N.; Chan, Tak Mao; Cheng, Ignatius K. P.; Lai, Kar Neng

1999-01-01

The optimal hepatitis B virus (HBV) DNA quantitative assay for clinical use remains to be determined. We examined the sensitivity, linearity, and variability of a novel second-generation antibody capture solution hybridization assay, the Digene Hybrid Capture II assay (HCII), and compared it with another widely used solution hybridization assay, the branched-DNA (bDNA) assay (Quantiplex; Chiron Corp.). Our results showed similar and satisfactory assay linearity values, as well as interassay and intra-assay variability values, for both HCII and bDNA assays across different ranges of HBV DNA. Ninety-one percent of 102 serum samples from hepatitis B surface antigen-positive patients showed concordant results with the two assays. The HCII assay was more sensitive than the bDNA assay by 1 dilution, with the lowest reading being 0.9 pg/ml (3.8 pg/ml by bDNA assay). The HBV DNA seropositivity rates for the 102 samples were 58, 67, and 97% by bDNA, HCII, and nested PCR, respectively. While the relationship between results obtained with the bDNA assay and those with the HCII assay was nonlinear, with the bDNA assay yielding values 2.83 ± 0.92-fold higher than those of the HCII assay, especially at high HBV DNA levels, a linear relationship was observed between the two sets of data after logarithmic conversion. The formula for interassay conversion of results was derived as follows: HBV DNA by HCII (picograms per milliliter) = 3.19 × [HBV DNA by bDNA (megaequivalents per milliliter)]0.866. The HCII assay was technically less complex and required a shorter assay time (4 h) than the bDNA assay (24 h). We conclude that the HCII assay compares favorably with the bDNA assay and offers the additional advantages of increased sensitivity and shorter assay time. The increased sensitivity should be particularly useful in monitoring the efficacy of antiviral therapies and detecting the emergence of drug-resistant HBV mutants. PMID:10405385

Comparison of the second-generation digene hybrid capture assay with the branched-DNA assay for measurement of hepatitis B virus DNA in serum.

PubMed

Ho, S K; Chan, T M; Cheng, I K; Lai, K N

1999-08-01

The optimal hepatitis B virus (HBV) DNA quantitative assay for clinical use remains to be determined. We examined the sensitivity, linearity, and variability of a novel second-generation antibody capture solution hybridization assay, the Digene Hybrid Capture II assay (HCII), and compared it with another widely used solution hybridization assay, the branched-DNA (bDNA) assay (Quantiplex; Chiron Corp.). Our results showed similar and satisfactory assay linearity values, as well as interassay and intra-assay variability values, for both HCII and bDNA assays across different ranges of HBV DNA. Ninety-one percent of 102 serum samples from hepatitis B surface antigen-positive patients showed concordant results with the two assays. The HCII assay was more sensitive than the bDNA assay by 1 dilution, with the lowest reading being 0.9 pg/ml (3.8 pg/ml by bDNA assay). The HBV DNA seropositivity rates for the 102 samples were 58, 67, and 97% by bDNA, HCII, and nested PCR, respectively. While the relationship between results obtained with the bDNA assay and those with the HCII assay was nonlinear, with the bDNA assay yielding values 2.83 +/- 0.92-fold higher than those of the HCII assay, especially at high HBV DNA levels, a linear relationship was observed between the two sets of data after logarithmic conversion. The formula for interassay conversion of results was derived as follows: HBV DNA by HCII (picograms per milliliter) = 3.19 x [HBV DNA by bDNA (megaequivalents per milliliter)](0.866). The HCII assay was technically less complex and required a shorter assay time (4 h) than the bDNA assay (24 h). We conclude that the HCII assay compares favorably with the bDNA assay and offers the additional advantages of increased sensitivity and shorter assay time. The increased sensitivity should be particularly useful in monitoring the efficacy of antiviral therapies and detecting the emergence of drug-resistant HBV mutants.

Mkit: A Cell Migration Assay Based on Microfluidic Device and Smartphone

PubMed Central

Yang, Ke; Wu, Jiandong; Peretz-Soroka, Hagit; Zhu, Ling; Li, Zhigang; Sang, Yaoshuo; Hipolito, Jolly; Zhang, Michael; Santos, Susy; Hillier, Craig; de Faria, Ricardo Lobato; Liu, Yong; Lin, Francis

2017-01-01

Mobile sensing based on the integration of microfluidic device and smartphone, so-called MS2 technology, has enabled many applications over recent years, and continues to stimulate growing interest in both research communities and industries. In particular, it has been envisioned that MS2 technology can be developed for various cell functional assays to enable basic research and clinical applications. Toward this direction, in this paper, we describe the development of a MS2-based cell functional assay for testing cell migration (the Mkit). The system is constructed as an integrated test kit, which includes microfluidic chips, a smartphone-based imaging platform, the phone apps for image capturing and data analysis, and a set of reagent and accessories for performing the cell migration assay. We demonstrated that the Mkit can effectively measure purified neutrophil and cancer cell chemotaxis. Furthermore, neutrophil chemotaxis can be tested from a drop of whole blood using the Mkit with red blood cell (RBC) lysis. The effects of chemoattractant dose and gradient profile on neutrophil chemotaxis were also tested using the Mkit. In addition to research applications, we demonstrated the effective use of the Mkit for on-site test at the hospital and for testing clinical samples from chronic obstructive pulmonary disease patient. Thus, this developed Mkit provides an easy and integrated experimental platform for cell migration related research and potential medical diagnostic applications. PMID:28772229

Quantification of cytomegalovirus (CMV) viral load by the hybrid capture assay allows for early detection of CMV disease in lung transplant recipients.

PubMed

Bhorade, S M; Sandesara, C; Garrity, E R; Vigneswaran, W T; Norwick, L; Alkan, S; Husain, A N; McCabe, M A; Yeldandi, V

2001-09-01

We prospectively compared the hybrid capture system (HCS) assay with conventional cell culture and shell vial assay for the detection of cytomegalovirus (CMV) infection and disease in the lung transplant population. Between January 1999 and February 2000, 34 lung transplant patients at Loyola University Medical Center, who were considered to be at risk for CMV disease, underwent surveillance testing for CMV cell culture, shell vial assay and HCS assay according to a pre-determined schedule. In addition, bronchoscopy with bronchoalveolar lavage (BAL) and transbronchial biopsy were performed at regular intervals and for clinical indications. All BAL samples were sent for CMV cultures and biopsy specimens were analyzed for histopathologic evidence of CMV by immunoperoxidase staining using antibody to early immediate nuclear antigen. Ten patients developed CMV disease/syndrome during the course of the study. The sensitivity, specificity, positive predictive value and negative predictive value were >90% for the HCS assay. The sensitivity of the HCS assay (90%) was statistically significantly higher than the sensitivity of either the SV assay (40%) or the cell culture (50%). In addition, the HCS assay was able to detect CMV 50 +/- 67 days prior to clinical evidence of CMV disease and an average of 36 days prior to the other detection techniques. The HCS assay is a sensitive diagnostic technique able to reliably detect CMV disease earlier than other diagnostic methods in the lung transplant population. Future studies may be able to evaluate whether pre-emptive anti-viral therapy targeted to specific viral loads using the HCS assay will be beneficial in preventing morbidity associated with CMV disease.

A lateral electrophoretic flow diagnostic assay

PubMed Central

Lin, Robert; Skandarajah, Arunan; Gerver, Rachel E.; Neira, Hector D.; Fletcher, Daniel A.

2015-01-01

Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a “lateral e-flow assay” and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to selectively immobilize unmodified capture antigen along the microchannel in a barcode-like pattern. The channel-filling polyacrylamide hydrogel incorporates a photoactive moiety (benzophenone) to immobilize capture antigen to the hydrogel without a priori antigen modification. We report a heterogeneous sandwich assay using low-power electrophoresis to drive biospecimen through the capture antigen barcode. Fluorescence barcode readout is collected via a low-resource appropriate imaging system (CellScope). We characterize lateral e-flow assay performance and demonstrate a serum assay for antibodies to the hepatitis C virus (HCV). In a pilot study, the lateral e-flow assay positively identifies HCV+ human sera in 60 min. The lateral e-flow assay provides a flexible format for conducting multiplexed immunoassays relevant to confirmatory diagnosis in near-patient settings. PMID:25608872

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

PubMed Central

Baker, Harold N.; Murphy, Robin; Lopez, Erica; Garcia, Carlos

2012-01-01

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results. In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample. Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and

Mkit: A cell migration assay based on microfluidic device and smartphone.

PubMed

Yang, Ke; Wu, Jiandong; Peretz-Soroka, Hagit; Zhu, Ling; Li, Zhigang; Sang, Yaoshuo; Hipolito, Jolly; Zhang, Michael; Santos, Susy; Hillier, Craig; de Faria, Ricardo Lobato; Liu, Yong; Lin, Francis

2018-01-15

Mobile sensing based on the integration of microfluidic device and smartphone, so-called MS 2 technology, has enabled many applications over recent years, and continues to stimulate growing interest in both research communities and industries. In particular, it has been envisioned that MS 2 technology can be developed for various cell functional assays to enable basic research and clinical applications. Toward this direction, in this paper, we describe the development of a MS 2 -based cell functional assay for testing cell migration (the M kit ). The system is constructed as an integrated test kit, which includes microfluidic chips, a smartphone-based imaging platform, the phone apps for image capturing and data analysis, and a set of reagent and accessories for performing the cell migration assay. We demonstrated that the M kit can effectively measure purified neutrophil and cancer cell chemotaxis. Furthermore, neutrophil chemotaxis can be tested from a drop of whole blood using the M kit with red blood cell (RBC) lysis. The effects of chemoattractant dose and gradient profile on neutrophil chemotaxis were also tested using the M kit . In addition to research applications, we demonstrated the effective use of the M kit for on-site test at the hospital and for testing clinical samples from chronic obstructive pulmonary disease patient. Thus, this developed M kit provides an easy and integrated experimental platform for cell migration related research and potential medical diagnostic applications. Copyright © 2017 Elsevier B.V. All rights reserved.

The stability of prostatic acid phosphatase, as measured by a capture immunoenzyme assay.

PubMed

Lin, T M; Chin-See, M W; Halbert, S P

1984-03-27

A capture immunoenzyme assay (CIEA) for prostatic acid phosphatase (PAP) was developed and used to study the stability of this isoenzyme. Immunospecifically purified goat antibodies to PAP were covalently bound to special discs and used to capture the enzyme in serum samples in a weakly acidic medium during the first incubation (2 h) at 37 degrees C. The capture enzyme was then measured by its catalytic activity with p-nitrophenyl phosphate as substrate during the second incubation (1 h) at 37 degrees C. As much as 98% of the PAP in test specimens was captured and measured by this CIEA. The test results were expressed as enzymatic activity (U/l), extrapolated from a standard curve which was linear between 0.026 and 70 U/l. In test sera stored at 4 degrees C, the PAP was variably stable for 7 to 70 days, but the enzyme was quite stable in serum when stored at -20 degrees C for at least 156 days. At room temperature, when the sera were appropriately acidified, there was no loss of enzymatic activity for periods of 15 days, and in some cases, a large proportion of activity was still intact after 70 days. At 4 degrees C, as well as -20 degrees C, acidified serum and the partially purified PAP standard showed complete stability for at least 7 months. The CIEA reactivity of positive test specimens was inhibited by L(+)-tartaric acid, but not by cupric sulfate. The acid phosphatases of blood cell extracts were non-reactive in the CIEA procedure. The CIEA results of 224 serum samples from patients with and without prostate cancer correlated very well with those obtained by two direct enzymatic and two commercial RIA procedures, with correlation coefficients between 0.960 and 0.993, and diagnostic agreement between 86% and 100%.

Scaling and automation of a high-throughput single-cell-derived tumor sphere assay chip.

PubMed

Cheng, Yu-Heng; Chen, Yu-Chih; Brien, Riley; Yoon, Euisik

2016-10-07

Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1-5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies. In this study, we present the scaling and automation of high-throughput single-cell-derived tumor sphere assay chips, facilitating the tracking of up to ∼10 000 cells on a chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single-cells with a variety of fluorescent intensities, a highly adaptable analysis program was developed for cell/sphere counting and size measurement. Using a Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment was created to test a controversial hypothesis: whether larger or smaller cells are more stem-like defined by the capability to form single-cell-derived spheres. Different cell lines showed different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, we identified heterogeneity in sphere growth dynamics, indicating the cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high-throughput and automation in CSC studies.

Detecting West Nile virus in owls and raptors by an antigen-capture assay.

PubMed

Gancz, Ady Y; Campbell, Douglas G; Barker, Ian K; Lindsay, Robbin; Hunter, Bruce

2004-12-01

We evaluated a rapid antigen-capture assay (VecTest) for detection of West Nile virus in oropharyngeal and cloacal swabs, collected at necropsy from owls (N = 93) and raptors (N = 27). Sensitivity was 93.5%-95.2% for northern owl species but <42.9% for all other species. Specificity was 100% for owls and 85.7% for raptors.

Improved Methods for Capture, Extraction, and Quantitative Assay of Environmental DNA from Asian Bigheaded Carp (Hypophthalmichthys spp.)

PubMed Central

Turner, Cameron R.; Miller, Derryl J.; Coyne, Kathryn J.; Corush, Joel

2014-01-01

Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species. PMID:25474207

Improved methods for capture, extraction, and quantitative assay of environmental DNA from Asian bigheaded carp (Hypophthalmichthys spp.).

PubMed

Turner, Cameron R; Miller, Derryl J; Coyne, Kathryn J; Corush, Joel

2014-01-01

Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.

Capturing CD4 cells using a functionalized circular microfluidic device and glutaraldehyde as biolinker for tuberculosis detection and diagnosis

NASA Astrophysics Data System (ADS)

Shih, Yeu-Farn; Huang, Nien-Tsu; Lee, Chih-Kung

2015-03-01

It is estimated that about one-third of the world's population has already been infected by tuberculosis. Mycobacterium tuberculosis, in general, can result in an active case of tuberculosis in approximately 5%-10% of those who suffer from latent tuberculosis and the chance of becoming ill is the highest within one of year of getting the disease. Although a newly developed methods called interferon gamma release assay (IGRA) can monitor CD4 cells secreted cytokine to diagnose tuberculosis (TB) condition. However, it is difficult to count total numbers of cytokine secreted CD4 cells, which make the diagnosis less accurate. Therefore, we develop a functionalized polydimethylsiloxane (PDMS) device using glutaraldehyde to capture CD4 cells. To enhance the capture efficiency, we use COMSOL simulation to optimize the arrangement of PDMS micro pillars to make cells uniformly distributed in the device. Our preliminary data showed the microfluidic configuration in a circular shape with HCP patterned micro pillars turned 30 degrees offers the highest cell capture rate.

Peptide-based protein capture agents with high affinity, selectivity, and stability as antibody replacements in biodetection assays

NASA Astrophysics Data System (ADS)

Coppock, Matthew B.; Farrow, Blake; Warner, Candice; Finch, Amethist S.; Lai, Bert; Sarkes, Deborah A.; Heath, James R.; Stratis-Cullum, Dimitra

2014-05-01

Current biodetection assays that employ monoclonal antibodies as primary capture agents exhibit limited fieldability, shelf life, and performance due to batch-to-batch production variability and restricted thermal stability. In order to improve upon the detection of biological threats in fieldable assays and systems for the Army, we are investigating protein catalyzed capture (PCC) agents as drop-in replacements for the existing antibody technology through iterative in situ click chemistry. The PCC agent oligopeptides are developed against known protein epitopes and can be mass produced using robotic methods. In this work, a PCC agent under development will be discussed. The performance, including affinity, selectivity, and stability of the capture agent technology, is analyzed by immunoprecipitation, western blotting, and ELISA experiments. The oligopeptide demonstrates superb selectivity coupled with high affinity through multi-ligand design, and improved thermal, chemical, and biochemical stability due to non-natural amino acid PCC agent design.

Detecting West Nile Virus in Owls and Raptors by an Antigen-capture Assay

PubMed Central

Campbell, Douglas G.; Barker, Ian K.; Lindsay, Robbin; Hunter, Bruce

2004-01-01

We evaluated a rapid antigen-capture assay (VecTest) for detection of West Nile virus in oropharyngeal and cloacal swabs, collected at necropsy from owls (N = 93) and raptors (N = 27). Sensitivity was 93.5%–95.2% for northern owl species but <42.9% for all other species. Specificity was 100% for owls and 85.7% for raptors. PMID:15663862

Development of a quantitative NS1-capture enzyme-linked immunosorbent assay for early detection of yellow fever virus infection.

PubMed

Ricciardi-Jorge, Taissa; Bordignon, Juliano; Koishi, Andrea; Zanluca, Camila; Mosimann, Ana Luiza; Duarte Dos Santos, Claudia Nunes

2017-11-24

Yellow fever is an arboviral disease that causes thousands of deaths every year in Africa and the Americas. However, few commercial diagnostic kits are available. Non-structural protein 1 (NS1) is an early marker of several flavivirus infections and is widely used to diagnose dengue virus (DENV) infection. Nonetheless, little is known about the dynamics of Yellow fever virus (YFV) NS1 expression and secretion, to encourage its use in diagnosis. To tackle this issue, we developed a quantitative NS1-capture ELISA specific for YFV using a monoclonal antibody and recombinant NS1 protein. This test was used to quantify NS1 in mosquito and human cell line cultures infected with vaccine and wild YFV strains. Our results showed that NS1 was detectable in the culture supernatants of both cell lines; however, a higher concentration was maintained as cell-associated rather than secreted into the extracellular milieu. A panel of 73 human samples was used to demonstrate the suitability of YFV NS1 as a diagnostic tool, resulting in 80% sensitivity, 100% specificity, a 100% positive predictive value and a 95.5% negative predictive value compared with RT-PCR. Overall, the developed NS1-capture ELISA showed potential as a promising assay for the detection of early YF infection.

Capture of mesothelioma cells with 'universal' CTC-chip.

PubMed

Yoneda, Kazue; Chikaishi, Yasuhiro; Kuwata, Taiji; Ohnaga, Takashi; Tanaka, Fumihiro

2018-02-01

Malignant mesothelioma (MM) is a highly aggressive malignant tumor, predominantly associated with job-related exposure to asbestos. Development of effective and non-invasive modalities for diagnosis is an important issue in occupational medicine. Circulating tumor cells (CTCs), which are tumor cells that are shed from primary tumors and circulate in the peripheral blood, may be detected at an earlier stage than malignant tumors, and detection of CTCs may provide a novel insight into the diagnosis of MM. In a previous study evaluating clinical utility of CTCs, detected with a widely used system 'CellSearch', the authors indicated a significant however insufficient capability in the diagnosis of MM, suggesting need for a more sensitive system. Accordingly, the authors developed a novel microfluidic system to capture CTCs (CTC-chip), and demonstrated that the CTC-chip effectively captured MM cells (ACC-MESO-4) spiked in the blood by conjugating an anti-podoplanin antibody. The results of the present study demonstrated that the CTC-chip coated with the anti-podoplanin antibody captured another MM cell (ACC-MESO-1). However, the capture efficiencies were lower than those for ACC-MESO-4. In addition, an anti-mesothelin antibody was used to capture CTCs, however the CTC-chip coated with the anti-mesothelin antibody failed to effectively capture MM cells, possibly due to low mesothelin expression. Overall, the CTC-chip may capture specific types of CTCs by conjugating any antibody against an antigen expressed on CTCs, and may be a useful system for the diagnosis of malignant tumors, including MM.

Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses.

PubMed

Almeida, Coral-Ann M; Roberts, Steven G; Laird, Rebecca; McKinnon, Elizabeth; Ahmed, Imran; Pfafferott, Katja; Turley, Joanne; Keane, Niamh M; Lucas, Andrew; Rushton, Ben; Chopra, Abha; Mallal, Simon; John, Mina

2009-05-15

The enzyme linked immunospot (ELISpot) assay is a fundamental tool in cellular immunology, providing both quantitative and qualitative information on cellular cytokine responses to defined antigens. It enables the comprehensive screening of patient derived peripheral blood mononuclear cells to reveal the antigenic restriction of T-cell responses and is an emerging technique in clinical laboratory investigation of certain infectious diseases. As with all cellular-based assays, the final results of the assay are dependent on a number of technical variables that may impact precision if not highly standardised between operators. When studies that are large scale or using multiple antigens are set up manually, these assays may be labour intensive, have many manual handling steps, are subject to data and sample integrity failure and may show large inter-operator variability. Here we describe the successful automated performance of the interferon (IFN)-gamma ELISpot assay from cell counting through to electronic capture of cytokine quantitation and present the results of a comparison between automated and manual performance of the ELISpot assay. The mean number of spot forming units enumerated by both methods for limiting dilutions of CMV, EBV and influenza (CEF)-derived peptides in six healthy individuals were highly correlated (r>0.83, p<0.05). The precision results from the automated system compared favourably with the manual ELISpot and further ensured electronic tracking, increased through-put and reduced turnaround time.

Standardization of a cytometric p24-capture bead-assay for the detection of main HIV-1 subtypes.

PubMed

Merbah, Mélanie; Onkar, Sayali; Grivel, Jean-Charles; Vanpouille, Christophe; Biancotto, Angélique; Bonar, Lydia; Sanders-Buell, Eric; Kijak, Gustavo; Michael, Nelson; Robb, Merlin; Kim, Jerome H; Tovanabutra, Sodsai; Chenine, Agnès-Laurence

2016-04-01

The prevailing method to assess HIV-1 replication and infectivity is to measure the production of p24 Gag protein by enzyme-linked immunosorbent assay (ELISA). Since fluorescent bead-based technologies offer a broader dynamic range and higher sensitivity, this study describes a p24 capture Luminex assay capable of detecting HIV-1 subtypes A-D, circulating recombinant forms (CRF) CRF01_AE and CRF02_AG, which together are responsible for over 90% of HIV-1 infections worldwide. The success of the assay lies in the identification and selection of a cross-reactive capture antibody (clone 183-H12-5C). Fifty-six isolates that belonged to six HIV-1 subtypes and CRFs were successfully detected with p-values below 0.021; limits of detection ranging from 3.7 to 3 × 104 pg/ml. The intra- and inter-assay variation gave coefficient of variations below 6 and 14%, respectively. The 183-bead Luminex assay also displayed higher sensitivity of 91% and 98% compared to commercial p24 ELISA and a previously described Luminex assay. The p24 concentrations measured by the 183-bead Luminex assay showed a significant correlation (R=0.92, p<0.0001) with the data obtained from quantitative real time PCR. This newly developed p24 assay leverages the advantages of the Luminex platform, which include smaller sample volume and simultaneous detection of up to 500 analytes in a single sample, and delivers a valuable tool for the field. Copyright © 2016 Elsevier B.V. All rights reserved.

Aptamer-aided target capturing with biocatalytic metal deposition: an electrochemical platform for sensitive detection of cancer cells.

PubMed

Yi, Zi; Li, Xiao-Yan; Gao, Qing; Tang, Li-Juan; Chu, Xia

2013-04-07

A novel aptamer biosensor for cancer cell assay has been reported on the basis of ultrasensitive electrochemical detection. Cancer cell capturing is first accomplished via aptamer-aided recognition, and the cell-aptamer binding events then mediate an alkaline phosphatase-catalyzed silver deposition reaction which can be probed by electrochemical detection. Following biocatalytic silver deposition, an efficient amplification approach for sensitive electrochemical measurements is demonstrated, for cell detection with high sensitivity. Ramos cell are used as a model case, a typical biomarker of the acute blood cell cancer, Burkitt's lymphoma. The results reveal that the developed technique displays desirable selectivity in Ramos cell discrimination, and linear response range from 10 to 10(6) cells with a detection limit as low as 10 cells. Due to the simple procedures, label-free and electrochemistry based detection format, this technique is simple and cost-effective, and exhibits excellent compatibility with miniaturization technologies. The electrochemical cell detection strategy may create an intrinsically specific and sensitive platform for cancer cell assay and associated studies.

Magnetizable stent-grafts enable endothelial cell capture

NASA Astrophysics Data System (ADS)

Tefft, Brandon J.; Uthamaraj, Susheil; Harburn, J. Jonathan; Hlinomaz, Ota; Lerman, Amir; Dragomir-Daescu, Dan; Sandhu, Gurpreet S.

2017-04-01

Emerging nanotechnologies have enabled the use of magnetic forces to guide the movement of magnetically-labeled cells, drugs, and other therapeutic agents. Endothelial cells labeled with superparamagnetic iron oxide nanoparticles (SPION) have previously been captured on the surface of magnetizable 2205 duplex stainless steel stents in a porcine coronary implantation model. Recently, we have coated these stents with electrospun polyurethane nanofibers to fabricate prototype stent-grafts. Facilitated endothelialization may help improve the healing of arteries treated with stent-grafts, reduce the risk of thrombosis and restenosis, and enable small-caliber applications. When placed in a SPION-labeled endothelial cell suspension in the presence of an external magnetic field, magnetized stent-grafts successfully captured cells to the surface regions adjacent to the stent struts. Implantation within the coronary circulation of pigs (n=13) followed immediately by SPION-labeled autologous endothelial cell delivery resulted in widely patent devices with a thin, uniform neointima and no signs of thrombosis or inflammation at 7 days. Furthermore, the magnetized stent-grafts successfully captured and retained SPION-labeled endothelial cells to select regions adjacent to stent struts and between stent struts, whereas the non-magnetized control stent-grafts did not. Early results with these prototype devices are encouraging and further refinements will be necessary in order to achieve more uniform cell capture and complete endothelialization. Once optimized, this approach may lead to more rapid and complete healing of vascular stent-grafts with a concomitant improvement in long-term device performance.

Magnetizable stent-grafts enable endothelial cell capture.

PubMed

Tefft, Brandon J; Uthamaraj, Susheil; Harburn, J Jonathan; Hlinomaz, Ota; Lerman, Amir; Dragomir-Daescu, Dan; Sandhu, Gurpreet S

2017-04-01

Emerging nanotechnologies have enabled the use of magnetic forces to guide the movement of magnetically-labeled cells, drugs, and other therapeutic agents. Endothelial cells labeled with superparamagnetic iron oxide nanoparticles (SPION) have previously been captured on the surface of magnetizable 2205 duplex stainless steel stents in a porcine coronary implantation model. Recently, we have coated these stents with electrospun polyurethane nanofibers to fabricate prototype stent-grafts. Facilitated endothelialization may help improve the healing of arteries treated with stent-grafts, reduce the risk of thrombosis and restenosis, and enable small-caliber applications. When placed in a SPION-labeled endothelial cell suspension in the presence of an external magnetic field, magnetized stent-grafts successfully captured cells to the surface regions adjacent to the stent struts. Implantation within the coronary circulation of pigs (n=13) followed immediately by SPION-labeled autologous endothelial cell delivery resulted in widely patent devices with a thin, uniform neointima and no signs of thrombosis or inflammation at 7 days. Furthermore, the magnetized stent-grafts successfully captured and retained SPION-labeled endothelial cells to select regions adjacent to stent struts and between stent struts, whereas the non-magnetized control stent-grafts did not. Early results with these prototype devices are encouraging and further refinements will be necessary in order to achieve more uniform cell capture and complete endothelialization. Once optimized, this approach may lead to more rapid and complete healing of vascular stent-grafts with a concomitant improvement in long-term device performance.

Microfluidic chip for isolation of viable circulating tumor cells of hepatocellular carcinoma for their culture and drug sensitivity assay.

PubMed

Zhang, Yu; Zhang, Xiaofeng; Zhang, Jinling; Sun, Bin; Zheng, Lulu; Li, Jun; Liu, Sixiu; Sui, Guodong; Yin, Zhengfeng

2016-11-01

Circulating tumor cells (CTCs) have been proposed to be an active source of metastasis or recurrence of hepatocellular carcinoma (HCC). The enumeration and characterization of CTCs has important clinical significance in recurrence prediction and treatment monitoring in HCC patients. We previously developed a unique method to separate HCC CTCs based on the interaction of the asialoglycoprotein receptor (ASGPR) expressed on their membranes with its ligand. The current study applied the ligand-receptor binding assay to a CTC-chip in a microfluidic device. Efficient capture of HCC CTCs originates from the small dimensions of microfluidic channels and enhanced local topographic interactions between the microfluidic channel and extracellular extensions. With the optimized conditions, a capture yield reached > 85% for artificial CTC blood samples. Clinical utility of the system was further validated. CTCs were detected in all the examined 36 patients with HCC, with an average of 14 ± 10/2 mL. On the contrary, no CTCs were detected in healthy, benign liver disease or non-HCC cancer subjects. The current study also successfully demonstrated that the captured CTCs on our CTC-chip were readily released with ethylene diamine tetraacetic acid (EDTA); released CTCs remained alive and could be expanded to form a spheroid-like structure in a 3-dimensional cell culture assay; furthermore, sensitivity of released CTCs to chemotherapeutic agents (sorafenib or oxaliplatin) could be effectively tested utilizing this culture assay. In conclusion, the methodologies presented here offer great promise for accurate enumeration and easy release of captured CTCs, and released CTCs could be cultured for further functional studies.

Penetration of multiple thin films in micrometeorite capture cells

NASA Technical Reports Server (NTRS)

Simon, Charles G.

1994-01-01

As part of a continuing effort to develop cosmic dust detectors/collectors for use in space, we performed a series of hypervelocity impact experiments on combined sensor/capture-cell assemblies using 10-200-micron-diameter glass projectiles and olivine crystals at velocities of 0.9-14.4 km/s. The design objective of the space-flight instrument is to measure the trajectories of individual particles with sufficient accuracy to permit identification of their parent bodies and to capture enough impactor material to allow chemical and isotopic analyses of samples returned to Earth. Three different multiple-film small-particle capture cell designs (0.1-100-micron-thick Al foils with approx. 10, 100, and 1800 micron spacing) were evaluated for their ability to capture impactor fragments and residue. Their performances were compared to two other types of capture cells, foil covered Ge crystals, and 0.50 and 0.120 g/cu cm aerogels. All capture cells were tested behind multifilm (1.4-6.0-micron-thick) polyvinylidene fluoride (PVDF) velocity/trajectory sensor devices. Several tests were also done without the PVDF sensors for comparison. The results of this study were reported by Simon in a comprehensive report in which the morphology of impacts and impactor residues in various types of capture cells after passage through two PVDF sensor films is discussed. Impactor fragments in selected capture cells from impacts at velocities up to 6.4 km/s were identified using scanning electron microscopy with energy dispersive spectroscopy (SEM/EDS).

Particle Capture Devices and Methods of Use Thereof

NASA Technical Reports Server (NTRS)

Voldman, Joel (Inventor); Skelley, Alison M. (Inventor); Kirak, Oktay (Inventor); Jaenisch, Rudolf (Inventor)

2015-01-01

The present invention provides a device and methods of use thereof in microscale particle capturing and particle pairing. This invention provides particle patterning device, which mechanically traps individual particles within first chambers of capture units, transfer the particles to second chambers of opposing capture units, and traps a second type of particle in the same second chamber. The device and methods allow for high yield assaying of trapped cells, high yield fusion of trapped, paired cells, for controlled binding of particles to cells and for specific chemical reactions between particle interfaces and particle contents. The device and method provide means of identification of the particle population and a facile route to particle collection.

Effects of nanopillar array diameter and spacing on cancer cell capture and cell behaviors

NASA Astrophysics Data System (ADS)

Wang, Shunqiang; Wan, Yuan; Liu, Yaling

2014-10-01

While substrates with nanopillars (NPs) have emerged as promising platforms for isolation of circulating tumor cells (CTCs), the influence of diameter and spacing of NPs on CTC capture is still unclear. In this paper, CTC-capture yield and cell behaviors have been investigated by using antibody functionalized NPs of various diameters (120-1100 nm) and spacings (35-800 nm). The results show a linear relationship between the cell capture yield and effective contact area of NP substrates where a NP array of small diameter and reasonable spacing is preferred; however, spacing that is too small or too large adversely impairs the capture efficiency and specificity, respectively. In addition, the formation of pseudopodia between captured cells and the substrate is found to be dependent not only on cell adhesion status but also on elution strength and shear direction. These findings provide essential guidance in designing NP substrates for more efficient capture of CTCs and manipulation of cytomorphology in future.While substrates with nanopillars (NPs) have emerged as promising platforms for isolation of circulating tumor cells (CTCs), the influence of diameter and spacing of NPs on CTC capture is still unclear. In this paper, CTC-capture yield and cell behaviors have been investigated by using antibody functionalized NPs of various diameters (120-1100 nm) and spacings (35-800 nm). The results show a linear relationship between the cell capture yield and effective contact area of NP substrates where a NP array of small diameter and reasonable spacing is preferred; however, spacing that is too small or too large adversely impairs the capture efficiency and specificity, respectively. In addition, the formation of pseudopodia between captured cells and the substrate is found to be dependent not only on cell adhesion status but also on elution strength and shear direction. These findings provide essential guidance in designing NP substrates for more efficient capture of CTCs

Nanobiotechnology for the capture and manipulation of circulating tumor cells.

PubMed

Hughes, Andrew D; King, Michael R

2012-01-01

A necessary step in metastasis is the dissemination of malignant cells into the bloodstream, where cancer cells travel throughout the body as circulating tumor cells (CTC) in search of an opportunity to seed a secondary tumor. CTC represent a valuable diagnostic tool: evidence indicates that the quantity of CTC in the blood has been shown to relate to the severity of the illness, and samples are readily obtained through routine blood draws. As such, there has been a push toward developing technologies to reliably detect CTC using a variety of molecular and immunocytochemical techniques. In addition to their use in diagnostics, CTC detection systems that isolate CTC in such a way that the cells remain viable will allow for the performance of live-cell assays to facilitate the development of personalized cancer therapies. Moreover, techniques for the direct manipulation of CTC in circulation have been developed, intending to block metastasis in situ. We review a number of current and emerging micro- and nanobiotechnology approaches for the detection, capture, and manipulation of rare CTC aimed at advancing cancer treatment. Copyright © 2011 Wiley Periodicals, Inc.

LINE-1 Cultured Cell Retrotransposition Assay

PubMed Central

Kopera, Huira C.; Larson, Peter A.; Moldovan, John B.; Richardson, Sandra R.; Liu, Ying; Moran, John V.

2016-01-01

Summary The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells. PMID:26895052

Controlled viable release of selectively captured label-free cells in microchannels.

PubMed

Gurkan, Umut Atakan; Anand, Tarini; Tas, Huseyin; Elkan, David; Akay, Altug; Keles, Hasan Onur; Demirci, Utkan

2011-12-07

Selective capture of cells from bodily fluids in microchannels has broadly transformed medicine enabling circulating tumor cell isolation, rapid CD4(+) cell counting for HIV monitoring, and diagnosis of infectious diseases. Although cell capture methods have been demonstrated in microfluidic systems, the release of captured cells remains a significant challenge. Viable retrieval of captured label-free cells in microchannels will enable a new era in biological sciences by allowing cultivation and post-processing. The significant challenge in release comes from the fact that the cells adhere strongly to the microchannel surface, especially when immuno-based immobilization methods are used. Even though fluid shear and enzymes have been used to detach captured cells in microchannels, these methods are known to harm cells and affect cellular characteristics. This paper describes a new technology to release the selectively captured label-free cells in microchannels without the use of fluid shear or enzymes. We have successfully released the captured CD4(+) cells (3.6% of the mononuclear blood cells) from blood in microfluidic channels with high specificity (89% ± 8%), viability (94% ± 4%), and release efficiency (59% ± 4%). We have further validated our system by specifically capturing and controllably releasing the CD34(+) stem cells from whole blood, which were quantified to be 19 cells per million blood cells in the blood samples used in this study. Our results also indicated that both CD4(+) and CD34(+) cells released from the microchannels were healthy and amenable for in vitro culture. Manual flow based microfluidic method utilizes inexpensive, easy to fabricate microchannels allowing selective label-free cell capture and release in less than 10 minutes, which can also be used at the point-of-care. The presented technology can be used to isolate and purify a broad spectrum of cells from mixed populations offering widespread applications in applied biological

Engineering cholesterol-based fibers for antibody immobilization and cell capture

NASA Astrophysics Data System (ADS)

Cohn, Celine

In 2015, the United States is expected to have nearly 600,000 deaths attributed to cancer. Of these 600,000 deaths, 90% will be a direct result of cancer metastasis, the spread of cancer throughout the body. During cancer metastasis, circulating tumor cells (CTCs) are shed from primary tumors and migrate through bodily fluids, establishing secondary cancer sites. As cancer metastasis is incredibly lethal, there is a growing emphasis on developing "liquid biopsies" that can screen peripheral blood, search for and identify CTCs. One popular method for capturing CTCs is the use of a detection platform with antibodies specifically suited to recognize and capture cancer cells. These antibodies are immobilized onto the platform and can then bind and capture cells of interest. However, current means to immobilize antibodies often leave them with drastically reduced function. The antibodies are left poorly suited for cell capture, resulting in low cell capture efficiencies. This body of work investigates the use of lipid-based fibers to immobilize proteins in a way that retains protein function, ultimately leading to increased cell capture efficiencies. The resulting increased efficiencies are thought to arise from the retained three-dimensional structure of the protein as well as having a complete coating of the material surface with antibodies that are capable of interacting with their antigens. It is possible to electrospin cholesterol-based fibers that are similar in design to the natural cell membrane, providing proteins a more natural setting during immobilization. Such fibers have been produced from cholesterol-based cholesteryl succinyl silane (CSS). These fibers have previously illustrated a keen aptitude for retaining protein function and increasing cell capture. Herein the work focuses on three key concepts. First, a model is developed to understand the immobilization mechanism used by electrospun CSS fibers. The antibody immobilization and cell capturing

Antigen-capture blocking enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen to differentiate Transmissible gastroenteritis virus from Porcine respiratory coronavirus antibodies.

PubMed

López, Lissett; Venteo, Angel; García, Marga; Camuñas, Ana; Ranz, Ana; García, Julia; Sarraseca, Javier; Anaya, Carmen; Rueda, Paloma

2009-09-01

A new commercially available antigen-capture, blocking enzyme-linked immunosorbent assay (antigen-capture b-ELISA), based on baculovirus truncated-S recombinant protein of Transmissible gastroenteritis virus (TGEV) and 3 specific monoclonal antibodies, was developed and evaluated by examining a panel of 453 positive Porcine respiratory coronavirus (PRCoV), 31 positive TGEV, and 126 negative field sera by using another commercially available differential coronavirus b-ELISA as the reference technique to differentiate TGEV- from PRCoV-induced antibodies. The recombinant S protein-based ELISA appeared to be 100% sensitive for TGEV and PRCoV detection and highly specific for TGEV and PRCoV detection (100% and 92.06%, respectively), when qualitative results (positive or negative) were compared with those of the reference technique. In variability experiments, the ELISA gave consistent results when the same serum was evaluated on different wells and different plates. These results indicated that truncated recombinant S protein is a suitable alternative to the complete virus as antigen in ELISA assays. The use of recombinant S protein as antigen offers great advantages because it is an easy-to-produce, easy-to-standardize, noninfectious antigen that does not require further purification or concentration. Those advantages represent an important improvement for antigen preparation, in comparison with other assays in which an inactivated virus from mammalian cell cultures is used.

Comparison of the Third Wave Invader Human Papillomavirus (HPV) Assay and the Digene HPV Hybrid Capture 2 Assay for Detection of High-Risk HPV DNA▿

PubMed Central

Ginocchio, C. C.; Barth, D.; Zhang, F.

2008-01-01

This study compared the clinical performance of the Digene Hybrid Capture 2 (HC2) assay to that of a prototype Third Wave Invader human papillomavirus (HPV) (IHPV) analyte-specific reagent-based assay for the detection of oncogenic or “high-risk” (HR) HPV DNA using liquid-based cytology specimens. In total, 821 ThinPrep vials were tested using both assays. In accordance with the type-specific probes contained within each test, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the IHPV assay were 95.9%, 97.6%, 97.5%, and 96.1%, respectively, and those for the HC2 assay were 98.1%, 86.2%, 87.1%, and 97.9%. Overall, the sensitivity and NPV were comparable between the assays, but the IHPV assay demonstrated a better specificity and PPV, since the IHPV assay had fewer false-positive HR HPV results. The incorporation of an internal control to evaluate the cellularity of the test material is an important feature of the IHPV assay and should reduce the risk of false-negative results due to insufficient sample collection rather than the lack of HR HPV DNA. An additional benefit of the IHPV assay was the smaller sample volume required (1 ml versus 4 ml for the HC2 assay). PMID:18367578

A Versatile Microarray Platform for Capturing Rare Cells

NASA Astrophysics Data System (ADS)

Brinkmann, Falko; Hirtz, Michael; Haller, Anna; Gorges, Tobias M.; Vellekoop, Michael J.; Riethdorf, Sabine; Müller, Volkmar; Pantel, Klaus; Fuchs, Harald

2015-10-01

Analyses of rare events occurring at extremely low frequencies in body fluids are still challenging. We established a versatile microarray-based platform able to capture single target cells from large background populations. As use case we chose the challenging application of detecting circulating tumor cells (CTCs) - about one cell in a billion normal blood cells. After incubation with an antibody cocktail, targeted cells are extracted on a microarray in a microfluidic chip. The accessibility of our platform allows for subsequent recovery of targets for further analysis. The microarray facilitates exclusion of false positive capture events by co-localization allowing for detection without fluorescent labelling. Analyzing blood samples from cancer patients with our platform reached and partly outreached gold standard performance, demonstrating feasibility for clinical application. Clinical researchers free choice of antibody cocktail without need for altered chip manufacturing or incubation protocol, allows virtual arbitrary targeting of capture species and therefore wide spread applications in biomedical sciences.

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects...

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...

Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

EPA Science Inventory

The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

Assaying Wnt5A-mediated Invasion in Melanoma Cells

PubMed Central

O'Connell, Michael P.; French, Amanda D.; Leotlela, Poloko D.; Weeraratna, Ashani T.

2009-01-01

Wnt5A has been implicated in melanoma metastasis, and the progression of other cancers including pancreatic, gastric, prostate and lung cancers. Assays to test motility and invasion include both in vivo assays, and in vitro assays. The former assays include the use of tail vein or footpad injections of metastatic cells, and are often laborious and expensive. In vitro invasion assays provide quick readouts that can help to establish conditions that either activate or inhibit melanoma cell motility, and to assess whether the conditions in question are worth translating into an in vivo model. Here we describe two standard methods for assaying motility and invasion in vitro including wound healing assays and Matrigel invasion assays (Boyden chamber assays). In addition, we and several other laboratories have previously shown that melanoma cells require MMP-2 for their invasion, and have recently shown that Wnt5A treatment can increase the levels of this enzyme in melanoma cells, as demonstrated by gelatin zymography. The use of these techniques can help to assess the migratory capacity of melanoma cells in response to Wnt treatment. PMID:19099260

RNA-Seq analysis to capture the transcriptome landscape of a single cell

PubMed Central

Tang, Fuchou; Barbacioru, Catalin; Nordman, Ellen; Xu, Nanlan; Bashkirov, Vladimir I; Lao, Kaiqin; Surani, M. Azim

2013-01-01

We describe here a protocol for digital transcriptome analysis in a single mouse blastomere using a deep sequencing approach. An individual blastomere was first isolated and put into lysate buffer by mouth pipette. Reverse transcription was then performed directly on the whole cell lysate. After this, the free primers were removed by Exonuclease I and a poly(A) tail was added to the 3′ end of the first-strand cDNA by Terminal Deoxynucleotidyl Transferase. Then the single cell cDNAs were amplified by 20 plus 9 cycles of PCR. Then 100-200 ng of these amplified cDNAs were used to construct a sequencing library. The sequencing library can be used for deep sequencing using the SOLiD system. Compared with the cDNA microarray technique, our assay can capture up to 75% more genes expressed in early embryos. The protocol can generate deep sequencing libraries within 6 days for 16 single cell samples. PMID:20203668

Integrated microfluidic devices for combinatorial cell-based assays.

PubMed

Yu, Zeta Tak For; Kamei, Ken-ichiro; Takahashi, Hiroko; Shu, Chengyi Jenny; Wang, Xiaopu; He, George Wenfu; Silverman, Robert; Radu, Caius G; Witte, Owen N; Lee, Ki-Bum; Tseng, Hsian-Rong

2009-06-01

The development of miniaturized cell culture platforms for performing parallel cultures and combinatorial assays is important in cell biology from the single-cell level to the system level. In this paper we developed an integrated microfluidic cell-culture platform, Cell-microChip (Cell-microChip), for parallel analyses of the effects of microenvironmental cues (i.e., culture scaffolds) on different mammalian cells and their cellular responses to external stimuli. As a model study, we demonstrated the ability of culturing and assaying several mammalian cells, such as NIH 3T3 fibroblast, B16 melanoma and HeLa cell lines, in a parallel way. For functional assays, first we tested drug-induced apoptotic responses from different cell lines. As a second functional assay, we performed "on-chip" transfection of a reporter gene encoding an enhanced green fluorescent protein (EGFP) followed by live-cell imaging of transcriptional activation of cyclooxygenase 2 (Cox-2) expression. Collectively, our Cell-microChip approach demonstrated the capability to carry out parallel operations and the potential to further integrate advanced functions and applications in the broader space of combinatorial chemistry and biology.

Integrated microfluidic devices for combinatorial cell-based assays

PubMed Central

Yu, Zeta Tak For; Kamei, Ken-ichiro; Takahashi, Hiroko; Shu, Chengyi Jenny; Wang, Xiaopu; He, George Wenfu; Silverman, Robert

2010-01-01

The development of miniaturized cell culture platforms for performing parallel cultures and combinatorial assays is important in cell biology from the single-cell level to the system level. In this paper we developed an integrated microfluidic cell-culture platform, Cell-microChip (Cell-μChip), for parallel analyses of the effects of microenvir-onmental cues (i.e., culture scaffolds) on different mammalian cells and their cellular responses to external stimuli. As a model study, we demonstrated the ability of culturing and assaying several mammalian cells, such as NIH 3T3 fibro-blast, B16 melanoma and HeLa cell lines, in a parallel way. For functional assays, first we tested drug-induced apoptotic responses from different cell lines. As a second functional assay, we performed "on-chip" transfection of a reporter gene encoding an enhanced green fluorescent protein (EGFP) followed by live-cell imaging of transcriptional activation of cyclooxygenase 2 (Cox-2) expression. Collectively, our Cell-μChip approach demonstrated the capability to carry out parallel operations and the potential to further integrate advanced functions and applications in the broader space of combinatorial chemistry and biology. PMID:19130244

Dielectrophoretic Capture and Genetic Analysis of Single Neuroblastoma Tumor Cells

PubMed Central

Carpenter, Erica L.; Rader, JulieAnn; Ruden, Jacob; Rappaport, Eric F.; Hunter, Kristen N.; Hallberg, Paul L.; Krytska, Kate; O’Dwyer, Peter J.; Mosse, Yael P.

2014-01-01

Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here, we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 106 white blood cells (WBCs). Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control WBCs. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples of patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here, we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients. PMID:25133137

Comparison of Onclarity Human Papillomavirus (HPV) Assay with Hybrid Capture II HPV DNA Assay for Detection of Cervical Intraepithelial Neoplasia Grade 2 and 3 Lesions

PubMed Central

Sideri, M.; Gulmini, C.; Igidbashian, S.; Tricca, A.; Casadio, C.; Carinelli, S.; Boveri, S.; Ejegod, D.; Bonde, J.; Sandri, M. T.

2015-01-01

Analytical and clinical performance validation is essential before introduction of a new human papillomavirus (HPV) assay into clinical practice. This study compares the new BD Onclarity HPV assay, which detects E6/E7 DNA from 14 high-risk HPV types, to the Hybrid Capture II (HC2) HPV DNA test, to concurrent cytology and histology results, in order to evaluate its performance in detecting high-grade cervical lesions. A population of 567 women, including 325 with ≥ASCUS (where ASCUS stands for atypical cells of undetermined significance) and any HC2 result and 242 with both negative cytology and negative HC2 results, were prospectively enrolled for the study. The overall agreement between Onclarity and HC2 was 94.6% (95% confidence intervals [CI], 92.3% to 96.2%). In this population with a high prevalence of disease, the relative sensitivities (versus adjudicated cervical intraepithelial neoplasia grades 2 and 3 [CIN2+] histology endpoints) of the Onclarity and HC2 tests were 95.2% (95% CI, 90.7% to 97.5%) and 96.9% (95% CI, 92.9% to 98.7%), respectively, and the relative specificities were 50.3% (95% CI, 43.2% to 57.4%) for BD and 40.8% (95% CI, 33.9%, 48.1%) for HC2. These results indicate that the BD Onclarity HPV assay has sensitivity comparable to that of the HC2 assay, with a trend to an increased specificity. Moreover, as Onclarity gives the chance to discriminate between the different genotypes, we calculated the genotype prevalence and the absolute risk of CIN2+: HPV 16 was the most prevalent genotype (19.8%) with an absolute risk of CIN2+ of 77.1%. PMID:25903574

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in...

Epithelial cells as alternative human biomatrices for comet assay.

PubMed

Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

2014-01-01

The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.

Epithelial cells as alternative human biomatrices for comet assay

PubMed Central

Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

2014-01-01

The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases. PMID:25506353

Liposome-based delivery of a boron-containing cholesteryl ester for high-LET particle-induced damage of prostate cancer cells: a boron neutron capture therapy study.

PubMed

Gifford, Ian; Vreeland, Wyatt; Grdanovska, Slavica; Burgett, Eric; Kalinich, John; Vergara, Vernieda; Wang, C-K Chris; Maimon, Eric; Poster, Dianne; Al-Sheikhly, Mohamad

2014-06-01

The efficacy of a boron-containing cholesteryl ester compound (BCH) as a boron neutron capture therapy (BNCT) agent for the targeted irradiation of PC-3 human prostate cancer cells was examined. Liposome-based delivery of BCH was quantified with inductively coupled plasma-mass spectrometry (ICP-MS) and high-performance liquid chromatography (HPLC). Cytotoxicity of the BCH-containing liposomes was evaluated with neutral red, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and lactate dehydrogenase assays. Colony formation assays were utilized to evaluate the decrease in cell survival due to high-linear energy transfer (LET) particles resulting from (10)B thermal neutron capture. BCH delivery by means of encapsulation in a lipid bilayer resulted in a boron uptake of 35.2 ± 4.3 μg/10(9) cells, with minimal cytotoxic effects. PC-3 cells treated with BCH and exposed to a 9.4 × 10(11) n/cm(2) thermal neutron fluence yielded a 20-25% decrease in clonogenic capacity. The decreased survival is attributed to the generation of high-LET α particles and (7)Li nuclei that deposit energy in densely ionizing radiation tracks. Liposome-based delivery of BCH is capable of introducing sufficient boron to PC-3 cells for BNCT. High-LET α particles and (7)Li nuclei generated from (10)B thermal neutron capture significantly decrease colony formation ability in the targeted PC-3 cells.

Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA

PubMed Central

Jahan, Munira; Lutful Moben, Ahmed; Tabassum, Shahina

2014-01-01

ABSTRACT Background Both real-time-polymerase chain reaction (PCR) and hybrid capture 2 (HC2) assay can detect and quantify hepatitis B virus (HBV) DNA. However, real-time-PCR can detect a wide range of HBV DNA, while HC2 assay could not detect lower levels of viremia. The present study was designed to detect and quantify HBV DNA by real-time-PCR and HC2 assay and compare the quantitative data of these two assays. Materials and methods A cross-sectional study was conducted in between July 2010 and June 2011. A total of 66 serologically diagnosed chronic hepatitis B (CHB) patients were selected for the study. Real-time-PCR and HC2 assay was done to detect HBV DNA. Data were analyzed by statistical Package for the social sciences (SPSS). Results Among 66 serologically diagnosed chronic hepatitis B patients 40 (60.61%) patients had detectable and 26 (39.39%) had undetectable HBV DNA by HC2 assay. Concordant results were obtained for 40 (60.61%) out of these 66 patients by real-time-PCR and HC2 assay with mean viral load of 7.06 ± 1.13 log10 copies/ml and 6.95 ± 1.08 log10 copies/ml, respectively. In the remaining 26 patients, HBV DNA was detectable by real-time-PCR in 20 patients (mean HBV DNA level was 3.67 ± 0.72 log10 copies/ml. However, HBV DNA could not be detectable in six cases by the both assays. The study showed strong correlation (r = 0.915) between real-time-PCR and HC2 assay for the detection and quantification of HBV DNA. Conclusion HC2 assay may be used as an alternative to real-time-PCR for CHB patients. How to cite this article: Majid F, Jahan M, Moben AL, Tabassum S. Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA. Euroasian J Hepato-Gastroenterol 2014;4(1):31-35. PMID:29264316

Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA.

PubMed

Majid, Farjana; Jahan, Munira; Lutful Moben, Ahmed; Tabassum, Shahina

2014-01-01

Both real-time-polymerase chain reaction (PCR) and hybrid capture 2 (HC2) assay can detect and quantify hepatitis B virus (HBV) DNA. However, real-time-PCR can detect a wide range of HBV DNA, while HC2 assay could not detect lower levels of viremia. The present study was designed to detect and quantify HBV DNA by real-time-PCR and HC2 assay and compare the quantitative data of these two assays. A cross-sectional study was conducted in between July 2010 and June 2011. A total of 66 serologically diagnosed chronic hepatitis B (CHB) patients were selected for the study. Real-time-PCR and HC2 assay was done to detect HBV DNA. Data were analyzed by statistical Package for the social sciences (SPSS). Among 66 serologically diagnosed chronic hepatitis B patients 40 (60.61%) patients had detectable and 26 (39.39%) had undetectable HBV DNA by HC2 assay. Concordant results were obtained for 40 (60.61%) out of these 66 patients by real-time-PCR and HC2 assay with mean viral load of 7.06 ± 1.13 log 10 copies/ml and 6.95 ± 1.08 log 10 copies/ml, respectively. In the remaining 26 patients, HBV DNA was detectable by real-time-PCR in 20 patients (mean HBV DNA level was 3.67 ± 0.72 log 10 copies/ml. However, HBV DNA could not be detectable in six cases by the both assays. The study showed strong correlation (r = 0.915) between real-time-PCR and HC2 assay for the detection and quantification of HBV DNA. HC2 assay may be used as an alternative to real-time-PCR for CHB patients. How to cite this article: Majid F, Jahan M, Moben AL, Tabassum S. Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA. Euroasian J Hepato-Gastroenterol 2014;4(1):31-35.

Detection of target staphylococcal enterotoxin B antigen in orange juice and popular carbonated beverages using antibody-dependent antigen-capture assays.

PubMed

Principato, MaryAnn; Njoroge, Joyce M; Perlloni, Andrei; O' Donnell, Michael; Boyle, Thomas; Jones, Robert L

2010-10-01

There is a critical need for qualitative and quantitative methodologies that provide the rapid and accurate detection of food contaminants in complex food matrices. However, the sensitivity of the assay can be affected when antigen-capture is applied to certain foods or beverages that are extremely acidic. This study was undertaken to assess the effects of orange juice and popular carbonated soft drink upon the fidelity of antibody-based antigen-capture assays and to develop simple approaches that could rescue assay performance without the introduction of additional or extensive extraction procedures. We examined the effects of orange juice and a variety of popular carbonated soft drink beverages upon a quantitative Interleukin-2 (IL-2) enzyme-linked immunosorbent assay (ELISA) assay system and a lateral flow device (LFD) adapted for the detection of staphylococcal enterotoxin B (SEB) in foods. Alterations in the performance and sensitivity of the assay were directly attributable to the food matrix, and alterations in pH were especially critical. The results demonstrate that approaches such as an alteration of pH and the use of milk as a blocking agent, either singly or in combination, will partially rescue ELISA performance. The same approaches permit lateral flow to efficiently detect antigen. Practical Application: The authors present ways to rescue an ELISA assay compromised by acidity in beverages and show that either the alteration of pH, or the use of milk as a blocking agent are not always capable of restoring the assay to its intended efficiency. However, the same methods, when employed with lateral flow technology, are rapid and extremely successful.

Fluorescein Diacetate Microplate Assay in Cell Viability Detection.

PubMed

Chen, Xi; Yang, Xiu-Ying; Fang, Lian-Hua; DU, Guan-Hua

2016-12-20

Objective To investigate the application of the fluorescein diacetate (FDA) microplate assay in cell viability detection. Methods Cells were seeded in a 96-well culture plate until detection. After incubated with FDA,the plate was detected by fluorescence microplate analyzer. The effects of FDA incubation duration,concentration,and other factors on the assay's accuracy and stability were assessed. We also compared the results of FDA with methyl thiazolyl(MTT) in terms of cell numbers and H 2 O 2 injury. Results Within 0-30 minutes,the fluorescence-cell number coefficient of FDA assay increased with duration and reached 0.99 in 27-30 minutes. The optimum concentration of final FDA in this study was 10-30 μg/ml. On cell viability detection,the result of FDA method was equivalent to MTT method. As to H 2 O 2 injury assay,the sensitivity of FDA method was superior to MTT on the higher concentration H 2 O 2 treatment due to a relative shorter duration for detection. Conclusion As a stable and reliable method,FDA is feasible for cell variability detection under varied conditions.

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

PubMed Central

Schotte, Lise; Rombaut, Bart; Thys, Bert

2012-01-01

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to

A microplate assay for measuring cell death in C2C12 cells.

PubMed

Lima, Tanes; Silveira, Leonardo

2018-03-22

The main goal of this study was to develop a straightforward and rapid microplate assay for measuring propidium iodide (PI) in C2C12 cells. The PI method proves to be an efficient quantitative assay for analyzing cell viability through PI fluorescence analysis. Importantly, the protocol takes less than 30 minutes, and the results are reproducible. C2C12 cells were exposed to an increasing concentration of palmitate for a period of 24 hours to induce cell death, and the PI fluorescence increased in a concentration-dependent manner. Evaluation of mitochondrial function and reactive oxygen species production validated the deleterious effects of palmitate treatment. Also, the microplate PI assay demonstrated high sensitivity as indicated by the detection of modest fluctuations in cell viability in response to catalase overexpression in palmitate-treated cells. The microplate PI assay, therefore, offers an accurate method to be used for in vitro studies.

A Reproducible Immunopotency Assay to Measure Mesenchymal Stromal Cell Mediated T cell Suppression

PubMed Central

Bloom, Debra D.; Centanni, John M.; Bhatia, Neehar; Emler, Carol A.; Drier, Diana; Leverson, Glen E.; McKenna, David H.; Gee, Adrian P.; Lindblad, Robert; Hei, Derek J.; Hematti, Peiman

2014-01-01

Background The T cell suppressive property of bone marrow derived mesenchymal stromal cells (MSCs) has been considered a major mode of action and basis for their utilization in a number of human clinical trials. However, there is no well-established reproducible assay to measure MSC-mediated T cell suppression. Methods At the University of Wisconsin-Madison Production Assistance for Cellular Therapy (PACT) Center we developed an in vitro quality control T cell suppression immunopotency assay (IPA) which utilizes anti-CD3 and anti-CD28 antibodies to stimulate T cell proliferation. We measured MSC-induced suppression of CD4+ T cell proliferation at various effector to target cell ratios using defined peripheral blood mononuclear cells and in parallel compared to a reference standard MSC product. We calculated an IPA value for suppression of CD4+ T cells for each MSC product. Results Eleven MSC products generated at three independent PACT centers were evaluated for cell surface phenotypic markers and T cell suppressive properties. Flow cytometry results demonstrated typical MSC cell surface marker profiles. There was significant variability in the level of suppression of T cell proliferation with IPA values ranging from 27% to 88%. However, MSC suppression did not correlate with HLA-DR expression. Discussion We have developed a reproducible immunopotency assay to measure allogeneic MSC-mediated suppression of CD4+ T cells. Additional studies may be warranted to determine how these in vitro assay results may correlate with other immunomodulatory properties of MSCs, in addition to evaluating the ability of this assay to predict in vivo efficacy. PMID:25455739

Monoclonal antibody passive hemagglutination and capture enzyme-linked immunosorbent assays for direct detection and quantitation of F41 and K99 fimbrial antigens in enterotoxigenic Escherichia coli.

PubMed Central

Raybould, T J; Crouch, C F; Acres, S D

1987-01-01

Production of diarrhea in neonatal calves by enterotoxigenic Escherichia coli depends on its ability to attach to the epithelial cells of the intestine via surface adhesins called pili or fimbriae and to secrete enterotoxins. The most important of these fimbriae are designated K99 and F41. We produced and characterized a murine monoclonal antibody specific to F41. This monoclonal antibody and a K99-specific monoclonal antibody were used to develop sensitive and specific passive hemagglutination and capture enzyme-linked immunosorbent assays (ELISAs) for detection and quantitation of F41 and K99 antigens in E. coli cultures and culture supernatants. The capture ELISA systems exhibited excellent sensitivity and specificity, whereas the passive hemagglutination systems appeared to be oversensitive. The ability of the capture ELISAs to detect K99 and F41 fimbrial antigens in fecal specimens from calves was evaluated. Fimbrial antigens were detected in six of six specimens from scouring calves but not in four of four specimens from nonscouring calves. PMID:2880866

Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells

PubMed Central

Chabaud, Mélanie; Heuzé, Mélina L.; Bretou, Marine; Vargas, Pablo; Maiuri, Paolo; Solanes, Paola; Maurin, Mathieu; Terriac, Emmanuel; Le Berre, Maël; Lankar, Danielle; Piolot, Tristan; Adelstein, Robert S.; Zhang, Yingfan; Sixt, Michael; Jacobelli, Jordan; Bénichou, Olivier; Voituriez, Raphaël; Piel, Matthieu; Lennon-Duménil, Ana-Maria

2015-01-01

The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space. PMID:26109323

Alpha 2-macroglobulin capture allows detection of mast cell chymase in serum and creates a reservoir of angiotensin II-generating activity.

PubMed

Raymond, Wilfred W; Su, Sharon; Makarova, Anastasia; Wilson, Todd M; Carter, Melody C; Metcalfe, Dean D; Caughey, George H

2009-05-01

Human chymase is a highly efficient angiotensin II-generating serine peptidase expressed by mast cells. When secreted from degranulating cells, it can interact with a variety of circulating antipeptidases, but is mostly captured by alpha(2)-macroglobulin, which sequesters peptidases in a cage-like structure that precludes interactions with large protein substrates and inhibitors, like serpins. The present work shows that alpha(2)-macroglobulin-bound chymase remains accessible to small substrates, including angiotensin I, with activity in serum that is stable with prolonged incubation. We used alpha(2)-macroglobulin capture to develop a sensitive, microtiter plate-based assay for serum chymase, assisted by a novel substrate synthesized based on results of combinatorial screening of peptide substrates. The substrate has low background hydrolysis in serum and is chymase-selective, with minimal cleavage by the chymotryptic peptidases cathepsin G and chymotrypsin. The assay detects activity in chymase-spiked serum with a threshold of approximately 1 pM (30 pg/ml), and reveals native chymase activity in serum of most subjects with systemic mastocytosis. alpha(2)-Macroglobulin-bound chymase generates angiotensin II in chymase-spiked serum, and it appears in native serum as chymostatin-inhibited activity, which can exceed activity of captopril-sensitive angiotensin-converting enzyme. These findings suggest that chymase bound to alpha(2)-macroglobulin is active, that the complex is an angiotensin-converting enzyme inhibitor-resistant reservoir of angiotensin II-generating activity, and that alpha(2)-macroglobulin capture may be exploited in assessing systemic release of secreted peptidases.

Nanotextured PDMS Substrates for Enhanced Roughness and Aptamer Immobilization for Cancer Cell Capture

NASA Astrophysics Data System (ADS)

Islam, Muhymin; Mahmood, Arif; Bellah, Md.; Kim, Young-Tae; Iqbal, Samir

2014-03-01

Detection of circulating tumor cells (CTCs) in the early stages of cancer is requires very sensitive approach. Nanotextured polydimethylsiloxane (PDMS) substrates were fabricated by micro reactive ion etching (Micro-RIE) to have better control on surface morphology and to improve the affinity of PDMS surfaces to capture cancer cells using surface immobilized aptamers. The aptamers were specific to epidermal growth factor receptors (EGFR) present in cell membranes, and overexpressed in tumor cells. We also investigated the effect of nano-scale features on cell capturing by implementing various surfaces of different roughnesses. Three different recipes were used to prepare nanotextured PDMS by micro-RIE using oxygen (O2) and carbon tetrafluoride (CF4). The measured average roughness of three nanotextured PDMS surfaces were found to impact average densities of captured cells. In all cases, nanotextured PDMS facilitated cell capturing possibly due to increased effective surface area of roughened substrates at nanoscale. It was also observed that cell capture efficiency was higher for higher surface roughness. The nanotextured PDMS substrates are thus useful for cancer cytology devices.

A photonic crystal hydrogel suspension array for the capture of blood cells from whole blood

NASA Astrophysics Data System (ADS)

Zhang, Bin; Cai, Yunlang; Shang, Luoran; Wang, Huan; Cheng, Yao; Rong, Fei; Gu, Zhongze; Zhao, Yuanjin

2016-02-01

Diagnosing hematological disorders based on the separation and detection of cells in the patient's blood is a significant challenge. We have developed a novel barcode particle-based suspension array that can simultaneously capture and detect multiple types of blood cells. The barcode particles are polyacrylamide (PAAm) hydrogel inverse opal microcarriers with characteristic reflection peak codes that remain stable during cell capture on their surfaces. The hydrophilic PAAm hydrogel scaffolds of the barcode particles can entrap various plasma proteins to capture different cells in the blood, with little damage to captured cells.Diagnosing hematological disorders based on the separation and detection of cells in the patient's blood is a significant challenge. We have developed a novel barcode particle-based suspension array that can simultaneously capture and detect multiple types of blood cells. The barcode particles are polyacrylamide (PAAm) hydrogel inverse opal microcarriers with characteristic reflection peak codes that remain stable during cell capture on their surfaces. The hydrophilic PAAm hydrogel scaffolds of the barcode particles can entrap various plasma proteins to capture different cells in the blood, with little damage to captured cells. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06368j

A photoelectrochemical platform for the capture and release of rare single cells.

PubMed

Parker, Stephen G; Yang, Ying; Ciampi, Simone; Gupta, Bakul; Kimpton, Kathleen; Mansfeld, Friederike M; Kavallaris, Maria; Gaus, Katharina; Gooding, J Justin

2018-06-12

For many normal and aberrant cell behaviours, it is important to understand the origin of cellular heterogeneity. Although powerful methods for studying cell heterogeneity have emerged, they are more suitable for common rather than rare cells. Exploring the heterogeneity of rare single cells is challenging because these rare cells must be first pre-concentrated and undergo analysis prior to classification and expansion. Here, a versatile capture & release platform consisting of an antibody-modified and electrochemically cleavable semiconducting silicon surface for release of individual cells of interest is presented. The captured cells can be interrogated microscopically and tested for drug responsiveness prior to release and recovery. The capture & release strategy was applied to identify rare tumour cells from whole blood, monitor the uptake of, and response to, doxorubicin and subsequently select cells for single-cell gene expression based on their response to the doxorubicin.

High-Density Dielectrophoretic Microwell Array for Detection, Capture, and Single-Cell Analysis of Rare Tumor Cells in Peripheral Blood.

PubMed

Morimoto, Atsushi; Mogami, Toshifumi; Watanabe, Masaru; Iijima, Kazuki; Akiyama, Yasuyuki; Katayama, Koji; Futami, Toru; Yamamoto, Nobuyuki; Sawada, Takeshi; Koizumi, Fumiaki; Koh, Yasuhiro

2015-01-01

Development of a reliable platform and workflow to detect and capture a small number of mutation-bearing circulating tumor cells (CTCs) from a blood sample is necessary for the development of noninvasive cancer diagnosis. In this preclinical study, we aimed to develop a capture system for molecular characterization of single CTCs based on high-density dielectrophoretic microwell array technology. Spike-in experiments using lung cancer cell lines were conducted. The microwell array was used to capture spiked cancer cells, and captured single cells were subjected to whole genome amplification followed by sequencing. A high detection rate (70.2%-90.0%) and excellent linear performance (R2 = 0.8189-0.9999) were noted between the observed and expected numbers of tumor cells. The detection rate was markedly higher than that obtained using the CellSearch system in a blinded manner, suggesting the superior sensitivity of our system in detecting EpCAM- tumor cells. Isolation of single captured tumor cells, followed by detection of EGFR mutations, was achieved using Sanger sequencing. Using a microwell array, we established an efficient and convenient platform for the capture and characterization of single CTCs. The results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.

Comparative study on antibody immobilization strategies for efficient circulating tumor cell capture.

PubMed

Ates, Hatice Ceren; Ozgur, Ebru; Kulah, Haluk

2018-03-23

Methods for isolation and quantification of circulating tumor cells (CTCs) are attracting more attention every day, as the data for their unprecedented clinical utility continue to grow. However, the challenge is that CTCs are extremely rare (as low as 1 in a billion of blood cells) and a highly sensitive and specific technology is required to isolate CTCs from blood cells. Methods utilizing microfluidic systems for immunoaffinity-based CTC capture are preferred, especially when purity is the prime requirement. However, antibody immobilization strategy significantly affects the efficiency of such systems. In this study, two covalent and two bioaffinity antibody immobilization methods were assessed with respect to their CTC capture efficiency and selectivity, using an anti-epithelial cell adhesion molecule (EpCAM) as the capture antibody. Surface functionalization was realized on plain SiO 2 surfaces, as well as in microfluidic channels. Surfaces functionalized with different antibody immobilization methods are physically and chemically characterized at each step of functionalization. MCF-7 breast cancer and CCRF-CEM acute lymphoblastic leukemia cell lines were used as EpCAM positive and negative cell models, respectively, to assess CTC capture efficiency and selectivity. Comparisons reveal that bioaffinity based antibody immobilization involving streptavidin attachment with glutaraldehyde linker gave the highest cell capture efficiency. On the other hand, a covalent antibody immobilization method involving direct antibody binding by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)-N-hydroxysuccinimide (NHS) reaction was found to be more time and cost efficient with a similar cell capture efficiency. All methods provided very high selectivity for CTCs with EpCAM expression. It was also demonstrated that antibody immobilization via EDC-NHS reaction in a microfluidic channel leads to high capture efficiency and selectivity.

Development of a lion-specific interferon-gamma assay.

PubMed

Maas, M; van Kooten, P J S; Schreuder, J; Morar, D; Tijhaar, E; Michel, A L; Rutten, V P M G

2012-10-15

The ongoing spread of bovine tuberculosis (BTB) in African free-ranging lion populations, for example in the Kruger National Park, raises the need for diagnostic assays for BTB in lions. These, in addition, would be highly relevant for zoological gardens worldwide that want to determine the BTB status of their lions, e.g. for translocations. The present study concerns the development of a lion-specific IFN-γ assay, following the production and characterization of monoclonal antibodies specific for lion interferon-gamma (IFN-γ). Recombinant lion IFN-γ (rLIFN-γ) was produced in mammalian cells and used to immunize mice to establish hybridoma cell lines producing monoclonal antibodies. These were used to develop a sensitive, lion IFN-γ-specific capture ELISA, able to detect rLIFN-γ to the level of 160 pg/ml. Recognition of native lion IFN-γ was shown in an initial assessment of supernatants of mitogen stimulated whole blood cultures of 11 known BTB-negative lions. In conclusion, the capture ELISA shows potential as a diagnostic assay for bovine tuberculosis in lions. Preliminary results also indicate the possible use of the test for other (feline) species. Copyright © 2012 Elsevier B.V. All rights reserved.

Antibody class capture assay (ACCA) for rubella-specific IgM antibody.

PubMed

Isaac, M; Payne, R A

1982-01-01

Enzyme-linked immunosorbent assays for IgM antirubella were carried out on 1,546 sera, using an IgM capture method with a F (ab')2 conjugate (ACCA). Under the conditions described, sera containing IgM antirubella bound up to 15 times as much enzyme activity as negative specimens. Paired serum specimens from 27 patients, serial serum specimens from 6 patients, and single serum specimens from 15 patients who had had recent rubella were examined by the haemagglutination inhibition test (HAI) in the presence and absence of 2-mercaptoethanol following sucrose density gradient centrifugation (SDGC). ACCA confirmed all the results found with HAI following SDGC. Specimens were examined from ten patients with congenital rubella; ACCA confirmed the results found with both immunofluorescence following SDGC and radioimmunoassay. Pre- and post-vaccination specimens from 123 patients who had been vaccinated against rubella were examined. An IgM response could only be demonstrated in the 57 cases when IgG was absent in the first specimen. The specificity of the assay was confirmed by testing 31 serum specimens from rubella immune patients that also contained rheumatoid factor, 163 serum specimens from patients with acute infections other than rubella, and 12 serum specimens from infants with miscellaneous neonatal abnormalities other than congenital rubella. The ACCA proved a simple, sensitive, and specific test for IgM antirubella and the results compared favourably with those obtained by the SDGC technique.

A novel label-free cell-based assay technology using biolayer interferometry.

PubMed

Verzijl, D; Riedl, T; Parren, P W H I; Gerritsen, A F

2017-01-15

Biolayer interferometry (BLI) is a well-established optical label-free technique to study biomolecular interactions. Here we describe for the first time a cell-based BLI (cBLI) application that allows label-free real-time monitoring of signal transduction in living cells. Human A431 epidermoid carcinoma cells were captured onto collagen-coated biosensors and serum-starved, followed by exposure to agonistic compounds targeting various receptors, while recording the cBLI signal. Stimulation of the epidermal growth factor receptor (EGFR) with EGF, the β 2 -adrenoceptor with dopamine, or the hepatocyte growth factor receptor (HGFR/c-MET) with an agonistic antibody resulted in distinct cBLI signal patterns. We show that the mechanism underlying the observed changes in cBLI signal is mediated by rearrangement of the actin cytoskeleton, a process referred to as dynamic mass redistribution (DMR). A panel of ligand-binding blocking and non-blocking anti-EGFR antibodies was used to demonstrate that this novel BLI application can be efficiently used as a label-free cellular assay for compound screening and characterization. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Single cell network profiling assay in bladder cancer.

PubMed

Covey, Todd M; Vira, Manish A; Westfall, Matt; Gulrajani, Michael; Cholankeril, Michelle; Okhunov, Zhamshid; Levey, Helen R; Marimpietri, Carol; Hawtin, Rachael; Fields, Scott Z; Cesano, Alessandra

2013-04-01

The aim of this study was to assess the feasibility of applying the single cell network profiling (SCNP) assay to the examination of signaling networks in epithelial cancer cells, using bladder washings from 29 bladder cancer (BC) and 15 nonbladder cancer (NC) subjects. This report describes the methods we developed to detect rare epithelial cells (within the cells we collected from bladder washings), distinguish cancer cells from normal epithelial cells, and reproducibly quantify signaling within these low frequency cancer cells. Specifically, antibodies against CD45, cytokeratin, EpCAM, and cleaved-PARP (cPARP) were used to differentiate nonapoptotic epithelial cells from leukocytes, while measurements of DNA content to determine aneuploidy (DAPI stain) allowed for distinction between tumor and normal epithelial cells. Signaling activity in the PI3K and MAPK pathways was assessed by measuring intracellular levels of p-AKT and p-ERK at baseline and in response to pathway modulation; 66% (N = 19) of BC samples and 27% (N = 4) of NC samples met the "evaluable" criteria, i.e., at least 400,000 total cells available upon sample receipt with >2% of cells showing an epithelial phenotype. The majority of epithelial cells detected in BC samples were nonapoptotic and all signaling data were generated from identified cPARP negative cells. In four of 19 BC samples but in none of the NC specimens, SCNP assay identified epithelial cancer cells with a quantifiable increase in epidermal growth factor-induced p-AKT and p-ERK levels. Furthermore, preincubation with the PI3K inhibitor GDC-0941 reduced or completely inhibited basal and epidermal growth factor-induced p-AKT but, as expected, had no effect on p-ERK levels. This study demonstrates the feasibility of applying SCNP assay using multiparametric flow cytometry to the functional characterization of rare, bladder cancer cells collected from bladder washing. Following assay standardization, this method could potentially serve

A Cell-Cell Fusion Assay to Assess Arenavirus Envelope Glycoprotein Membrane-Fusion Activity.

PubMed

York, Joanne; Nunberg, Jack H

2018-01-01

For many viruses that enter their target cells through pH-dependent fusion of the viral and endosomal membranes, cell-cell fusion assays can provide an experimental platform for investigating the structure-function relationships that promote envelope glycoprotein membrane-fusion activity. Typically, these assays employ effector cells expressing the recombinant envelope glycoprotein on the cell surface and target cells engineered to quantitatively report fusion with the effector cell. In the protocol described here, Vero cells are transfected with a plasmid encoding the arenavirus envelope glycoprotein complex GPC and infected with the vTF7-3 vaccinia virus expressing the bacteriophage T7 RNA polymerase. These effector cells are mixed with target cells infected with the vCB21R-lacZ vaccinia virus encoding a β-galactosidase reporter under the control of the T7 promoter. Cell-cell fusion is induced upon exposure to low-pH medium (pH 5.0), and the resultant expression of the β-galactosidase reporter is quantitated using a chemiluminescent substrate. We have utilized this robust microplate cell-cell fusion assay extensively to study arenavirus entry and its inhibition by small-molecule fusion inhibitors.

Comparison of the CMV brite turbo assay and the digene hybrid capture CMV DNA (Version 2.0) assay for quantitation of cytomegalovirus in renal transplant recipients.

PubMed

Ho, S K; Li, F K; Lai, K N; Chan, T M

2000-10-01

We compared the CMV Brite Turbo Kit (BT) and the Digene Hybrid Capture CMV DNA (version 2.0) assay (HC2) in the quantitation of pp65 antigenemia and cytomegalovirus (CMV) DNA levels in immunosuppressed renal transplant recipients. Of 123 blood specimens collected from 24 renal transplant recipients, BT and HC2 assays detected 35 and 39 positive samples, respectively. The overall concordance rate between the two assays was 90%. Discordant results were observed at low levels of viremia, so that 8 samples were HC2 positive but BT negative and another 4 were BT positive but HC2 negative. There was good correlation (R(2) = 0.766; P<0.01) between the levels of CMV DNA and pp65 antigenemia in the 31 concordant positive samples. Correlation between results obtained with the two assays was confirmed by longitudinal studies for a patient who developed clinical CMV disease. HC2 may be more sensitive at low viremia levels and allow earlier detection of impending CMV disease. The BT assay offered the advantage of a rapid (2-h) turnaround time. We conclude that BT and HC2 assays have similar sensitivity and efficacy in the diagnosis and monitoring of CMV infection and disease in renal transplant recipients. While the HC2 assay would be appropriate for centers that handle a large number of samples, the BT test may be more suitable for small sample numbers or when results are needed urgently.

Single Cell Assay for Analyzing Single Cell Exosome and Endocrine Secretion and Cancer Markers

NASA Astrophysics Data System (ADS)

Chiu, Yu-Jui

To understand the inhomogeneity of cells in biological systems, there is a growing demand for the capability to characterize the properties of individual single cells. Since single cell studies require continuous monitoring of the cell behaviors instead of a snapshot test at a single time point, an effective single-cell assay that can support time lapsed studies in a high throughput manner is desired. Most currently available single-cell technologies cannot provide proper environments to sustain cell growth and cannot provide, for appropriate cell types, proliferation of single cells and convenient, non-invasive tests of single cell behaviors from molecular markers. In this dissertation, I present a highly versatile single-cell assay that can accommodate different cellular types, enable easy and efficient single cell loading and culturing, and be suitable for the study of effects of in-vitro environmental factors in combination with drug screening. The salient features of the assay are the non-invasive collection and surveying of single cell secretions at different time points and massively parallel translocation of single cells by user defined criteria, producing very high compatibility to the downstream process such as single cell qPCR and sequencing. Above all, the acquired information is quantitative -- for example, one of the studies is measured by the number of exosomes each single cell secretes for a given time period. Therefore, our single-cell assay provides a convenient, low-cost, and enabling tool for quantitative, time lapsed studies of single cell properties.

Development of Antigen Detection Assay for Diagnosis of Tuberculosis Using Sputum Samples

PubMed Central

Pereira Arias-Bouda, Lenka M.; Nguyen, Lan N.; Ho, Ly M.; Kuijper, Sjoukje; Jansen, Henk M.; Kolk, Arend H. J.

2000-01-01

The rising incidence of tuberculosis worldwide means an increasing burden on diagnostic facilities, so tests simpler than Ziehl-Neelsen staining are needed. Such tests should be objective, reproducible, and have at least as good a detection limit as 104 bacteria/ml. A capture enzyme-linked immunosorbent assay (ELISA) was developed for detection of lipoarabinomannan (LAM) in human sputum samples. As a capture antibody, we used a murine monoclonal antibody against LAM, with rabbit antiserum against Mycobacterium tuberculosis as a source of detector antibodies. The sensitivity of the capture ELISA was evaluated by using purified LAM and M. tuberculosis whole cells. We were able to detect 1 ng of purified LAM/ml and 104 M. tuberculosis whole cells/ml. LAM could also be detected in culture filtrate of a 3-week-old culture of M. tuberculosis. The culture filtrate contained approximately 100 μg of LAM/ml. The detection limit in sputum pretreated with N-acetyl-l-cysteine and proteinase K was 104 M. tuberculosis whole cells per ml. Thirty-one (91%) of 34 sputum samples from 18 Vietnamese patients with tuberculosis (32 smear positive and 2 smear negative) were positive in the LAM detection assay. In contrast, none of the 25 sputum samples from 21 nontuberculous patients was positive. This specific and sensitive assay for the detection of LAM in sputum is potentially useful for the diagnosis of tuberculosis. PMID:10834989

Single-cell immunofluorescence assay for terminal transferase: human leukaemic and non-leukaemic cells.

PubMed

Okamura, S; Crane, F; Jamal, N; Messner, H A; Mak, T W

1980-02-01

The characteristics of a single-cell immunofluorescence assay for terminal deoxynucleotidyl transferase (terminal transferase, TdT) is described. The data indicate that the single-cell immunofluorescence assay is highly efficient and specific for the detection of cells containing TdT. Using this assay, we have examined 124 marrow or peripheral-blood samples from 104 patients with or without haematological malignancies. Results indicate that TdT(+) cells from 6% to 100% were found in the following patients: 34/40 samples from patients with ALL at the time of diagnosis or during relapse; 2/3 patients with acute undifferentiated leukaemia; 2/3 patients with acute myelomonocytic leukaemia; 1/24 patients with acute myeloblastic leukaemia; 1/5 patients with chronic myelocytic leukaemia (CML) in blastic crisis; and 2/2 patients with diffuse lymphoblastic lymphoma. In contrast less than 1% of TdT(+) cells were found in 20 marrow or peripheral-blood samples from ALL patients in complete remission; 8 patients with CML in chronic phase; 2 patients with myeloma; 1 sample from a patient with Hodgkin's disease, peripheral-blood samples from 7 normal donors and marrow samples from 6 patients without haematological malignancies. TdT(+) cells were also found in association with cells with lymphoblast morphology. The TdT(+) cells in marrow were shown to be directly correlated with the percentage of morphological lymphoblasts, with a Spearman rank coefficient of 0·81, significant at a 0·001 level. In 2 longitudinal studies of 2 ALL patients with TdT(+) cells at diagnosis, the percentage TdT(+) cells also changed in parallel with the proportion of lymphoblasts. However, studies of 2 other patients with morphologically diagnosed ALL with < 1% TdT(+) cells at diagnosis also showed < 1% TdT(+) cells throughout the period studied, indicating a stable phenotype of blast cells in these patients. The single-cell immunofluorescence assay for TdT, which requires < 0·1% of the cells used in

Fibrinogen Motif Discriminates Platelet and Cell Capture in Peptide-Modified Gold Micropore Arrays.

PubMed

Adamson, Kellie; Spain, Elaine; Prendergast, Una; Moran, Niamh; Forster, Robert J; Keyes, Tia E

2018-01-16

Human blood platelets and SK-N-AS neuroblastoma cancer-cell capture at spontaneously adsorbed monolayers of fibrinogen-binding motifs, GRGDS (generic integrin adhesion), HHLGGAKQAGDV (exclusive to platelet integrin α IIb β 3 ), or octanethiol (adhesion inhibitor) at planar gold and ordered 1.6 μm diameter spherical cap gold cavity arrays were compared. In all cases, arginine/glycine/aspartic acid (RGD) promoted capture, whereas alkanethiol monolayers inhibited adhesion. Conversely only platelets adhered to alanine/glycine/aspartic acid (AGD)-modified surfaces, indicating that the AGD motif is recognized preferentially by the platelet-specific integrin, α IIb β 3 . Microstructuring of the surface effectively eliminated nonspecific platelet/cell adsorption and dramatically enhanced capture compared to RGD/AGD-modified planar surfaces. In all cases, adhesion was reversible. Platelets and cells underwent morphological change on capture, the extent of which depended on the topography of the underlying substrate. This work demonstrates that both the nature of the modified interface and its underlying topography influence the capture of cancer cells and platelets. These insights may be useful in developing cell-based cancer diagnostics as well as in identifying strategies for the disruption of platelet cloaks around circulating tumor cells.

A microfluidic platform for 3-dimensional cell culture and cell-based assays.

PubMed

Kim, Minseok S; Yeon, Ju Hun; Park, Je-Kyun

2007-02-01

This paper reports a novel microfluidic platform introducing peptide hydrogel to make biocompatible microenvironment as well as realizing in situ cell-based assays. Collagen composite, OPLA and Puramatrix scaffolds are compared to select good environment for human hepatocellular carcinoma cells (HepG2) by albumin measurement. The selected biocompatible self-assembling peptide hydrogel, Puramatrix, is hydrodynamically focused in the middle of main channel of a microfluidic device, and at the same time the cells are 3-dimensionally immobilized and encapsulated without any additional surface treatment. HepG2 cells have been 3-dimensionally cultured in a poly(dimethylsiloxane) (PDMS) microfluidic device for 4 days. The cells cultured in micro peptide scaffold are compared with those cultured by conventional petri dish in morphology and the rate of albumin secretion. By injection of different reagents into either side of the peptide scaffold, the microfluidic device also forms a linear concentration gradient profile across the peptide scaffold due to molecular diffusion. Based on this characteristic, toxicity tests are performed by Triton X-100. As the higher toxicant concentration gradient forms, the wider dead zone of cells in the peptide scaffold represents. This microfluidic platform facilitates in vivo-like 3-dimensional microenvironment, and have a potential for the applications of reliable cell-based screening and assays including cytotoxicity test, real-time cell viability monitoring, and continuous dose-response assay.

Comparison of glutathione levels measured using optimized monochlorobimane assay with those from ortho-phthalaldehyde assay in intact cells.

PubMed

Čapek, Jan; Hauschke, Martina; Brůčková, Lenka; Roušar, Tomáš

2017-11-01

Fluorometric glutathione assays have been generally preferred for their high specificity and sensitivity. An additional advantage offered by fluorescent bimane dyes is their ability to penetrate inside the cell. Their ability to react with glutathione within intact cells is frequently useful in flow cytometry and microscopy. Hence, the aims of our study were to use monochlorobimane for optimizing a spectrofluorometric glutathione assay in cells and then to compare that assay with the frequently used ortho-phthalaldehyde assay. We used glutathione-depleting agents (e.g., cisplatin and diethylmalonate) to induce cell impairment. For glutathione assessment, monochlorobimane (40μM) was added to cells and fluorescence was detected at 394/490nm. In addition to the regularly used calculation of glutathione levels from fluorescence change after 60min, we used an optimized calculation from the linear part of the fluorescence curve after 10min of measurement. We found that 10min treatment of cells with monochlorobimane is sufficient for evaluating cellular glutathione concentration and provides results entirely comparable with those from the standard ortho-phthalaldehyde assay. In contrast, the results obtained by the standardly used evaluation after 60min of monochlorobimane treatment provided higher glutathione values. We conclude that measuring glutathione using monochlorobimane with the here-described optimized evaluation of fluorescence signal could be a simple and useful method for routine and rapid assessment of glutathione within intact cells in large numbers of samples. Copyright © 2017 Elsevier Inc. All rights reserved.

Analysis of Histone Deacetylase-Dependent Effects on Cell Migration Using the Stripe Assay.

PubMed

Mertsch, Sonja; Thanos, Solon

2017-01-01

For normal embryonic development/morphogenesis, cell migration and homing are well-orchestrated and important events requiring specific cellular mechanisms. In diseases such as cancer deregulated cell migration represents a major problem. Therefore, numerous efforts are under way to understand the molecular mechanisms of tumor cell migration and to generate more efficient tumor therapies. Cell migration assays are one of the most commonly used functional assays. The wound-healing assay or the Boyden chamber assay are variations of these assays. Nearly all of them are two-dimensional assays and the cells can only migrate on one substrate at a time. This is in contrast to the in vivo situation where the cells are faced simultaneously with different surfaces and interact with different cell types. To approach this in vivo situation we used a modified version of the stripe assay designed by Bonhoeffer and colleagues to examine mechanisms of axonal guidance. The design of this assay allows cells to decide between two different substrates offered at the same time. Utilizing alternating neuronal substrates for migration analyses we can partially mimic the complex in vivo situation for brain tumor cells. Here we describe the detailed protocol to perform a modified version of the stripe assay in order to observe substrate-dependent migration effects in vitro, to analyze the effect of Rho-dependent kinases (ROCKS), of histone deacetylases (HDACs) and of other molecules on glioma cells.

Nanostructured Substrates for Capturing Circulating Tumor Cells in Whole Blood

NASA Astrophysics Data System (ADS)

Tseng, Hsian-Rong

2009-03-01

Over the past decade, circulating tumor cells (CTCs) has become an emerging ``biomarker'' for detecting early-stage cancer metastasis, predicting patient prognosis, as well as monitoring disease progression and therapeutic outcomes. However, isolation of CTCs has been technically challenging due to the extremely low abundance (a few to hundreds per ml) of CTCs among a high number of hematologic cells (109 per mL) in the blood. Our joint research team at UCLA has developed a new cell capture technology for quantification of CTCs in whole blood samples. Similar to most of the existing approaches, epithelial cell adhesion molecule antibody (anti-EpCAM) was grafted onto the surfaces to distinguish CTCs from the surrounding hematologic cells. The uniqueness of our technology is the use of nanostructured surfaces, which facilitates local topographical interactions between CTCs and substrates at the very first cell/substrate contacting time point. We demonstrated the ability of these nanostructured substrates to capture CTCs in whole blood samples with significantly improved efficiency and selectivity. The successful demonstration of this cell capture technology using brain, breast and prostate cancer cell lines encouraged us to test this approach in clinical setting. We have been able to bond our first validation study with a commercialized technology based on the use of immunomagnetic nanoparticles. A group of clinically well-characterized prostate cancer patients at UCLA hospital have been recruited and tested in parallel by these two technologies.

Enrichment of cancer cells using aptamers immobilized on a microfluidic channel

PubMed Central

Phillips, Joseph A.; Xu, Ye; Xia, Zheng

2009-01-01

This work describes the development and investigation of an aptamer modified microfluidic device that captures rare cells to achieve a rapid assay without pre-treatment of cells. To accomplish this, aptamers are first immobilized on the surface of a poly (dimethylsiloxane) microchannel, followed by pumping a mixture of cells through the device. This process permits the use of optical microscopy to measure the cell-surface density from which we calculate the percentage of cells captured as a function of cell and aptamer concentration, flow velocity, and incubation time. This aptamer-based device was demonstrated to capture target cells with > 97% purity and > 80% efficiency. Since the cell capture assay is completed within minutes and requires no pre-treatment of cells, the device promises to play a key role in the early detection and diagnosis of cancer where rare diseased cells can first be enriched and then captured for detection. PMID:19115856

Self-Sterilizing and Regeneratable Microchip for the Precise Capture and Recovery of Viable Circulating Tumor Cells from Patients with Cancer.

PubMed

Hui, Lanlan; Su, Yi; Ye, Tingting; Liu, Zhao; Tian, Qingchang; He, Chuanjiang; Zhao, Yueqi; Chen, Pu; Wang, Xiaojia; Han, Weidong; Luo, Yan; Wang, Ben

2018-01-10

Cancer cells metastasize and are transported in the bloodstream, easily reaching any site in the body through the blood circulation. A method designed to assess the number of circulating tumor cells (CTCs) should be validated as a clinical tool for predicting the response to therapy and monitoring the disease progression in patients with cancer. Although CTCs are detectable in many cases, they remain unavailable for clinic usage because of their high testing cost, tedious operation, and poor clinical relevance. Herein, we developed a regeneratable microchip for isolating CTCs, which is available for robust cell heterogeneity assays on-site without the need for a sterile environment. The ivy-like hierarchical roughened zinc oxide (ZnO) nanograss interface was synthesized and directly integrated into the microfluidic devices and enables effective CTC capture and flexible, nontoxic CTC release during incubation in a mildly acidic solution, thus enabling cellular and molecular analyses. The microchip can be regenerated and recycled to capture CTCs with the remaining ZnO without affecting the efficiency, even after countless cycles of cell release. Moreover, microbial infection is avoided during its storage, distribution, and even in the open space usage, which ideally appeals to the demands of point-of-care (POC) and home testing and meets to the requirements for blood examinations in undeveloped or resource-limited settings. Furthermore, the findings generated using this platform based on the cocktail of antiepithelial cell adhesion molecule and antivimentin antibodies indicate that CTC capture was more precise and reasonable for patients with advanced cancer.

Evaluation of agglutination strength by a flow-induced cell movement assay based surface plasmon resonance (SPR) technique.

PubMed

Sudprasert, Krisda; Peungthum, Patjaree; Vongsakulyanon, Apirom; Amarit, Ratthasart; Somboonkaew, Armote; Sutapun, Boonsong; Kitpoka, Pimpun; Kunakorn, Mongkol; Srikhirin, Toemsak

2015-02-07

A flow-induced cell movement assay combined with a surface plasmon resonance (SPR) technique was developed to quantify the agglutination strength, derived from the standard tube-agglutination test. Red blood cells (RBCs), based on the ABO blood group system, were specifically captured by anti-A and/or anti-B antibodies immobilized on a sensor surface. The agglutination strength corresponds to the amount of antigen-antibody interactions or the strength of RBC adhesion. Under a shear flow, the adherent RBCs were forced to move out of the region of interest with different average cell velocities (vc) depending upon the adhesion strength and wall shear stress (WSS). That is, a higher adhesion strength (higher agglutination strength) or lower WSS represents a lower vc or vice versa. In this work, the agglutination strength was derived from the vc that was calculated from the time derivative of the relative SPR signal by using a simple model of cell movement response, whose validity was verified. The vc values of different samples were correlated with their agglutination strengths at a given WSS and antibody surface density. The vc decreased as the agglutination strength increased, which can be considered as a linear regression. The coefficient of variation of the calculated vc decreased to 0.1 as vc increased to 30 μm min(-1). The sensitivity of this assay can be controlled by optimizing the antibody surface density or the WSS. This assay has the capability to resolve the antigen density of A1 and B RBCs from that of A1B RBCs.

Automated image-based assay for evaluation of HIV neutralization and cell-to-cell fusion inhibition.

PubMed

Sheik-Khalil, Enas; Bray, Mark-Anthony; Özkaya Şahin, Gülsen; Scarlatti, Gabriella; Jansson, Marianne; Carpenter, Anne E; Fenyö, Eva Maria

2014-08-30

Standardized techniques to detect HIV-neutralizing antibody responses are of great importance in the search for an HIV vaccine. Here, we present a high-throughput, high-content automated plaque reduction (APR) assay based on automated microscopy and image analysis that allows evaluation of neutralization and inhibition of cell-cell fusion within the same assay. Neutralization of virus particles is measured as a reduction in the number of fluorescent plaques, and inhibition of cell-cell fusion as a reduction in plaque area. We found neutralization strength to be a significant factor in the ability of virus to form syncytia. Further, we introduce the inhibitory concentration of plaque area reduction (ICpar) as an additional measure of antiviral activity, i.e. fusion inhibition. We present an automated image based high-throughput, high-content HIV plaque reduction assay. This allows, for the first time, simultaneous evaluation of neutralization and inhibition of cell-cell fusion within the same assay, by quantifying the reduction in number of plaques and mean plaque area, respectively. Inhibition of cell-to-cell fusion requires higher quantities of inhibitory reagent than inhibition of virus neutralization.

Cell-of-Origin of Cancer versus Cancer Stem Cells: Assays and Interpretations.

PubMed

Rycaj, Kiera; Tang, Dean G

2015-10-01

A tumor originates from a normal cell that has undergone tumorigenic transformation as a result of genetic mutations. This transformed cell is the cell-of-origin for the tumor. In contrast, an established clinical tumor is sustained by subpopulations of self-renewing cancer cells operationally called cancer stem cells (CSC) that can generate, intraclonally, both tumorigenic and nontumorigenic cells. Identifying and characterizing tumor cell-of-origin and CSCs should help elucidate tumor cell heterogeneity, which, in turn, should help understand tumor cell responses to clinical treatments, drug resistance, tumor relapse, and metastatic spread. Both tumor transplantation and lineage-tracing assays have been helpful in characterizing these cancer cell populations, although each system has its strengths and caveats. In this article, we briefly review and summarize advantages and limitations of both assays in support of a combinatorial approach to accurately define the roles of both cancer-initiating and cancer-propagating cells. As an aside, we also wish to clarify the definitions of cancer cell-of-origin and CSCs, which are often interchangeably used by mistake. ©2015 American Association for Cancer Research.

A PDMS Device Coupled with Culture Dish for In Vitro Cell Migration Assay.

PubMed

Lv, Xiaoqing; Geng, Zhaoxin; Fan, Zhiyuan; Wang, Shicai; Pei, WeiHua; Chen, Hongda

2018-04-30

Cell migration and invasion are important factors during tumor progression and metastasis. Wound-healing assay and the Boyden chamber assay are efficient tools to investigate tumor development because both of them could be applied to measure cell migration rate. Therefore, a simple and integrated polydimethylsiloxane (PDMS) device was developed for cell migration assay, which could perform quantitative evaluation of cell migration behaviors, especially for the wound-healing assay. The integrated device was composed of three units, which included cell culture dish, PDMS chamber, and wound generation mold. The PDMS chamber was integrated with cell culture chamber and could perform six experiments under different conditions of stimuli simultaneously. To verify the function of this device, it was utilized to explore the tumor cell migration behaviors under different concentrations of fetal bovine serum (FBS) and transforming growth factor (TGF-β) at different time points. This device has the unique capability to create the "wound" area in parallel during cell migration assay and provides a simple and efficient platform for investigating cell migration assay in biomedical application.

Assessment of biological effectiveness of boron neutron capture therapy in primary and metastatic melanoma cell lines.

PubMed

Rossini, Andrés E; Dagrosa, Maria A; Portu, Agustina; Saint Martin, Giselle; Thorp, Silvia; Casal, Mariana; Navarro, Aimé; Juvenal, Guillermo J; Pisarev, Mario A

2015-01-01

In order to optimize the effectiveness of Boron Neutron Capture Therapy (BNCT), Relative Biological Effectiveness (RBE) and Compound Biological Effectiveness (CBE) were determined in two human melanoma cell lines, M8 and Mel-J cells, using the amino acid p-boronophenylalanine (BPA) as boron carrier. The effects of BNCT on the primary amelanotic cell line M8 and on the metastatic pigmented melanoma cell line Mel-J were studied using colony formation assay. The RBE values were determined using both a gamma ray source, and the neutron beam from the Nuclear Reactor of the National Atomic Energy Commission (RA-3). For the determination of the RBE, cells were irradiated with increasing doses of both sources, between 1 and 8 Gy; and for the determination of CBE factors, the cells were pre-incubated with BPA before irradiation. Afterwards, the cell surviving fraction (SF) was determined for each treatment. Marked differences were observed between both cell lines. Mel-J cells were more radioresistant than the M8 cell line. The clonogenic assays showed that for a SF of 1%, the RBE values were 1.3 for M8 cells and 1.5 for Mel-J cells. Similarly, the CBE values for a 1% SF were 2.1 for M8 and 3 for Mel-J cell lines. For the endpoint of 0.1% of SF the RBE values obtained were 1.2 for M8 and 1.4 for Mel-J cells. Finally, CBE values calculated for a 0.1% were 2 and 2.6 for M8 and Mel-J cell lines respectively. In order to estimate the uptake of the non-radioactive isotope Boron 10 ((10)B), a neutron induced autoradiographic technique was performed showing discrepancies in (10)B uptake between both cell lines. These obtained in vitro results are the first effectiveness factors determined for human melanoma at the RA-3 nuclear reactor and show that BNCT dosimetry planning for patients could be successfully performed using these new factors.

Electrochemical Responsive Superhydrophilic Surfaces of Polythiophene Derivatives towards Cell Capture and Release.

PubMed

Hao, Yuwei; Li, Yingying; Zhang, Feilong; Cui, Haijun; Hu, Jinsong; Meng, Jingxin; Wang, Shutao

2018-03-23

Highly efficient cell capture and release with low background are urgently required for early diagnosis of diseases such as cancer. Herein, we report an electrochemical responsive superhydrophilic surface exhibiting specific cell capture and release with high yields and extremely low nonspecific adhesion. Through electrochemical deposition, 3-substituted thiophene derivatives are deposited onto indium tin oxide (ITO) nanowire arrays with 4-n-nonylbenzeneboronic acid (BA) as dopant, fabricating the electrochemical responsive superhydrophilic surfaces. The molecular recognition between sialic acids over-expressed on the cell membrane and doped BAs endows the electrochemical responsive surfaces with the ability to capture and release targeted cancer cells. By adjusting the substituent group of thiophene derivatives, the surface wettability can be readily regulated and further utilized for reducing nonspecific cell adhesion. Significantly, the released cells still maintain a high proliferation ability, which indicates that the applied potential does not significantly harm the cells. Therefore, these results may provide a new strategy to achieve advanced functions of biomedical materials, such as low nonspecific adhesion. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microfluidics cell sample preparation for analysis: Advances in efficient cell enrichment and precise single cell capture

PubMed Central

Bian, Shengtai; Cheng, Yinuo; Shi, Guanya; Liu, Peng; Ye, Xiongying

2017-01-01

Single cell analysis has received increasing attention recently in both academia and clinics, and there is an urgent need for effective upstream cell sample preparation. Two extremely challenging tasks in cell sample preparation—high-efficiency cell enrichment and precise single cell capture—have now entered into an era full of exciting technological advances, which are mostly enabled by microfluidics. In this review, we summarize the category of technologies that provide new solutions and creative insights into the two tasks of cell manipulation, with a focus on the latest development in the recent five years by highlighting the representative works. By doing so, we aim both to outline the framework and to showcase example applications of each task. In most cases for cell enrichment, we take circulating tumor cells (CTCs) as the target cells because of their research and clinical importance in cancer. For single cell capture, we review related technologies for many kinds of target cells because the technologies are supposed to be more universal to all cells rather than CTCs. Most of the mentioned technologies can be used for both cell enrichment and precise single cell capture. Each technology has its own advantages and specific challenges, which provide opportunities for researchers in their own area. Overall, these technologies have shown great promise and now evolve into real clinical applications. PMID:28217240

High-content adhesion assay to address limited cell samples†

PubMed Central

Warrick, Jay W.; Young, Edmond W. K.; Schmuck, Eric G.; Saupe, Kurt W.

2013-01-01

Cell adhesion is a broad topic in cell biology that involves physical interactions between cells and other cells or the surrounding extracellular matrix, and is implicated in major research areas including cancer, development, tissue engineering, and regenerative medicine. While current methods have contributed significantly to our understanding of cell adhesion, these methods are unsuitable for tackling many biological questions requiring intermediate numbers of cells (102–105), including small animal biopsies, clinical samples, and rare cell isolates. To overcome this fundamental limitation, we developed a new assay to quantify the adhesion of ~102–103 cells at a time on engineered substrates, and examined the adhesion strength and population heterogeneity via distribution-based modeling. We validated the platform by testing adhesion strength of cancer cells from three different cancer types (breast, prostate, and multiple myeloma) on both IL-1β activated and non-activated endothelial monolayers, and observed significantly increased adhesion for each cancer cell type upon endothelial activation, while identifying and quantifying distinct subpopulations of cell-substrate interactions. We then applied the assay to characterize adhesion of primary bone marrow stromal cells to different cardiac fibroblast-derived matrix substrates to demonstrate the ability to study limited cell populations in the context of cardiac cell-based therapies. Overall, these results demonstrate the sensitivity and robustness of the assay as well as its ability to enable extraction of high content, functional data from limited and potentially rare primary samples. We anticipate this method will enable a new class of biological studies with potential impact in basic and translational research. PMID:23426645

α2-Macroglobulin Capture Allows Detection of Mast Cell Chymase in Serum and Creates a Circulating Reservoir of Angiotensin II-generating Activity1

PubMed Central

Raymond, Wilfred W.; Su, Sharon; Makarova, Anastasia; Wilson, Todd M.; Carter, Melody C.; Metcalfe, Dean D.; Caughey, George H.

2009-01-01

Human chymase is a highly efficient angiotensin II-generating serine peptidase expressed by the MCTC subset of mast cells. When secreted from degranulating cells, it can interact with a variety of circulating anti-peptidases, but is mostly captured by α2-macroglobulin, which sequesters peptidases in a cage-like structure that precludes interactions with large protein substrates and inhibitors, like serpins. The present work shows that α2-macroglobulin-bound chymase remains accessible to small substrates, including angiotensin I, with activity in serum that is stable with prolonged incubation. We used α2-macroglobulin capture to develop a sensitive, microtiter plate-based assay for serum chymase, assisted by a novel substrate synthesized based on results of combinatorial screening of peptide substrates. The substrate has low background hydrolysis in serum and is chymase-selective, with minimal cleavage by the chymotryptic peptidases cathepsin G and chymotrypsin. The assay detects activity in chymase-spiked serum with a threshold of ~1 pM (30 pg/ml), and reveals native chymase activity in serum of most subjects with systemic mastocytosis. α2-Macroglobulin-bound chymase generates angiotensin II in chymase-spiked serum, and appears in native serum as chymostatin-inhibited activity, which can exceed activity of captopril-sensitive angiotensin converting enzyme. These findings suggest that chymase bound to α2-macroglobulin is active, that the circulating complex is an angiotensin-converting enzyme inhibitor-resistant reservoir of angiotensin II-generating activity, and that α2-macroglobulin capture may be exploited in assessing systemic release of secreted peptidases. PMID:19380825

Biochemical Assays of Cultured Cells

NASA Technical Reports Server (NTRS)

Barlow, G. H.

1985-01-01

Subpopulations of human embryonic kidney cells isolated from continuous flow electrophoresis experiments performed at McDonnell Douglas and on STS-8 have been analyzed. These analyses have included plasminogen activator assays involving indirect methodology on fibrin plated and direct methodology using chromogenic substrates. Immunological studies were performed and the conditioned media for erythropoietin activity and human granulocyte colony stimulating (HGCSF) activity was analyzed.

Simultaneous capture and in situ analysis of circulating tumor cells using multiple hybrid nanoparticles.

PubMed

Lee, Hun Joo; Cho, Hyeon-Yeol; Oh, Jin Ho; Namkoong, Kak; Lee, Jeong Gun; Park, Jong-Myeon; Lee, Soo Suk; Huh, Nam; Choi, Jeong-Woo

2013-09-15

Using hybrid nanoparticles (HNPs), we demonstrate simultaneous capture, in situ protein expression analysis, and cellular phenotype identification of circulating tumor cells (CTCs). Each HNP consists of three parts: (i) antibodies that bind specifically to a known biomarker for CTCs, (ii) a quantum dot that emits fluorescence signals, and (iii) biotinylated DNA that allows capture and release of CTC-HNP complex to an in-house developed capture & recovery chip (CRC). To evaluate our approach, cells representative of different breast cancer subtypes (MCF-7: luminal; SK-BR-3: HER2; and MDA-MB-231: basal-like) were captured onto CRC and expressions of EpCAM, HER2, and EGFR were detected concurrently. The average capture efficiency of CTCs was 87.5% with identification accuracy of 92.4%. Subsequently, by cleaving the DNA portion with restriction enzymes, captured cells were released at efficiencies of 86.1%. Further studies showed that these recovered cells are viable and can proliferate in vitro. Using HNPs, it is possible to count, analyze in situ protein expression, and culture CTCs, all from the same set of cells, enabling a wide range of molecular- and cellular-based studies using CTCs. Copyright © 2013 Elsevier B.V. All rights reserved.

Prostate cancer marker panel with single cell sensitivity in urine.

PubMed

Nickens, Kristen P; Ali, Amina; Scoggin, Tatiana; Tan, Shyh-Han; Ravindranath, Lakshmi; McLeod, David G; Dobi, Albert; Tacha, David; Sesterhenn, Isabell A; Srivastava, Shiv; Petrovics, Gyorgy

2015-06-15

Over one million men undergo prostate biopsies annually in the United States, a majority of whom due to elevated serum PSA. More than half of the biopsies turn out to be negative for prostate cancer (CaP). The limitations of both the PSA test and the biopsy procedure have led to the development for more precise CaP detection assays in urine (e.g., PCA3, TMPRSS2-ERG) or blood (e.g., PHI, 4K). Here, we describe the development and evaluation of the Urine CaP Marker Panel (UCMP) assay for sensitive and reproducible detection of CaP cells in post-digital rectal examination (post-DRE) urine. The cellular content of the post-DRE urine was captured on a translucent filter membrane, which is placed on Cytoclear slides for direct evaluation by microscopy and immuno-cytochemistry (ICC). Cells captured on the membrane were assayed for PSA and Prostein expression to identify prostate epithelial cells, and for ERG and AMACR to identify prostate tumor cells. Immunostained cells were analyzed for quantitative and qualitative features and correlated with biopsy positive and negative status for malignancy. The assay was optimized for single cell capture sensitivity and downstream evaluations by spiking a known number of cells from established CaP cell lines, LNCaP and VCaP, into pre-cleared control urine. The cells captured from the post-DRE urine of subjects, obtained prior to biopsy procedure, were co-stained for ERG, AMACR (CaP specific), and Prostein or PSA (prostate epithelium specific) rendering a whole cell based analysis and characterization. A feasibility cohort of 63 post-DRE urine specimens was assessed. Comparison of the UCMP results with blinded biopsy results showed an assay sensitivity of 64% (16 of 25) and a specificity of 68.8% (22 of 32) for CaP detection by biopsy. This pilot study assessing a minimally invasive CaP detection assay with single cell sensitivity cell-capture and characterization from the post-DRE urine holds promise for further development of this

Real-time quantification of antibody-short interfering RNA conjugate in serum by antigen capture reverse transcription-polymerase chain reaction.

PubMed

Tan, Martha; Vernes, Jean-Michel; Chan, Joyce; Cuellar, Trinna L; Asundi, Aarati; Nelson, Christopher; Yip, Victor; Shen, Ben; Vandlen, Richard; Siebel, Christian; Meng, Y Gloria

2012-11-15

Short interfering RNA (siRNA) has therapeutic potential. However, efficient delivery is a formidable task. To facilitate delivery of siRNA into cells, we covalently conjugated siRNA to antibodies that bind to cell surface proteins and internalize. Understanding how these antibody-siRNA conjugates function in vivo requires pharmacokinetic analysis. Thus, we developed a simple real-time antigen capture reverse transcription-polymerase chain reaction (RT-PCR) assay to detect intact antibody-siRNA conjugates. Biotinylated antigen bound to streptavidin-coated PCR tubes was used to capture antibody-siRNA conjugate. The captured antibody-siRNA conjugate was then reverse-transcribed in the same tube, avoiding a sample transfer step. This reproducible assay had a wide standard curve range of 0.029 to 480ng/ml and could detect as low as 0.58ng/ml antibody-siRNA conjugates in mouse serum. The presence of unconjugated antibody that could be generated from siRNA degradation in vivo did not affect the assay as long as the total antibody concentration in the antigen capture step did not exceed 480ng/ml. Using this assay, we observed a more rapid decrease in serum antibody-siRNA conjugate concentrations than the total antibody concentrations in mice dosed with antibody-siRNA conjugates, suggesting loss of siRNA from the antibody. This assay is useful for optimizing antibody-siRNA and likely aptamer-siRNA conjugates to improve pharmacokinetics and aid siRNA delivery. Copyright © 2012 Elsevier Inc. All rights reserved.

In vitro immunotherapy potency assays using real-time cell analysis

PubMed Central

Cerignoli, Fabio; Abassi, Yama A.; Lamarche, Brandon J.; Guenther, Garret; Santa Ana, David; Guimet, Diana; Zhang, Wen; Zhang, Jing

2018-01-01

A growing understanding of the molecular interactions between immune effector cells and target tumor cells, coupled with refined gene therapy approaches, are giving rise to novel cancer immunotherapeutics with remarkable efficacy in the clinic against both solid and liquid tumors. While immunotherapy holds tremendous promise for treatment of certain cancers, significant challenges remain in the clinical translation to many other types of cancers and also in minimizing adverse effects. Therefore, there is an urgent need for functional potency assays, in vitro and in vivo, that could model the complex interaction of immune cells with tumor cells and can be used to rapidly test the efficacy of different immunotherapy approaches, whether it is small molecule, biologics, cell therapies or combinations thereof. Herein we report the development of an xCELLigence real-time cytolytic in vitro potency assay that uses cellular impedance to continuously monitor the viability of target tumor cells while they are being subjected to different types of treatments. Specialized microtiter plates containing integrated gold microelectrodes enable the number, size, and surface attachment strength of adherent target tumor cells to be selectively monitored within a heterogeneous mixture that includes effector cells, antibodies, small molecules, etc. Through surface-tethering approach, the killing of liquid cancers can also be monitored. Using NK92 effector cells as example, results from RTCA potency assay are very well correlated with end point data from image-based assays as well as flow cytometry. Several effector cells, i.e., PBMC, NK, CAR-T were tested and validated as well as biological molecules such as Bi-specific T cell Engagers (BiTEs) targeting the EpCAM protein expressed on tumor cells and blocking antibodies against the immune checkpoint inhibitor PD-1. Using the specifically designed xCELLigence immunotherapy software, quantitative parameters such as KT50 (the amount of time

A portable circulating tumor cell capture microdevice

NASA Astrophysics Data System (ADS)

Datar, Ram H.

2009-03-01

Sensitive detection of earliest metastatic spread of tumors in a minimally invasive and user-friendly manner will revolutionize the clinical management of cancer patients. The current methodologies for circulating tumor cell (CTC) capture and identification have significant limitations including time, cost, limited capture efficiency and lack of standardization. We have developed and optimized a novel parylene membrane filter-based portable microdevice for size-based isolation of CTC from human peripheral blood. Following characterization with a model system to study the recovery rate and enrichment factor, a comparison of the microdevice with the commercially available system using blood from cancer patients demonstrated superior recovery rate and the promise of clinical utility of the microdevice. The development of the microdevice and its potential clinical applicability will be discussed.

A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells.

PubMed

O'Brien, Martha; Moehring, Danielle; Muñoz-Planillo, Raúl; Núñez, Gabriel; Callaway, Justin; Ting, Jenny; Scurria, Mike; Ugo, Tim; Bernad, Laurent; Cali, James; Lazar, Dan

2017-08-01

Inflammasomes are protein complexes induced by diverse inflammatory stimuli that activate caspase-1, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent, plate-based assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized lytic reagent added directly to cultured cells. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor in the lytic reagent and by use of a caspase-1 inhibitor to confirm activity. This approach enables a specific and rapid determination of caspase-1 activation. Caspase-1 activity is stable in the reagent thereby providing assay convenience and flexibility. Using this assay system, caspase-1 activation has been determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, gramicidin, MSU, R848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages (BMDMs) from mice, as well as in human primary monocytes. Caspase-1 activity was not detected in treated BMDMs derived from Casp1 -/- mice, further confirming the specificity of the assay. Caspase-1 activity can be measured directly in cultured cells using the lytic reagent, or caspase-1 activity released into medium can be monitored by assay of transferred supernatant. The caspase-1 assay can be multiplexed with other assays to monitor additional parameters from the same cells, such as IL-1β release or cell death. The caspase-1 assay in combination with a sensitive real-time monitor of cell death allows one to accurately establish pyroptosis. This assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective

Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

PubMed

Pu, Xinzhu; Wang, Zemin; Klaunig, James E

2015-08-06

Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. Copyright © 2015 John Wiley & Sons, Inc.

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4 h 51Cr-release assay

PubMed Central

Kim, GG; Donnenberg, VS; Donnenberg, AD; Gooding, W; Whiteside, TL

2007-01-01

Natural killer (NK) cell- or T cell-mediated cytotoxicity traditionally is measured in 4-16h 51Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3−CD16+CD56+). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8–13% and reliably measures NK cell- or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies. PMID:17617419

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

PubMed

Kim, G G; Donnenberg, V S; Donnenberg, A D; Gooding, W; Whiteside, T L

2007-08-31

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

A Novel Assay for Antibody-Dependent Cell-Mediated Cytotoxicity against HIV-1- or SIV-Infected Cells Reveals Incomplete Overlap with Antibodies Measured by Neutralization and Binding Assays

PubMed Central

Alpert, Michael D.; Heyer, Lisa N.; Williams, David E. J.; Harvey, Jackson D.; Greenough, Thomas; Allhorn, Maria

2012-01-01

The resistance of human immunodeficiency virus type 1 (HIV-1) to antibody-mediated immunity often prevents the detection of antibodies that neutralize primary isolates of HIV-1. However, conventional assays for antibody functions other than neutralization are suboptimal. Current methods for measuring the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC) are limited by the number of natural killer (NK) cells obtainable from individual donors, donor-to-donor variation, and the use of nonphysiological targets. We therefore developed an ADCC assay based on NK cell lines that express human or macaque CD16 and a CD4+ T-cell line that expresses luciferase from a Tat-inducible promoter upon HIV-1 or simian immunodeficiency virus (SIV) infection. NK cells and virus-infected targets are mixed in the presence of serial plasma dilutions, and ADCC is measured as the dose-dependent loss of luciferase activity. Using this approach, ADCC titers were measured in plasma samples from HIV-infected human donors and SIV-infected macaques. For the same plasma samples paired with the same test viruses, this assay was approximately 2 orders of magnitude more sensitive than optimized assays for neutralizing antibodies—frequently allowing the measurement of ADCC in the absence of detectable neutralization. Although ADCC correlated with other measures of Env-specific antibodies, neutralizing and gp120 binding titers did not consistently predict ADCC activity. Hence, this assay affords a sensitive method for measuring antibodies capable of directing ADCC against HIV- or SIV-infected cells expressing native conformations of the viral envelope glycoprotein and reveals incomplete overlap of the antibodies that direct ADCC and those measured in neutralization and binding assays. PMID:22933282

Quantifying rates of cell migration and cell proliferation in co-culture barrier assays reveals how skin and melanoma cells interact during melanoma spreading and invasion.

PubMed

Haridas, Parvathi; Penington, Catherine J; McGovern, Jacqui A; McElwain, D L Sean; Simpson, Matthew J

2017-06-21

Malignant spreading involves the migration of cancer cells amongst other native cell types. For example, in vivo melanoma invasion involves individual melanoma cells migrating through native skin, which is composed of several distinct subpopulations of cells. Here, we aim to quantify how interactions between melanoma and fibroblast cells affect the collective spreading of a heterogeneous population of these cells in vitro. We perform a suite of circular barrier assays that includes: (i) monoculture assays with fibroblast cells; (ii) monoculture assays with SK-MEL-28 melanoma cells; and (iii) a series of co-culture assays initiated with three different ratios of SK-MEL-28 melanoma cells and fibroblast cells. Using immunostaining, detailed cell density histograms are constructed to illustrate how the two subpopulations of cells are spatially arranged within the spreading heterogeneous population. Calibrating the solution of a continuum partial differential equation to the experimental results from the monoculture assays allows us to estimate the cell diffusivity and the cell proliferation rate for the melanoma and the fibroblast cells, separately. Using the parameter estimates from the monoculture assays, we then make a prediction of the spatial spreading in the co-culture assays. Results show that the parameter estimates obtained from the monoculture assays lead to a reasonably accurate prediction of the spatial arrangement of the two subpopulations in the co-culture assays. Overall, the spatial pattern of spreading of the melanoma cells and the fibroblast cells is very similar in monoculture and co-culture conditions. Therefore, we find no clear evidence of any interactions other than cell-to-cell contact and crowding effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Three-Dimensional Inverse Opal Photonic Crystal Substrates toward Efficient Capture of Circulating Tumor Cells.

PubMed

Xu, Hongwei; Dong, Biao; Xiao, Qiaoqin; Sun, Xueke; Zhang, Xinran; Lyu, Jiekai; Yang, Yudan; Xu, Lin; Bai, Xue; Zhang, Shuang; Song, Hongwei

2017-09-13

Artificial fractal structures have attracted considerable scientific interest in circulating tumor cells (CTCs) detection and capture, which plays a pivotal role in the diagnosis and prognosis of cancer. Herein, we designed a bionic TiO 2 inverse opal photonic crystal (IOPC) structure for highly efficient immunocapture of CTCs by combination of a magnetic Fe 3 O 4 @C6@silane nanoparticles with anti-EpCAM (antiepithelial cell adhesion molecule) and microchannel structure. Porous structure and dimension of IOPC TiO 2 can be precisely controlled for mimicking cellular components, and anti-EpCAM antibody was further modified on IOPC interface by conjugating with polydopamine (PDA). The improvement of CTCs capture efficiency reaches a surprising factor of 20 for the IOPC interface compared to that on flat glass, suggesting that the IOPCs are responsible for the dramatic enhancement of the capture efficiency of MCF-7 cells. IOPC substrate with pore size of 415 nm leads to the optimal CTCs capture efficiency of 92% with 1 mL/h. Besides the cell affinity, IOPCs also have the advantage of light scattering property which can enhance the excitation and emission light of fluorescence labels, facilitating the real-time monitoring of CTCs capture. The IOPC-based platform demonstrates excellent performance in CTCs capture, which will take an important step toward specific recognition of disease-related rare cells.

Comparison of the Sanofi Diagnostics Pasteur Chlamydia Microplate EIA shortened assay with the original standard assay and cell culture.

PubMed

Chan, E L; Brandt, K; Stoneham, H; Horsman, G

1997-08-01

The new Sanofi Diagnostics Pasteur Chlamydia Microplate EIA shortened assay was evaluated by comparison with the original standard assay and cell culture. A total of 853 paired male and female genital tract specimens was tested with both Sanofi Chlamydia Microplate EIA shortened and standard assays and the results were compared with those of cell culture. For confirmation, a blocking assay run in the shortened format was used. Discrepancies between the three methods were resolved by a direct fluorescent antibody (DFA) test on the EIA samples or the culture retentate, or both. After resolution of discrepant results, the standard assay had a sensitivity, specificity, positive predictive value and negative predictive value of 98.5%, 100%, 100% and 99.9%, respectively. The shortened assay results were 100%, 100%, 100% and 100%, respectively. The shortened assay takes approximately 1.5 h less time than the standard assay and this study demonstrated that they have equivalent sensitivity and specificity. The improvement in turnaround time enables results to be reported on the same day.

A Rapid Survival Assay to Measure Drug-Induced Cytotoxicity and Cell Cycle Effects

PubMed Central

Valiathan, Chandni; McFaline, Jose L.

2012-01-01

We describe a rapid method to accurately measure the cytotoxicity of mammalian cells upon exposure to various drugs. Using this assay, we obtain survival data in a fraction of the time required to perform the traditional clonogenic survival assay, considered the gold standard. The dynamic range of the assay allows sensitivity measurements on a multi-log scale allowing better resolution of comparative sensitivities. Moreover, the results obtained contain additional information on cell cycle effects of the drug treatment. Cell survival is obtained from a quantitative comparison of proliferation between drug-treated and untreated cells. During the assay, cells are treated with a drug and, following a recovery period, allowed to proliferate in the presence of BrdU. Cells that synthesize DNA in the presence of bromodeoxyuridine (BrdU) exhibit quenched Hoechst fluorescence easily detected by flow cytometry; quenching is used to determine relative proliferation in treated versus untreated cells. Finally, the multi-well setup of this assay allows the simultaneous screening of multiple cell lines, multiple doses, or multiple drugs to accurately measure cell survival and cell cycle changes after drug treatment. PMID:22133811

A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase 1 Not Found Using a Cell-Free Enzyme Assay.

PubMed

Kashem, Mohammed A; Kennedy, Charles A; Fogarty, Kylie E; Dimock, Janice R; Zhang, Yunlong; Sanville-Ross, Mary L; Skow, Donna J; Brunette, Steven R; Swantek, Jennifer L; Hummel, Heidi S; Swindle, John; Nelson, Richard M

2016-01-01

Sphingosine kinase 1 (SphK1) is a lipid kinase that phosphorylates sphingosine to produce the bioactive sphingolipid, sphingosine-1-phosphate (S1P), and therefore represents a potential drug target for a variety of pathological processes such as fibrosis, inflammation, and cancer. We developed two assays compatible with high-throughput screening to identify small-molecule inhibitors of SphK1: a purified component enzyme assay and a genetic complementation assay in yeast cells. The biochemical enzyme assay measures the phosphorylation of sphingosine-fluorescein to S1P-fluorescein by recombinant human full-length SphK1 using an immobilized metal affinity for phosphochemicals (IMAP) time-resolved fluorescence resonance energy transfer format. The yeast assay employs an engineered strain of Saccharomyces cerevisiae, in which the human gene encoding SphK1 replaced the yeast ortholog and quantitates cell viability by measuring intracellular adenosine 5'-triphosphate (ATP) using a luciferase-based luminescent readout. In this assay, expression of human SphK1 was toxic, and the resulting yeast cell death was prevented by SphK1 inhibitors. We optimized both assays in a 384-well format and screened ∼10(6) compounds selected from the Boehringer Ingelheim library. The biochemical IMAP high-throughput screen identified 5,561 concentration-responsive hits, most of which were ATP competitive and not selective over sphingosine kinase 2 (SphK2). The yeast screen identified 205 concentration-responsive hits, including several distinct compound series that were selective against SphK2 and were not ATP competitive.

Research Techniques Made Simple: Analysis of Collective Cell Migration Using the Wound Healing Assay.

PubMed

Grada, Ayman; Otero-Vinas, Marta; Prieto-Castrillo, Francisco; Obagi, Zaidal; Falanga, Vincent

2017-02-01

Collective cell migration is a hallmark of wound repair, cancer invasion and metastasis, immune responses, angiogenesis, and embryonic morphogenesis. Wound healing is a complex cellular and biochemical process necessary to restore structurally damaged tissue. It involves dynamic interactions and crosstalk between various cell types, interaction with extracellular matrix molecules, and regulated production of soluble mediators and cytokines. In cutaneous wound healing, skin cells migrate from the wound edges into the wound to restore skin integrity. Analysis of cell migration in vitro is a useful assay to quantify alterations in cell migratory capacity in response to experimental manipulations. Although several methods exist to study cell migration (such as Boyden chamber assay, barrier assays, and microfluidics-based assays), in this short report we will explain the wound healing assay, also known as the "in vitro scratch assay" as a simple, versatile, and cost-effective method to study collective cell migration and wound healing. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Monochromosomal hybrid cell assay for evaluating the genotoxicity of environmental chemicals

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

Sandhu, S.S.; Gudi, R.D.; Athwal, R.S.

1988-12-01

The development and utilization of a monochromosomal hybrid cell assay for detecting aneuploidy and chromosomal aberrations are described. The monochromosomal hybrid cell lines were produced by a two-step process involving transfer of a marker bacterial gene to a human chromosome and then by integration of that human chromosome into a mouse complement of chromosomes through microcell fusion. For chemically induced aneuploidy, the segregation of a single human chromosome among mouse chromosomes is used as a cytogenetic marker. The genetic assay for aneuploidy is based on the ability of the cells to grow in a medium that selects for the lossmore » of the human chromosome. The assay for clastogenicity is based on survival of the cells after treatment with the chemicals in medium that selects for retention of the human chromosome but loss of its segment containing diphtheria toxin locus. The assays greatly simplify the detection of chromosomal aberrations induced by environmental factors at low-dose levels.« less

Microfluidic Cell-based Assays in Stem Cell and Other Rare Cell Type Research

DOE PAGES

Wu, Meiye

2015-03-23

Microfluidics is a technology defined by the engineered precise manipulation of minute amount of liquids through channels with dimensions in the micron scale. Much of microfluidic devices used for biomedical purposes are produced in the form of so called “lab-on-a-chip” format, where multiple steps of conventional biochemical analyses such as staining, washing, and signal collection are miniaturized and integrated into chips fabricated from polymer or glass. Cell-based microfluidic lab-on-achip technology provides some obvious advantages: 1) drastically reduced sample and reagent requirement, and 2) separation and detection with improved sensitivity due to fluid properties at the microscale, i.e. laminar flow. Basedmore » on these two advantages, the obvious place where microfluidic cell assays will provide the most benefit is wherescientists must gather much information from precious little sample. Stem cells and other precious cell types such as circulating tumor cells (CTCs), and rare immune subsets are the perfect match for microfluidic multiplex assays. The recent demonstration that multiple cellular changes such as surface receptor activation, protein translocation, long and short RNA, and DNA changes can all be extracted from intact single cells paves the way to systems level understanding of cellular states during development or disease. Finally, with the ability to preserve cell integrity in a microfluidic device during multiplexed analysis, one also preserves the single cell resolution, where information regarding the cell-to-cell heterogeneity during differentiation or response to stimuli is vitally important.« less

High-content image analysis (HCIA) assay has the highest correlation with direct counting cell suspension compared to the ATP, WST-8 and Alamar blue assays for measurement of cytotoxicity.

PubMed

Tahara, Haruna; Matsuda, Shun; Yamamoto, Yusuke; Yoshizawa, Hiroe; Fujita, Masaharu; Katsuoka, Yasuhiro; Kasahara, Toshihiko

2017-11-01

Various cytotoxicity assays measuring indicators such as enzyme activity, dye uptake, or cellular ATP content are often performed using 96-well microplates. However, recent reports show that cytotoxicity assays such as the ATP assay and MTS assay underestimate cytotoxicity when compounds such as anti-cancer drugs or mutagens induce cell hypertrophy whilst increasing intracellular ATP content. Therefore, we attempted to evaluate the reliability of a high-content image analysis (HCIA) assay to count cell number in a 96-well microplate automatically without using a cell-number indicator. We compared cytotoxicity results of 25 compounds obtained from ATP, WST-8, Alamar blue, and HCIA assays with those directly measured using an automatic cell counter, and repeating individual experiments thrice. The number of compounds showing low correlation in cell viability measured using cytotoxicity assays compared to automatic cell counting (r 2 <0.8, at least 2 of 3 experiments) were follows: ATP assay; 7; WST-8 assay, 2; Alamar blue assay, 3; HCIA cytotoxicity assay, 0. Compounds for which correlation was poor in 3 assays, except the HCIA assay, induced an increase in nuclear and cell size. However, correlation between cell viability measured by automatic cell counter and the HCIA assay was strong regardless of nuclear and cell size. Additionally, correlation coefficients between IC 50 values obtained from automatic cell counter and from cytotoxicity assays were as follows: ATP assay, 0.80; WST-8 assay, 0.84; Alamar blue assay, 0.84; and HCIA assay, 0.98. From the above, we showed that the HCIA cytotoxicity assay produces similar data to the automatic cell counter and is highly accurate in measuring cytotoxicity. Copyright © 2017 Elsevier Inc. All rights reserved.

Single cell kinase signaling assay using pinched flow coupled droplet microfluidics.

PubMed

Ramji, Ramesh; Wang, Ming; Bhagat, Ali Asgar S; Tan Shao Weng, Daniel; Thakor, Nitish V; Teck Lim, Chwee; Chen, Chia-Hung

2014-05-01

Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel (5 mm) to separate cell aggregates and to form a uniform cell distribution in a droplet-generating platform that encapsulated single cells with >55% encapsulation efficiency beating Poisson encapsulation statistics. Using this platform and commercially available Sox substrates (8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline), we have demonstrated a high throughput dynamic single cell signaling assay to measure the activity of receptor tyrosine kinases (RTKs) in lung cancer cells triggered by cell surface ligand binding. The phosphorylation of the substrates resulted in fluorescent emission, showing a sigmoidal increase over a 12 h period. The result exhibited a heterogeneous signaling rate in individual cells and showed various levels of drug resistance when treated with the tyrosine kinase inhibitor, gefitinib.

A Fully Automated High-Throughput Zebrafish Behavioral Ototoxicity Assay.

PubMed

Todd, Douglas W; Philip, Rohit C; Niihori, Maki; Ringle, Ryan A; Coyle, Kelsey R; Zehri, Sobia F; Zabala, Leanne; Mudery, Jordan A; Francis, Ross H; Rodriguez, Jeffrey J; Jacob, Abraham

2017-08-01

Zebrafish animal models lend themselves to behavioral assays that can facilitate rapid screening of ototoxic, otoprotective, and otoregenerative drugs. Structurally similar to human inner ear hair cells, the mechanosensory hair cells on their lateral line allow the zebrafish to sense water flow and orient head-to-current in a behavior called rheotaxis. This rheotaxis behavior deteriorates in a dose-dependent manner with increased exposure to the ototoxin cisplatin, thereby establishing itself as an excellent biomarker for anatomic damage to lateral line hair cells. Building on work by our group and others, we have built a new, fully automated high-throughput behavioral assay system that uses automated image analysis techniques to quantify rheotaxis behavior. This novel system consists of a custom-designed swimming apparatus and imaging system consisting of network-controlled Raspberry Pi microcomputers capturing infrared video. Automated analysis techniques detect individual zebrafish, compute their orientation, and quantify the rheotaxis behavior of a zebrafish test population, producing a powerful, high-throughput behavioral assay. Using our fully automated biological assay to test a standardized ototoxic dose of cisplatin against varying doses of compounds that protect or regenerate hair cells may facilitate rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

Comparison of the Roche Cobas(®) 4800 HPV assay to Digene Hybrid Capture 2, Roche Linear Array and Roche Amplicor for Detection of High-Risk Human Papillomavirus Genotypes in Women undergoing treatment for cervical dysplasia.

PubMed

Phillips, Samuel; Garland, Suzanne M; Tan, Jeffery H; Quinn, Michael A; Tabrizi, Sepehr N

2015-01-01

The recently FDA (U.S. food and drug administration) approved Roche Cobas(®) 4800 (Cobas) human papillomavirus (HPV) has limited performance data compared to current HPV detection methods for test of cure in women undergoing treatment for high grade lesions. Evaluation of Cobas HPV assay using historical samples from women undergoing treatment for cervical dysplasia. A selection of 407 samples was tested on the Cobas assay and compared to previous results from Hybrid Capture 2, HPV Amplicor and Roche Linear Array. Overall, a correlation between high-risk HPV positivity and high grade histological diagnosis was 90.6% by the Cobas, 86.1% by Hybrid Capture 2, 92.9% by HPV Amplicor and 91.8% by Roche Linear Array. The Cobas HPV assay is comparative to both the HPV Amplicor and Roche Linear Array assays and better than Hybrid capture 2 assay in the detection of High-Risk HPV in women undergoing treatment for cervical dysplasia. Copyright © 2014 Elsevier B.V. All rights reserved.

Real time monitoring and quantification of reactive oxygen species in breast cancer cell line MCF-7 by 2',7'-dichlorofluorescin diacetate (DCFDA) assay.

PubMed

Figueroa, Daniela; Asaduzzaman, Mohammad; Young, Fiona

2018-04-07

The detection of reactive oxygen species (ROS) using 2',7'-dichlorofluorescin diacetate (DCFDA) is commonly performed by a single measurement of fluorescence but this fails to capture a profile of ROS generation over time. This study aimed to develop a real-time monitoring method to increase the utility of the assay, to incorporate cytotoxicity screening and to describe the combined effects of DCFDA and the ROS generator, Ter-butyl hydrogen peroxide (TBHP). Breast cancer MCF-7 cells were loaded with DCFDA (0-50 μM) for 45 min, and then exposed to TBHP (0-50 μM). Fluorescence was recorded according to three different schedules: every hour for 6 h, or once after 6 h or 24 h. Viability was assessed in a crystal violet assay and cell morphology was examined by microscopy. TBHP caused a time and dose-dependent increase in ROS and the magnitude of the fluorescent signal was affected by the loading concentration of DCFDA. Reading the fluorescence every hour for 6 h did not diminish the emission signal. The most sensitive and reliable combination for this ROS assay was 10 μM DCFDA with 25 μM TBHP; since higher concentrations of DCFDA compromised cell viability. In conclusion we adapted a single point ROS assay to enable production of a profile of ROS generation over an extended 6 h period, and related this to cell viability and morphology. Published by Elsevier Inc.

Hematopoietic Stem Cell Capture and Directional Differentiation into Vascular Endothelial Cells for Metal Stent-Coated Chitosan/Hyaluronic Acid Loading CD133 Antibody

PubMed Central

Zhang, Fan; Feng, Bo; Fan, Qingyu; Yang, Feng; Shang, Debin; Sui, Jinghan; Zhao, Hong

2015-01-01

A series of metal stents coated with chitosan/hyaluronic acid (CS/HA) loading antibodies by electrostatic self-assembled method were prepared, and the types of cells captured by antibodies and their differentiation in vascular endothelial cells (ECs) evaluated by molecular biology and scanning electron microscope. The results showed that CD133 stent can selectively capture hematopoietic stem cells (HSC),which directionally differentiate into vascular ECs in peripheral blood by (CS/HA) induction, and simultaneously inhibit migration and proliferation of immune cells and vascular smooth muscle cells (MCs). CD34 stent can capture HSC, hematopoietic progenitor cells that differentiate into vascular ECs and immune cells, promoting smooth MCs growth, leading to thrombosis, inflammation, and rejection. CD133 stent can be implanted into miniature pig heart coronary and can repair vascular damage by capturing own HSC, thus contributing to the rapid natural vascular repair, avoiding inflammation and rejection, thrombosis and restenosis. These studies demonstrated that CD133 stent of HSC capture will be an ideal coated metal stent providing a new therapeutic approach for cardiovascular and cerebrovascular disease. PMID:25404533

Hematopoietic stem cell capture and directional differentiation into vascular endothelial cells for metal stent-coated chitosan/hyaluronic acid loading CD133 antibody.

PubMed

Zhang, Shixuan; Zhang, Fan; Feng, Bo; Fan, Qingyu; Yang, Feng; Shang, Debin; Sui, Jinghan; Zhao, Hong

2015-03-01

A series of metal stents coated with chitosan/hyaluronic acid (CS/HA) loading antibodies by electrostatic self-assembled method were prepared, and the types of cells captured by antibodies and their differentiation in vascular endothelial cells (ECs) evaluated by molecular biology and scanning electron microscope. The results showed that CD133 stent can selectively capture hematopoietic stem cells (HSC),which directionally differentiate into vascular ECs in peripheral blood by (CS/HA) induction, and simultaneously inhibit migration and proliferation of immune cells and vascular smooth muscle cells (MCs). CD34 stent can capture HSC, hematopoietic progenitor cells that differentiate into vascular ECs and immune cells, promoting smooth MCs growth, leading to thrombosis, inflammation, and rejection. CD133 stent can be implanted into miniature pig heart coronary and can repair vascular damage by capturing own HSC, thus contributing to the rapid natural vascular repair, avoiding inflammation and rejection, thrombosis and restenosis. These studies demonstrated that CD133 stent of HSC capture will be an ideal coated metal stent providing a new therapeutic approach for cardiovascular and cerebrovascular disease.

A Dual-Responsive Self-Assembled Monolayer for Specific Capture and On-Demand Release of Live Cells.

PubMed

Gao, Xia; Li, Qiang; Wang, Fengchao; Liu, Xuehui; Liu, Dingbin

2018-06-22

We report a dual-responsive self-assembled monolayer (SAM) on a well-defined rough gold substrate for dynamic capture and release of live cells. By incorporating 5'-triphosphate (ATP) aptamer into a SAM, we can accurately isolate specific cell types and subsequently release captured cells at either population or desired-group (or even single-cell) levels. On one hand, the whole SAMs can be disassembled through addition of ATP solution, leading to the entire release of the captured cells from the supported substrate. On the other hand, desired cells can be selectively released by using near-infrared light (NIR) irradiation, with relatively high spatial and temporal precision. The proposed dual-responsive cell capture-and-release system is biologically friendly and is reusable with another round of modification, showing great usefulness in cancer diagnosis and molecular analysis.

Fundamentals of rapid injection molding for microfluidic cell-based assays.

PubMed

Lee, Ulri N; Su, Xiaojing; Guckenberger, David J; Dostie, Ashley M; Zhang, Tianzi; Berthier, Erwin; Theberge, Ashleigh B

2018-01-30

Microscale cell-based assays have demonstrated unique capabilities in reproducing important cellular behaviors for diagnostics and basic biological research. As these assays move beyond the prototyping stage and into biological and clinical research environments, there is a need to produce microscale culture platforms more rapidly, cost-effectively, and reproducibly. 'Rapid' injection molding is poised to meet this need as it enables some of the benefits of traditional high volume injection molding at a fraction of the cost. However, rapid injection molding has limitations due to the material and methods used for mold fabrication. Here, we characterize advantages and limitations of rapid injection molding for microfluidic device fabrication through measurement of key features for cell culture applications including channel geometry, feature consistency, floor thickness, and surface polishing. We demonstrate phase contrast and fluorescence imaging of cells grown in rapid injection molded devices and provide design recommendations to successfully utilize rapid injection molding methods for microscale cell-based assay development in academic laboratory settings.

Discrete and Continuum Approximations for Collective Cell Migration in a Scratch Assay with Cell Size Dynamics.

PubMed

Matsiaka, Oleksii M; Penington, Catherine J; Baker, Ruth E; Simpson, Matthew J

2018-04-01

Scratch assays are routinely used to study the collective spreading of cell populations. In general, the rate at which a population of cells spreads is driven by the combined effects of cell migration and proliferation. To examine the effects of cell migration separately from the effects of cell proliferation, scratch assays are often performed after treating the cells with a drug that inhibits proliferation. Mitomycin-C is a drug that is commonly used to suppress cell proliferation in this context. However, in addition to suppressing cell proliferation, mitomycin-C also causes cells to change size during the experiment, as each cell in the population approximately doubles in size as a result of treatment. Therefore, to describe a scratch assay that incorporates the effects of cell-to-cell crowding, cell-to-cell adhesion, and dynamic changes in cell size, we present a new stochastic model that incorporates these mechanisms. Our agent-based stochastic model takes the form of a system of Langevin equations that is the system of stochastic differential equations governing the evolution of the population of agents. We incorporate a time-dependent interaction force that is used to mimic the dynamic increase in size of the agents. To provide a mathematical description of the average behaviour of the stochastic model we present continuum limit descriptions using both a standard mean-field approximation and a more sophisticated moment dynamics approximation that accounts for the density of agents and density of pairs of agents in the stochastic model. Comparing the accuracy of the two continuum descriptions for a typical scratch assay geometry shows that the incorporation of agent growth in the system is associated with a decrease in accuracy of the standard mean-field description. In contrast, the moment dynamics description provides a more accurate prediction of the evolution of the scratch assay when the increase in size of individual agents is included in the model.

Microfluidic devices with permeable polymer barriers for capture and transport of biomolecules and cells.

PubMed

Lee, Ho Suk; Chu, Wai Keung; Zhang, Kun; Huang, Xiaohua

2013-09-07

We report a method for fabricating permeable polymer microstructure barriers in polydimethylsiloxane (PDMS) microfluidic devices and the use of the devices to capture and transport DNA and cells. The polymer microstructure in a desired location in a fluidic channel is formed in situ by the polymerization of acrylamide and polyethylene diacrylate cross-linker (PEG-DA) monomer in a solution which is trapped in the location using a pair of PDMS valves. The porous polymer microstructure provides a mechanical barrier to convective fluid flow in the channel or between two microfluidic chambers while it still conducts ions or small charged species under an electric field, allowing for the rapid capture and transport of biomolecules and cells by electrophoresis. We have demonstrated the application of the devices for the rapid capture and efficient release of bacteriophage λ genomic DNA, solution exchange and for the transport and capture of HeLa cells. Our devices will enable the multi-step processing of biomolecules and cells or individual cells within a single microfluidic chamber.

Evaluation of royal jelly as an alternative to fetal bovine serum in cell culture using cell proliferation assays and live cell imaging.

PubMed

Musa, Marahaini; Nasir, Nurul Fatihah Mohamad; Thirumulu, Kannan Ponnuraj

2014-01-01

Royal jelly is a nutritious substance produced by the young nurse bees and contains significant amounts of proteins which are important for cell growth and proliferation. The aim of this study was to evaluate the effect of royal jelly as an alternative to fetal bovine serum (FBS) in cell culture using cell proliferation assays and live cell imaging. MRC-5 cells were treated with various concentrations of royal jelly extract in MTT assay. The control groups were comprised of Alpha-Minimal Essential Medium (α-MEM) alone and α-MEM with 10% FBS. Subsequently, the cell proliferation was studied for 10 days using Alamar Blue assay and live cell imaging from 48 to 72 h. The population doubling time (PDT) was determined using trypan blue assay after live cell imaging. In MTT assay, 0.156 and 0.078 mg/ml of royal jelly produced higher cell viability compared to positive control group but were not significantly different (P > 0.05). In the Alamar Blue assay, 0.156 and 0.078 mg/ml of royal jelly produced greater percentage of reduction at day 3 even though no significant difference was found (P > 0.05). Based on live cell imaging, the PDT for positive, negative, 0.156 and 0.078 mg/ml of royal jelly groups were 29.09, 62.50, 41.67 and 41.67 h respectively. No significant difference was found in the PDT between all the groups (P > 0.05). Royal jelly does not exhibit similar ability like FBS to facilitate cell growth under the present test conditions.

Honey is cytotoxic towards prostate cancer cells but interacts with the MTT reagent: Considerations for the choice of cell viability assay.

PubMed

Abel, Sean D A; Baird, Sarah K

2018-02-15

Honey is a complex biological substance, consisting mainly of sugars, phenolic compounds and enzymes. Using five quick and accessible assays for measuring honey's cytotoxicity in vitro, we found honey is cytotoxic towards prostate cancer cells PC3 and DU145. However, the level of cell death varied with assay. The MTT assay was confounded by the reduction of the MTT reagent by honey's reducing sugars and phenolic compounds, and the lactate dehydrogenase assay was invalidated by honey oxidising the enzyme cofactor NADH. The sulforhodamine B assay gave valid results, but measures only protein content, providing no information about cell death in the remaining cells. The trypan blue assay and a microscope-based propidium iodide/Hoechst staining assay assess only late stage membrane permeability. However, the propidium iodide/Hoechst assay gives morphological information about cell death mechanism. A combination of the sulforhodamine B and propidium iodide/Hoechst assays would provide the most accurate quantification of honey cytotoxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development of a rapid cell-fusion-based phenotypic HIV-1 tropism assay

PubMed Central

Teeranaipong, Phairote; Hosoya, Noriaki; Kawana-Tachikawa, Ai; Fujii, Takeshi; Koibuchi, Tomohiko; Nakamura, Hitomi; Koga, Michiko; Kondo, Naoyuki; Gao, George F; Hoshino, Hiroo; Matsuda, Zene; Iwamoto, Aikichi

2013-01-01

Introduction A dual split reporter protein system (DSP), recombining Renilla luciferase (RL) and green fluorescent protein (GFP) split into two different constructs (DSP1–7 and DSP8–11), was adapted to create a novel rapid phenotypic tropism assay (PTA) for HIV-1 infection (DSP-Pheno). Methods DSP1–7 was stably expressed in the glioma-derived NP-2 cell lines, which expressed CD4/CXCR4 (N4X4) or CD4/CCR5 (N4R5), respectively. An expression vector with DSP8–11 (pRE11) was constructed. The HIV-1 envelope genes were subcloned in pRE11 (pRE11-env) and transfected into 293FT cells. Transfected 293FT cells were incubated with the indicator cell lines independently. In developing the assay, we selected the DSP1–7-positive clones that showed the highest GFP activity after complementation with DSP8–11. These cell lines, designated N4R5-DSP1–7, N4X4-DSP1–7 were used for subsequent assays. Results The env gene from the reference strains (BaL for R5 virus, NL4-3 for X4 virus, SF2 for dual tropic virus) subcloned in pRE11 and tested, was concordant with the expected co-receptor usage. Assay results were available in two ways (RL or GFP). The assay sensitivity by RL activity was comparable with those of the published phenotypic assays using pseudovirus. The shortest turnaround time was 5 days after obtaining the patient's plasma. All clinical samples gave positive RL signals on R5 indicator cells in the fusion assay. Median RLU value of the low CD4 group was significantly higher on X4 indicator cells and suggested the presence of more dual or X4 tropic viruses in this group of patients. Comparison of representative samples with Geno2Pheno [co-receptor] assay was concordant. Conclusions A new cell-fusion-based, high-throughput PTA for HIV-1, which would be suitable for in-house studies, was developed. Equipped with two-way reporter system, RL and GFP, DSP-Pheno is a sensitive test with short turnaround time. Although maintenance of cell lines and laboratory

[Detection of viable metabolically active yeast cells using a colorimetric assay].

PubMed

Růzicka, F; Holá, V

2008-02-01

The increasing concern of yeasts able to form biofilm brings about the need for susceptibility testing of both planktonic and biofilm cells. Detection of viability or metabolic activity of yeast cells after exposure to antimicrobials plays a key role in the assessment of susceptibility testing results. Colorimetric assays based on the color change of the medium in the presence of metabolically active cells proved suitable for this purpose. In this study, the usability of a colorimetric assay with the resazurin redox indicator for monitoring the effect of yeast inoculum density on the reduction rate was tested. As correlation between the color change rate and inoculum density was observed, approximate quantification of viable cells was possible. The assay would be of relevance to antifungal susceptibility testing in both planktonic and biofilm yeasts.

Biodegradable nano-films for capture and non-invasive release of circulating tumor cells.

PubMed

Li, Wei; Reátegui, Eduardo; Park, Myoung-Hwan; Castleberry, Steven; Deng, Jason Z; Hsu, Bryan; Mayner, Sarah; Jensen, Anne E; Sequist, Lecia V; Maheswaran, Shyamala; Haber, Daniel A; Toner, Mehmet; Stott, Shannon L; Hammond, Paula T

2015-10-01

Selective isolation and purification of circulating tumor cells (CTCs) from whole blood is an important capability for both clinical medicine and biological research. Current techniques to perform this task place the isolated cells under excessive stresses that reduce cell viability, and potentially induce phenotype change, therefore losing valuable information about the isolated cells. We present a biodegradable nano-film coating on the surface of a microfluidic chip, which can be used to effectively capture as well as non-invasively release cancer cell lines such as PC-3, LNCaP, DU 145, H1650 and H1975. We have applied layer-by-layer (LbL) assembly to create a library of ultrathin coatings using a broad range of materials through complementary interactions. By developing an LbL nano-film coating with an affinity-based cell-capture surface that is capable of selectively isolating cancer cells from whole blood, and that can be rapidly degraded on command, we are able to gently isolate cancer cells and recover them without compromising cell viability or proliferative potential. Our approach has the capability to overcome practical hurdles and provide viable cancer cells for downstream analyses, such as live cell imaging, single cell genomics, and in vitro cell culture of recovered cells. Furthermore, CTCs from cancer patients were also captured, identified, and successfully released using the LbL-modified microchips. Published by Elsevier Ltd.

The Use of Nanotrap Particles Technology in Capturing HIV-1 Virions and Viral Proteins from Infected Cells

PubMed Central

Sampey, Gavin; Shafagati, Nazly; Van Duyne, Rachel; Iordanskiy, Sergey; Kehn-Hall, Kylene; Liotta, Lance; Petricoin, Emanuel; Young, Mary; Lepene, Benjamin; Kashanchi, Fatah

2014-01-01

HIV-1 infection results in a chronic but incurable illness since long-term HAART can keep the virus to an undetectable level. However, discontinuation of therapy rapidly increases viral burden. Moreover, patients under HAART frequently develop various metabolic disorders and HIV-associated neuronal disease. Today, the main challenge of HIV-1 research is the elimination of the residual virus in infected individuals. The current HIV-1 diagnostics are largely comprised of serological and nucleic acid based technologies. Our goal is to integrate the nanotrap technology into a standard research tool that will allow sensitive detection of HIV-1 infection. This study demonstrates that majority of HIV-1 virions in culture supernatants and Tat/Nef proteins spiked in culture medium can be captured by nanotrap particles. To determine the binding affinities of different baits, we incubated target molecules with nanotrap particles at room temperature. After short sequestration, materials were either eluted or remained attached to nanotrap particles prior to analysis. The unique affinity baits of nanotrap particles preferentially bound HIV-1 materials while excluded albumin. A high level capture of Tat or Tat peptide by NT082 and NT084 particles was measured by western blot (WB). Intracellular Nef protein was captured by NT080, while membrane-associated Nef was captured by NT086 and also detected by WB. Selective capture of HIV-1 particles by NT073 and NT086 was measured by reverse transcriptase assay, while capture of infectious HIV-1 by these nanoparticles was demonstrated by functional transactivation in TZM-bl cells. We also demonstrated specific capture of HIV-1 particles and exosomes-containing TAR-RNA in patients' serum by NT086 and NT082 particles, respectively, using specific qRT-PCR. Collectively, our data indicate that certain types of nanotrap particles selectively capture specific HIV-1 molecules, and we propose to use this technology as a platform to enhance HIV-1

Detection and capture of single circulating melanoma cells using photoacoustic flowmetry

NASA Astrophysics Data System (ADS)

O'Brien, Christine; Mosley, Jeffrey; Goldschmidt, Benjamin S.; Viator, John A.

2010-02-01

Photoacoustic flowmetry has been used to detect single circulating melanoma cells in vitro. Circulating melanoma cells are those cells that travel in the blood and lymph systems to create secondary tumors and are the hallmark of metastasis. This technique involves taking blood samples from patients, separating the white blood and melanoma cells from whole blood and irradiating them with a pulsed laser in a flowmetry set up. Rapid, visible wavelength laser pulses on the order of 5 ns can induce photoacoustic waves in melanoma cells due to their melanin content, while surrounding white blood cells remain acoustically passive. We have developed a system that identifies rare melanoma cells and captures them in 50 microliter volumes using suction applied near the photoacoustic detection chamber. The 50 microliter sample is then diluted and the experiment is repeated using the new sample until only a melanoma cell remains. We have tested this system on dyed microspheres ranging in size from 300 to 500 microns. Capture of circulating melanoma cells may provide the opportunity to study metastatic cells for basic understanding of the spread of cancer and to optimize patient specific therapies.

Enhanced Cell Capture on Functionalized Graphene Oxide Nanosheets through Oxygen Clustering.

PubMed

Bardhan, Neelkanth M; Kumar, Priyank V; Li, Zeyang; Ploegh, Hidde L; Grossman, Jeffrey C; Belcher, Angela M; Chen, Guan-Yu

2017-02-28

With the global rise in incidence of cancer and infectious diseases, there is a need for the development of techniques to diagnose, treat, and monitor these conditions. The ability to efficiently capture and isolate cells and other biomolecules from peripheral whole blood for downstream analyses is a necessary requirement. Graphene oxide (GO) is an attractive template nanomaterial for such biosensing applications. Favorable properties include its two-dimensional architecture and wide range of functionalization chemistries, offering significant potential to tailor affinity toward aromatic functional groups expressed in biomolecules of interest. However, a limitation of current techniques is that as-synthesized GO nanosheets are used directly in sensing applications, and the benefits of their structural modification on the device performance have remained unexplored. Here, we report a microfluidic-free, sensitive, planar device on treated GO substrates to enable quick and efficient capture of Class-II MHC-positive cells from murine whole blood. We achieve this by using a mild thermal annealing treatment on the GO substrates, which drives a phase transformation through oxygen clustering. Using a combination of experimental observations and MD simulations, we demonstrate that this process leads to improved reactivity and density of functionalization of cell capture agents, resulting in an enhanced cell capture efficiency of 92 ± 7% at room temperature, almost double the efficiency afforded by devices made using as-synthesized GO (54 ± 3%). Our work highlights a scalable, cost-effective, general approach to improve the functionalization of GO, which creates diverse opportunities for various next-generation device applications.

Single Cell Multiplex Protein Measurements through Rare Earth Element Immunolabeling, Laser Capture Microdissection and Inductively Coupled Mass Spectrometry.

PubMed

Liba, Amir; Wanagat, Jonathan

2014-11-01

Complex diseases such as heart disease, stroke, cancer, and aging are the primary causes of death in the US. These diseases cause heterogeneous conditions among cells, conditions that cannot be measured in tissue homogenates and require single cell approaches. Understanding protein levels within tissues is currently assayed using various molecular biology techniques (e.g., Western blots) that rely on milligram to gram quantities of tissue homogenates or immunofluorescent (IF) techniques that are limited by spectral overlap. Tissue homogenate studies lack references to tissue structure and mask signals from individual or rare cellular events. Novel techniques are required to bring protein measurement sensitivity to the single cell level and offer spatiotemporal resolution and scalability. We are developing a novel approach to protein quantification by exploiting the inherently low concentration of rare earth elements (REE) in biological systems. By coupling REE-antibody immunolabeling of cells with laser capture microdissection (LCM) and ICP-QQQ, we are achieving multiplexed protein measurement in histological sections of single cells. This approach will add to evolving single cell techniques and our ability to understand cellular heterogeneity in complex biological systems and diseases.

A self-contained 48-well fatty acid oxidation assay.

PubMed

Wang, Xiaojun; Wang, Rose; Nemcek, Thomas A; Cao, Ning; Pan, Jeffrey Y; Frevert, Ernst U

2004-02-01

The modulation of fatty acid metabolism and especially the stimulation of fatty acid oxidation in liver or skeletal muscle are attractive therapeutic approaches for the treatment of obesity and the associated insulin resistance. However, current beta-oxidation assays are run in very low throughput, which represents an obstacle for drug discovery in this area. Here we describe results for a 48-well beta-oxidation assay using a new instrument design. A connecting chamber links two adjacent wells to form an experimental unit, in which one well contains the beta-oxidation reaction and the other captures CO(2). The experimental units are sealed from each other and from the outside to prevent release of radioactivity from the labeled substrate. CO(2) capture in this instrument is linear with time and over the relevant experimental range of substrate concentration. Cellular viability is maintained in the sealed environment, and cells show the expected responses to modulators of beta-oxidation, such as the AMP kinase activator 5-aminoimidazole carboxamide riboside. Data are presented for different lipid substrates and cell lines. The increased throughput of this procedure compared with previously described methods should facilitate the evaluation of compounds that modulate fatty acid metabolism.

Objective scoring of transformed foci in BALB/c 3T3 cell transformation assay by statistical image descriptors.

PubMed

Urani, C; Corvi, R; Callegaro, G; Stefanini, F M

2013-09-01

In vitro cell transformation assays (CTAs) have been shown to model important stages of in vivo carcinogenesis and have the potential to predict carcinogenicity in humans. Advantages of CTAs are their ability of revealing both genotoxic and non-genotoxic carcinogens while reducing both experimental costs and the number of animals used. The endpoint of the CTA is foci formation, and requires classification under light microscopy based on morphology. Thus current limitations for the wide adoption of the assay partially depend on a fair degree of subjectivity in foci scoring. An objective evaluation may be obtained after separating foci from background monolayer in the digital image, and quantifying values of statistical descriptors which are selected to capture eye-scored morphological features. The aim of this study was to develop statistical descriptors to be applied to transformed foci of BALB/c 3T3, which cover foci size, multilayering and invasive cell growth into the background monolayer. Proposed descriptors were applied to a database of 407 foci images to explore the numerical features, and to illustrate open problems and potential solutions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Capture of endothelial cells under flow using immobilized vascular endothelial growth factor

PubMed Central

Smith, Randall J.; Koobatian, Maxwell T.; Shahini, Aref; Swartz, Daniel D.; Andreadis, Stelios T.

2015-01-01

We demonstrate the ability of immobilized vascular endothelial growth factor (VEGF) to capture endothelial cells (EC) with high specificity under fluid flow. To this end, we engineered a surface consisting of heparin bound to poly-L-lysine to permit immobilization of VEGF through the C-terminal heparin-binding domain. The immobilized growth factor retained its biological activity as shown by proliferation of EC and prolonged activation of KDR signaling. Using a microfluidic device we assessed the ability to capture EC under a range of shear stresses from low (0.5 dyne/cm2) to physiological (15 dyne/cm2). Capture was significant for all shear stresses tested. Immobilized VEGF was highly selective for EC as evidenced by significant capture of human umbilical vein and ovine pulmonary artery EC but no capture of human dermal fibroblasts, human hair follicle derived mesenchymal stem cells, or mouse fibroblasts. Further, VEGF could capture EC from mixtures with non-EC under low and high shear conditions as well as from complex fluids like whole human blood under high shear. Our findings may have far reaching implications, as they suggest that VEGF could be used to promote endothelialization of vascular grafts or neovascularization of implanted tissues by rare but continuously circulating EC. PMID:25771020

The radiosensitivity of a murine fibrosarcoma as measured by three cell survival assays.

PubMed Central

Rice, L.; Urano, M.; Suit, H. D.

1980-01-01

The radiation sensitivity of a weakly immunogenic spontaneous fibrosarcoma of the C3Hf/Sed mouse (designated FSa-II) was assessed by three in vivo cell survival methods: end-point dilution (TD50) assay, lung colony (LC) assay, and agar diffusion chamber (ADC) assay. The hypoxic fraction of this tumour was also determined by the ADC method. Although there was a good agreement of the cell survival data between the ADC and LC methods, the TD50 method yielded a considerably less steep cell survival curve. Beneficial aspects and limitations of each assay are discussed. In addition, the use of the ADC method for the growth of xenogeneic cell lines and a preliminary experiment with human tumour cells in non-immunosuppressed hosts suggest that this method may be a valuable adjunct for studying the growth and therapeutic responses of human tumour cells. PMID:6932931

Electrochemical detection of microRNAs via gap hybridization assay.

PubMed

Pöhlmann, Christopher; Sprinzl, Mathias

2010-06-01

MicroRNAs have recently been associated with cancer development by acting as tumor suppressors or oncogenes and could therefore be applied as molecular markers for early diagnosis of cancer. In this work, we established a rapid, selective, and sensitive gap hybridization assay for detection of mature microRNAs based on four components DNA/RNA hybridization and electrochemical detection using esterase 2-oligodeoxynucleotide conjugates. Complementary binding of microRNA to a gap built of capture and detector oligodeoxynucleotide, the reporter enzyme is brought to the vicinity of the electrode and produces enzymatically an electrochemical signal. In the absence of microRNA, the gap between capture and detector oligodeoxynucleotide is not filled, and missing base stacking energy destabilizes the hybridization complex. The gap hybridization assay demonstrates selective detection of miR-16 within a mixture of other miRNAs, including the feasibility of single mismatch discrimination. Applying the biosensor assay, a detection limit of 2 pM or 2 amol of miR-16 was obtained. Using isolated total RNA from human breast adenocarcinoma MCF-7 cells, the assay detected specifically miR-21 and miR-16 in parallel, and higher expression of oncogene miR-21 compared to miR-16 was demonstrated. Including RNA isolation, the gap hybridization assay was developed with a total assay time of 60 min and without the need for reverse transcription PCR amplification of the sample. The characteristics of the assay developed in this work could satisfy the need for rapid and easy methods for early cancer marker detection in clinical diagnostics.

Mutation assays involving blood cells that metabolize toxic substances

DOEpatents

Crespi, Charles L.; Thilly, William G.

1985-01-01

A line of human blood cells which have high levels of oxidative activity (such as oxygenase, oxidase, peroxidase, and hydroxylase activity) is disclosed. Such cells grow in suspension culture, and are useful to determine the mutagenicity of xenobiotic substances that are metabolized into toxic or mutagenic substances. Mutation assays using these cells, and other cells with similar characteristics, are also disclosed.

Assay of Vitamins and Amino Acids With Cultured Tissue Cells and Antimetabolites

PubMed Central

Savchuck, W. B.; Merriman, M. E.; Lockhart, W. L.

1964-01-01

A survey of growth responses of tissue-culture cells to vitamins and amino acids was undertaken to explore the potentialities of tissue culture in the assay of growth factors. An antagonist of the nutrient was included in each test system to improve its sensitivity. Addition of an antimetabolite was advantageous in the thiamine and phenylalanine assays. Tissue-culture assays of tryptophan and of phenylalanine supplemented with β-2-thienylalanine compared favorably with microbial assays, and may serve as confirmatory or supplementary test systems. The sensitivity of cultured tissue cells to minute amounts of a variety of physiologically active substances suggests their employment in hormone and toxic compound assays. Images FIG. 7 PMID:14199020

A simple non-perturbing cell migration assay insensitive to proliferation effects.

PubMed

Glenn, Honor L; Messner, Jacob; Meldrum, Deirdre R

2016-08-18

Migration is a fundamental cellular behavior that plays an indispensable role in development and homeostasis, but can also contribute to pathology such as cancer metastasis. Due to its relevance to many aspects of human health, the ability to accurately measure cell migration is of broad interest, and numerous approaches have been developed. One of the most commonly employed approaches, because of its simplicity and throughput, is the exclusion zone assay in which cells are allowed to migrate into an initially cell-free region. A major drawback of this assay is that it relies on simply counting cells in the exclusion zone and therefore cannot distinguish the effects of proliferation from migration. We report here a simple modification to the exclusion zone migration assay that exclusively measures cell migration and is not affected by proliferation. This approach makes use of a lineage-tracing vital stain that is retained through cell generations and effectively reads out migration relative to the original, parental cell population. This modification is simple, robust, non-perturbing, and inexpensive. We validate the method in a panel of cell lines under conditions that inhibit or promote migration and demonstrate its use in normal and cancer cell lines as well as primary cells.

A simple non-perturbing cell migration assay insensitive to proliferation effects

PubMed Central

Glenn, Honor L.; Messner, Jacob; Meldrum, Deirdre R.

2016-01-01

Migration is a fundamental cellular behavior that plays an indispensable role in development and homeostasis, but can also contribute to pathology such as cancer metastasis. Due to its relevance to many aspects of human health, the ability to accurately measure cell migration is of broad interest, and numerous approaches have been developed. One of the most commonly employed approaches, because of its simplicity and throughput, is the exclusion zone assay in which cells are allowed to migrate into an initially cell-free region. A major drawback of this assay is that it relies on simply counting cells in the exclusion zone and therefore cannot distinguish the effects of proliferation from migration. We report here a simple modification to the exclusion zone migration assay that exclusively measures cell migration and is not affected by proliferation. This approach makes use of a lineage-tracing vital stain that is retained through cell generations and effectively reads out migration relative to the original, parental cell population. This modification is simple, robust, non-perturbing, and inexpensive. We validate the method in a panel of cell lines under conditions that inhibit or promote migration and demonstrate its use in normal and cancer cell lines as well as primary cells. PMID:27535324

Selective Individual Primary Cell Capture Using Locally Bio-Functionalized Micropores

PubMed Central

Liu, Jie; Bombera, Radoslaw; Leroy, Loïc; Roupioz, Yoann; Baganizi, Dieudonné R.; Marche, Patrice N.; Haguet, Vincent; Mailley, Pascal; Livache, Thierry

2013-01-01

Background Solid-state micropores have been widely employed for 6 decades to recognize and size flowing unlabeled cells. However, the resistive-pulse technique presents limitations when the cells to be differentiated have overlapping dimension ranges such as B and T lymphocytes. An alternative approach would be to specifically capture cells by solid-state micropores. Here, the inner wall of 15-µm pores made in 10 µm-thick silicon membranes was covered with antibodies specific to cell surface proteins of B or T lymphocytes. The selective trapping of individual unlabeled cells in a bio-functionalized micropore makes them recognizable just using optical microscopy. Methodology/Principal Findings We locally deposited oligodeoxynucleotide (ODN) and ODN-conjugated antibody probes on the inner wall of the micropores by forming thin films of polypyrrole-ODN copolymers using contactless electro-functionalization. The trapping capabilities of the bio-functionalized micropores were validated using optical microscopy and the resistive-pulse technique by selectively capturing polystyrene microbeads coated with complementary ODN. B or T lymphocytes from a mouse splenocyte suspension were specifically immobilized on micropore walls functionalized with complementary ODN-conjugated antibodies targeting cell surface proteins. Conclusions/Significance The results showed that locally bio-functionalized micropores can isolate target cells from a suspension during their translocation throughout the pore, including among cells of similar dimensions in complex mixtures. PMID:23469221

Prevalence estimation of tick-borne encephalitis virus (TBEV) antibodies in dogs from Finland using novel dog anti-TBEV IgG MAb-capture and IgG immunofluorescence assays based on recombinant TBEV subviral particles.

PubMed

Levanov, Lev; Vera, Cristina Pérez; Vapalahti, Olli

2016-07-01

Tick-borne encephalitis (TBE) is one of the most dangerous human neurological infections occurring in Europe and Northern parts of Asia with thousands of cases and millions vaccinated against it. The risk of TBE might be assessed through analyses of the samples taken from wildlife or from animals which are in close contact with humans. Dogs have been shown to be a good sentinel species for these studies. Serological assays for diagnosis of TBE in dogs are mainly based on purified and inactivated TBEV antigens. Here we describe novel dog anti-TBEV IgG monoclonal antibody (MAb)-capture assay which is based on TBEV prME subviral particles expressed in mammalian cells from Semliki Forest virus (SFV) replicon as well as IgG immunofluorescence assay (IFA) which is based on Vero E6 cells transfected with the same SFV replicon. We further demonstrate their use in a small-scale TBEV seroprevalence study of dogs representing different regions of Finland. Altogether, 148 dog serum samples were tested by novel assays and results were compared to those obtained with a commercial IgG enzyme immunoassay (EIA), hemagglutination inhibition test and IgG IFA with TBEV infected cells. Compared to reference tests, the sensitivities of the developed assays were 90-100% and the specificities of the two assays were 100%. Analysis of the dog serum samples showed a seroprevalence of 40% on Åland Islands and 6% on Southwestern archipelago of Finland. In conclusion, a specific and sensitive EIA and IFA for the detection of IgG antibodies in canine sera were developed. Based on these assays the seroprevalence of IgG antibodies in dogs from different regions of Finland was assessed and was shown to parallel the known human disease burden as the Southwestern archipelago and Åland Islands in particular had considerable dog TBEV antibody prevalence and represent areas with high risk of TBE for humans. Copyright © 2016 Elsevier GmbH. All rights reserved.

A novel honeycomb cell assay kit designed for evaluating horizontal cell migration in response to functionalized self-assembling peptide hydrogels

NASA Astrophysics Data System (ADS)

Guan, Fengyi; Lu, Jiaju; Wang, Xiumei

2017-03-01

A clear understanding on cell migration behaviors contributes to designing novel biomaterials in tissue engineering and elucidating related tissue regeneration processes. Many traditional evaluation methods on cell migration including scratch assay and transwell migration assay possess all kinds of limitations. In this study, a novel honeycomb cell assay kit was designed and made of photosensitive resin by 3D printing. This kit has seven hexagonal culture chambers so that it can evaluate the horizontal cell migration behavior in response to six surrounding environments simultaneously, eliminating the effect of gravity on cells. Here this cell assay kit was successfully applied to evaluate endothelial cell migration cultured on self-assembling peptide (SAP) RADA (AcN-RADARADARADARADA-CONH2) nanofiber hydrogel toward different functionalized SAP hydrogels. Our results indicated that the functionalized RADA hydrogels with different concentration of bioactive motifs of KLT or PRG could induce cell migration in a dose-dependent manner. The total number and migration distance of endothelial cells on functionalized SAP hydrogels significantly increased with increasing concentration of bioactive motif PRG or KLT. Therefore, the honeycomb cell assay kit provides a simple, efficient and convenient tool to investigate cell migration behavior in response to multi-environments simultaneously.

Hybrid capture-II and LCR-E7 PCR assays for HPV typing in cervical cytologic samples.

PubMed

Yamazaki, H; Sasagawa, T; Basha, W; Segawa, T; Inoue, M

2001-10-15

As part of an ongoing cohort study in the Hokuriku region of Japan, cervical cell samples from histologically confirmed normal (n = 114) or abnormal (n = 286) women were examined for the presence of HPV DNA using a second-generation hybrid capture assay (HCA-II) and LCR-E7 PCR. HCA-II detected low-risk (HPV-6, -11, -42, 43 and -44) and high-risk (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59 and -68) HPV types, while LCR-E7 PCR detected an additional 7 HPV types and some uncharacterized types. In screening of high-grade squamous intraepithelial lesions (HSILs) and invasive cervical cancer, the sensitivities of HCA-II and LCR-E7 PCR testing the high-risk HPV types were 83% and 81%, respectively, while the specificity of both assays was 93%. The sensitivity of LCR-E7 PCR increased to 87%, which was significantly higher than that in HCA-II, when testing both high-risk and other HPV types. Sixty-eight inconsistent results (17% of total tested) from HCA-II and LCR-E7 PCR were due to (i) low copy number of HPV genome (false-negative for HCA-II, 5.3% and for LCR-E7 PCR, 1.3%), (ii) infection with HPV types undetectable by HCA-II (4.8%), (iii) multiple HPV infections (5%) or (iv) unknown reasons (0.8%). LCR-E7 PCR revealed that infections with HPV-16, -18, -31, -33, -35, -51, -52, -56, -58 or -67 was a high risk for cancer since these types predominated in HSIL and invasive cervical cancer. Samples showing high relative light units (>20) with a high-risk probe in HCA-II also gave positive results in LCR-E7 PCR and were generally associated with abnormal cervical lesions. Thus, we propose that both HCA-II and LCR-E7 PCR are valuable screening tests for premalignant and malignant cervical lesions. Copyright 2001 Wiley-Liss, Inc.

Deep magnetic capture of magnetically loaded cells for spatially targeted therapeutics.

PubMed

Huang, Zheyong; Pei, Ning; Wang, Yanyan; Xie, Xinxing; Sun, Aijun; Shen, Li; Zhang, Shuning; Liu, Xuebo; Zou, Yunzeng; Qian, Juying; Ge, Junbo

2010-03-01

Magnetic targeting has recently demonstrated potential in promoting magnetically loaded cell delivery to target lesion, but its application is limited by magnetic attenuation. For deep magnetic capture of cells for spatial targeting therapeutics, we designed a magnetic pole, in which the magnetic field density can be focused at a distance from the pole. As flowing through a tube served as a model of blood vessels, the magnetically loaded mesenchymal stem cells (MagMSCs) were highly enriched at the site distance from the magnetic pole. The cell capture efficiency was positively influenced by the magnetic flux density, and inversely influenced by the flow velocity, and well-fitted with the deductive value by theoretical considerations. It appeared to us that the spatially-focused property of the magnetic apparatus promises a new deep targeting strategy to promote homing and engraftment for cellular therapy. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

In vitro Cell Viability by CellProfiler® Software as Equivalent to MTT Assay.

PubMed

Gasparini, Luciana S; Macedo, Nayana D; Pimentel, Elisângela F; Fronza, Marcio; Junior, Valdemar L; Borges, Warley S; Cole, Eduardo R; Andrade, Tadeu U; Endringer, Denise C; Lenz, Dominik

2017-07-01

This study evaluated in vitro cell viability by the colorimetric MTT stands for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay compared to image analysis by CellProfiler ® software. Hepatoma (Hepa-1c1c7) and fibroblast (L929) cells were exposed to isolated substances, camptothecin, lycorine, tazettine, albomaculine, 3-epimacronine, trispheridine, galanthine and Padina gymnospora , Sargassum sp. methanolic extract, and Habranthus itaobinus Ravenna ethyl acetate in different concentrations. After MTT assay, cells were stained with Panotic dye kit. Cell images were obtained with an inverted microscope equipped with a digital camera. The images were analyzed by CellProfiler ® . No cytotoxicity at the highest concentration analyzed for 3-epimacronine, albomaculine, galanthine, trispheridine, P. gymnospora extract and Sargassum sp. extract where detected. Tazettine offered cytotoxicity only against the Hepa1c1c7 cell line. Lycorine, camptothecin, and H. itaobinus extract exhibited cytotoxic effects in both cell lines. The viability methods tested were correlated demonstrated by Bland-Atman test with normal distribution with mean difference between the two methods close to zero, bias value 3.0263. The error was within the limits of the confidence intervals and these values had a narrow difference. The correlation between the two methods was demonstrated by the linear regression plotted as R 2 . CellProfiler ® image analysis presented similar results to the MTT assay in the identification of viable cells, and image analysis may assist part of biological analysis procedures. The presented methodology is inexpensive and reproducible. In vitro cell viability assessment with MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay may be replaced by image analysis by CellProfiler ® . The viability methods

Multiplex High-Throughput Targeted Proteomic Assay To Identify Induced Pluripotent Stem Cells.

PubMed

Baud, Anna; Wessely, Frank; Mazzacuva, Francesca; McCormick, James; Camuzeaux, Stephane; Heywood, Wendy E; Little, Daniel; Vowles, Jane; Tuefferd, Marianne; Mosaku, Olukunbi; Lako, Majlinda; Armstrong, Lyle; Webber, Caleb; Cader, M Zameel; Peeters, Pieter; Gissen, Paul; Cowley, Sally A; Mills, Kevin

2017-02-21

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine, disease modeling, and drug screening. However, their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research, only the best, competent clones should be used. The standard assays for pluripotency are based on genomic approaches, which take up to 1 week to perform and incur significant cost. Therefore, there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here, we describe a novel multiplexed, high-throughput, and sensitive peptide-based multiple reaction monitoring mass spectrometry assay, allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests.

Buccal Micronucleus Cytome Assay in Sickle Cell Disease.

PubMed

Naga, Mallika Bokka Sri Satya; Gour, Shreya; Nallagutta, Nalini; Ealla, Kranti Kiran Reddy; Velidandla, Surekha; Manikya, Sangameshwar

2016-06-01

Sickle Cell Anaemia (SCA) is a commonly inherited blood disorder preceded by episodes of pain, chronic haemolytic anaemia and severe infections. The underlying phenomenon which causes this disease is the point mutation in the haemoglobin beta gene (Hbβ) found on chromosome 11 p. Increased oxidative stress leads to DNA damage. DNA damage occurring in such conditions can be studied by the buccal micronucleus cytome assay, which is a minimally invasive method for studying chromosomal instability, cell death and regenerative potential of human buccal tissue. To evaluate genomic instability in patients with sickle cell disease by buccal micronucleus cytome assay. The study included 40 sickle cell anemia patients (Group A) and 40 age and sex matched controls (Group B). Buccal swabs were collected and stained with Papanicolaou (PAP). Number of cells with micronucleus, binuclei, nuclear bud, pyknosis and karyolysis were counted in two groups as parameters for the evaluation of genome stability. All the analysis was done using t-test. A p-value of <0.001 was considered statistically significant. There was a statistically significant increase in micronuclei number in SCA patients when compared with controls. Karyolytic (un-nucleated) cell number in Group A was more than to those of the controls. The results might suggest that patients with sickle cell anaemia have genome instability which is represented by the presence of micronuclei in the somatic cells. Presence of apoptotic cells might only indicate the bodily damage to the tissue as a result of the disease.

Cell-based Assays for Assessing Toxicity: A Basic Guide.

PubMed

Parboosing, Raveen; Mzobe, Gugulethu; Chonco, Louis; Moodley, Indres

2016-01-01

Assessment of toxicity is an important component of the drug discovery process. Cellbased assays are a popular choice for assessing cytotoxicity. However, these assays are complex because of the wide variety of formats and methods that are available, lack of standardization, confusing terminology and the inherent variability of biological systems and measurement. This review is intended as a guide on how to take these factors into account when planning, conducting and/or interpreting cell based toxicity assays. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Mutation assays involving blood cells that metabolize toxic substances

DOEpatents

Crespi, C.L.; Thilly, W.G.

1999-08-10

The present invention pertains to a line of human blood cells which have high levels of oxidative activity (such as oxygenase, oxidase, peroxidase, and hydroxylase activity). Such cells grow in suspension culture, and are useful to determine the mutagenicity of xenobiotic substances that are metabolized into toxic or mutagenic substances. The invention also includes mutation assays using these cells, and other cells with similar characteristics. 3 figs.

Mutation assays involving blood cells that metabolize toxic substances

DOEpatents

Crespi, Charles L.; Thilly, William G.

1999-01-01

The present invention pertains to a line of human blood cells which have high levels of oxidative activity (such as oxygenase, oxidase, peroxidase, and hydroxylase activity). Such cells grow in suspension culture, and are useful to determine the mutagenicity of xenobiotic substances that are metabolized into toxic or mutagenic substances. The invention also includes mutation assays using these cells, and other cells with similar characteristics.

Cross-reactivity profiles of hybrid capture II, cobas, and APTIMA human papillomavirus assays: split-sample study.

PubMed

Preisler, Sarah; Rebolj, Matejka; Ejegod, Ditte Møller; Lynge, Elsebeth; Rygaard, Carsten; Bonde, Jesper

2016-07-20

High-risk Human Papillomavirus (HPV) testing is replacing cytology in cervical cancer screening as it is more sensitive for preinvasive cervical lesions. However, the bottleneck of HPV testing is the many false positive test results (positive tests without cervical lesions). Here, we evaluated to what extent these can be explained by cross-reactivity, i.e. positive test results without evidence of high-risk HPV genotypes. The patterns of cross-reactivity have been thoroughly studied for hybrid capture II (HC2) but not yet for newer HPV assays although the manufacturers claimed no or limited frequency of cross-reactivity. In this independent study we evaluated the frequency of cross-reactivity for HC2, cobas, and APTIMA assays. Consecutive routine cervical screening samples from 5022 Danish women, including 2859 from women attending primary screening, were tested with the three evaluated DNA and mRNA HPV assays. Genotyping was undertaken using CLART HPV2 assay, individually detecting 35 genotypes. The presence or absence of cervical lesions was determined with histological examinations; women with abnormal cytology were managed as per routine recommendations; those with normal cytology and positive high-risk HPV test results were invited for repeated testing in 18 months. Cross-reactivity to low-risk genotypes was detected in 109 (2.2 %) out of 5022 samples on HC2, 62 (1.2 %) on cobas, and 35 (0.7 %) on APTIMA with only 10 of the samples cross-reacting on all 3 assays. None of the 35 genotypes was detected in 49 (1.0 %), 162 (3.2 %), and 56 (1.1 %) samples, respectively. In primary screening at age 30 to 65 years (n = 2859), samples of 72 (25 %) out of 289 with high-risk infections on HC2 and < CIN2 histology were due to cross-reactivity. On cobas, this was 106 (26 %) out of 415, and on APTIMA 48 (21 %) out of 224. Despite manufacturer claims, all three assays showed cross-reactivity. In primary cervical screening at age ≥30 years, cross

A high-throughput assay of NK cell activity in whole blood and its clinical application

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

Lee, Saet-byul; Cha, Junhoe; Kim, Im-kyung

2014-03-14

Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate themore » status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.« less

DNA-barcode directed capture and electrochemical metabolic analysis of single mammalian cells on a microelectrode array.

PubMed

Douglas, Erik S; Hsiao, Sonny C; Onoe, Hiroaki; Bertozzi, Carolyn R; Francis, Matthew B; Mathies, Richard A

2009-07-21

A microdevice is developed for DNA-barcode directed capture of single cells on an array of pH-sensitive microelectrodes for metabolic analysis. Cells are modified with membrane-bound single-stranded DNA, and specific single-cell capture is directed by the complementary strand bound in the sensor area of the iridium oxide pH microelectrodes within a microfluidic channel. This bifunctional microelectrode array is demonstrated for the pH monitoring and differentiation of primary T cells and Jurkat T lymphoma cells. Single Jurkat cells exhibited an extracellular acidification rate of 11 milli-pH min(-1), while primary T cells exhibited only 2 milli-pH min(-1). This system can be used to capture non-adherent cells specifically and to discriminate between visually similar healthy and cancerous cells in a heterogeneous ensemble based on their altered metabolic properties.

DNA-barcode directed capture and electrochemical metabolic analysis of single mammalian cells on a microelectrode array

PubMed Central

Douglas, Erik S.; Hsiao, Sonny C.; Onoe, Hiroaki; Bertozzi, Carolyn R.; Francis, Matthew B.; Mathies, Richard A.

2010-01-01

A microdevice is developed for DNA-barcode directed capture of single cells on an array of pH-sensitive microelectrodes for metabolic analysis. Cells are modified with membrane-bound single-stranded DNA, and specific single-cell capture is directed by the complementary strand bound in the sensor area of the iridium oxide pH microelectrodes within a microfluidic channel. This bifunctional microelectrode array is demonstrated for the pH monitoring and differentiation of primary T cells and Jurkat T lymphoma cells. Single Jurkat cells exhibited an extracellular acidification rate of 11 milli-pH min−1, while primary T cells exhibited only 2 milli-pH min−1. This system can be used to capture non-adherent cells specifically and to discriminate between visually similar healthy and cancerous cells in a heterogeneous ensemble based on their altered metabolic properties. PMID:19568668

Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem-like cells.

PubMed

Fedr, Radek; Pernicová, Zuzana; Slabáková, Eva; Straková, Nicol; Bouchal, Jan; Grepl, Michal; Kozubík, Alois; Souček, Karel

2013-05-01

The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples. Copyright © 2013 International Society for Advancement of Cytometry.

Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling*

PubMed Central

Poetz, Oliver; Henzler, Tanja; Hartmann, Michael; Kazmaier, Cornelia; Templin, Markus F.; Herget, Thomas; Joos, Thomas O.

2010-01-01

Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibody-coated, magnetic suspension bead arrays. As a miniaturized immunoassay format, suspension bead array-based assays fulfill the criteria of the ambient analyte theory, and our experiments reveal that the analyte concentrations are not significantly changed. The value of sequential multiplex analyte capturing was demonstrated by probing tumor cell line lysates for the abundance of seven different receptor tyrosine kinases and their degree of phosphorylation and by measuring the complex phosphorylation pattern of the epidermal growth factor receptor in the same sample from the same cavity. PMID:20682761

Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.

PubMed

de Oliveira, Edson R A; Lima, Bruna M M P; de Moura, Wlamir C; Nogueira, Ana Cristina M de A

2013-12-31

Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the

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.

Microfluidic assay of circulating endothelial cells in coronary artery disease patients with angina pectoris.

PubMed

Chen, Shuiyu; Sun, Yukun; Neoh, Kuang Hong; Chen, Anqi; Li, Weiju; Yang, Xiaorui; Han, Ray P S

2017-01-01

Circulating endothelial cells (CECs) are widely reported as a promising biomarker of endothelial damage/dysfunction in coronary artery disease (CAD). The two popular methods of CEC quantification include the use of immunomagnetic beads separation (IB) and flow cytometry analysis (FC); however, they suffer from two main shortcomings that affect their diagnostic and prognostic responses: non-specific bindings of magnetic beads to non-target cells and a high degree of variability in rare cell identification, respectively. We designed a microfluidic chip with spatially staggered micropillars for the efficient harvesting of CECs with intact cellular morphology in an attempt to revisit the diagnostic goal of CEC counts in CAD patients with angina pectoris. A label-free microfluidic assay that involved an in-situ enumeration and immunofluorescent identification (DAPI+/CD146+/VEGFR1+/CD45-) of CECs was carried out to assess the CEC count in human peripheral blood samples. A total of 55 CAD patients with angina pectoris [16 with chronic stable angina (CSA) and 39 with unstable angina (UA)], together with 15 heathy controls (HCs) were enrolled in the study. CEC counts are significantly higher in both CSA and UA groups compared to the HC group [respective medians of 6.9, 10.0 and 1.5 cells/ml (p < 0.01)]. Further, a significant elevation of CEC count was observed in the three UA subgroups [low risk (5.3) vs. intermediate risk (10.8) vs. high risk (18.0) cells/ml, p < 0.001) classified in accordance to the TIMI NSTEMI/UA risk score system. From the receiver-operating characteristic curve analysis, the AUCs for distinguishing CSA and UA from HC were 0.867 and 0.938, respectively. The corresponding sensitivities were 87.5% and 84.6% and the specificities were 66.7% and 86.7%, respectively. Our microfluidic assay system is efficient and stable for CEC capture and enumeration. The results showed that the CEC count has the potential to be a promising clinical biomarker for the

Drosophila hemocyte migration: an in vivo assay for directional cell migration.

PubMed

Moreira, Carolina G A; Regan, Jennifer C; Zaidman-Rémy, Anna; Jacinto, Antonio; Prag, Soren

2011-01-01

This protocol describes an in vivo assay for random and directed hemocyte migration in Drosophila. Drosophila is becoming an increasingly powerful model system for in vivo cell migration analysis, combining unique genetic tools with translucency of the embryo and pupa, which allows direct imaging and traceability of different cell types. In the assay we present here, we make use of the hemocyte response to epithelium wounding to experimentally induce a transition from random to directed migration. Time-lapse confocal microscopy of hemocyte migration in untreated conditions provides a random cell migration assay that allows identification of molecular mechanisms involved in this complex process. Upon laser-induced wounding of the thorax epithelium, a rapid chemotactic response changes hemocyte migratory behavior into a directed migration toward the wound site. This protocol provides a direct comparison of cells during both types of migration in vivo, and combined with recently developed resources such as transgenic RNAi, is ideal for forward genetic screens.

A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis.

PubMed

Collins, Tony J; Ylanko, Jarkko; Geng, Fei; Andrews, David W

2015-11-01

A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose-response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds.

A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis

PubMed Central

Collins, Tony J.; Ylanko, Jarkko; Geng, Fei

2015-01-01

Abstract A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose–response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds. PMID:26422066

Buccal Micronucleus Cytome Assay in Sickle Cell Disease

PubMed Central

Naga, Mallika Bokka Sri Satya; Gour, Shreya; Nallagutta, Nalini; Velidandla, Surekha; Manikya, Sangameshwar

2016-01-01

Introduction Sickle Cell Anaemia (SCA) is a commonly inherited blood disorder preceded by episodes of pain, chronic haemolytic anaemia and severe infections. The underlying phenomenon which causes this disease is the point mutation in the haemoglobin beta gene (Hbβ) found on chromosome 11 p. Increased oxidative stress leads to DNA damage. DNA damage occurring in such conditions can be studied by the buccal micronucleus cytome assay, which is a minimally invasive method for studying chromosomal instability, cell death and regenerative potential of human buccal tissue. Aim To evaluate genomic instability in patients with sickle cell disease by buccal micronucleus cytome assay. Materials and Methods The study included 40 sickle cell anemia patients (Group A) and 40 age and sex matched controls (Group B). Buccal swabs were collected and stained with Papanicolaou (PAP). Number of cells with micronucleus, binuclei, nuclear bud, pyknosis and karyolysis were counted in two groups as parameters for the evaluation of genome stability. Results All the analysis was done using t-test. A p-value of <0.001 was considered statistically significant. There was a statistically significant increase in micronuclei number in SCA patients when compared with controls. Karyolytic (un-nucleated) cell number in Group A was more than to those of the controls. Conclusion The results might suggest that patients with sickle cell anaemia have genome instability which is represented by the presence of micronuclei in the somatic cells. Presence of apoptotic cells might only indicate the bodily damage to the tissue as a result of the disease. PMID:27504413

Nerve Growth Factor-Induced Angiogenesis: 1. Endothelial Cell Tube Formation Assay.

PubMed

Lazarovici, Philip; Lahiani, Adi; Gincberg, Galit; Haham, Dikla; Fluksman, Arnon; Benny, Ofra; Marcinkiewicz, Cezary; Lelkes, Peter I

2018-01-01

Nerve growth factor (NGF) is a neurotrophin promoting survival, proliferation, differentiation, and neuroprotection in the embryonal and adult nervous system. NGF also induces angiogenic effects in the cardiovascular system, which may be beneficial in engineering new blood vessels and for developing novel anti-angiogenesis therapies for cancer. Angiogenesis is a cellular process characterized by a number of events, including endothelial cell migration, invasion, and assembly into capillaries. In vitro endothelial tube formation assays are performed using primary human umbilical vein endothelial cells, human aortic endothelial cells, and other human or rodent primary endothelial cells isolated from the vasculature of both tumors and normal tissues. Immortalized endothelial cell lines are also used for these assays. When seeded onto Matrigel, these cells reorganize to create tubelike structure, which may be used as models for studying some aspects of in vitro angiogenesis. Image acquisition by light and fluorescence microscopy and/or quantification of fluorescently labeled cells can be carried out manually or digitally, using commercial software and automated image processing. Here we detail materials, procedure, assay conditions, and cell labeling for quantification of endothelial cell tube formation. This model can be applied to study cellular and molecular mechanisms by which NGF or other neurotrophins promote angiogenesis. This model may also be useful for the development of potential angiogenic and/or anti-angiogenic drugs targeting NGF receptors.

ViriChip: a solid phase assay for detection and identification of viruses by atomic force microscopy

NASA Astrophysics Data System (ADS)

Nettikadan, Saju R.; Johnson, James C.; Vengasandra, Srikanth G.; Muys, James; Henderson, Eric

2004-03-01

Bionanotechnology can be viewed as the integration of tools and concepts in nanotechnology with the attributes of biomolecules. We report here on an atomic force microscopy-immunosensor assay (AFMIA) that couples AFM with solid phase affinity capture of biological entities for the rapid detection and identification of group B coxsackievirus particles. Virus identification is based on type-specific immunocapture and the morphological properties of the captured viruses as obtained by the AFM. Representatives of the six group B coxsackieviruses have been specifically captured from 1 µl volumes of clarified cell lysates, body fluids and environmental samples. Concentration and kinetic profiles for capture indicate that detection is possible at 103 TCID50 µl-1 and the dynamic range of the assay spans three logs. The results demonstrate that the melding of a nanotechnological tool (AFM) with biotechnology (solid phase immunocapture of virus particles) can create a clinically relevant platform, useful for the detection and identification of enterovirus particles in a variety of samples.

Robust detection of chromosomal interactions from small numbers of cells using low-input Capture-C

PubMed Central

Oudelaar, A. Marieke; Davies, James O.J.; Downes, Damien J.; Higgs, Douglas R.

2017-01-01

Abstract Chromosome conformation capture (3C) techniques are crucial to understanding tissue-specific regulation of gene expression, but current methods generally require large numbers of cells. This hampers the investigation of chromatin architecture in rare cell populations. We present a new low-input Capture-C approach that can generate high-quality 3C interaction profiles from 10 000–20 000 cells, depending on the resolution used for analysis. We also present a PCR-free, sequencing-free 3C technique based on NanoString technology called C-String. By comparing C-String and Capture-C interaction profiles we show that the latter are not skewed by PCR amplification. Furthermore, we demonstrate that chromatin interactions detected by Capture-C do not depend on the degree of cross-linking by performing experiments with varying formaldehyde concentrations. PMID:29186505

Micromachined nanocalorimetric sensor for ultra-low-volume cell-based assays.

PubMed

Johannessen, Erik A; Weaver, John M R; Bourova, Lenka; Svoboda, Petr; Cobbold, Peter H; Cooper, Jonathan M

2002-05-01

Current strategies for cell-based screening generally focus on the development of highly specific assays, which require an understanding of the nature of the signaling molecules and cellular pathways involved. In contrast, changes in temperature of cells provides a measure of altered cellular metabolism that is not stimulus specific and hence could have widespread applications in cell-based screening of receptor agonists and antagonists, as well as in the assessment of toxicity of new lead compounds. Consequently, we have developed a micromachined nanocalorimetric biological sensor using a small number of isolated living cells integrated within a subnanoliter format, which is capable of detecting 13 nW of generated power from the cells, upon exposure to a chemical or pharmaceutical stimulus. The sensor comprises a 10-junction gold and nickel thermopile, integrated on a silicon chip which was back-etched to span a 800-nm-thick membrane of silicon nitride. The thin-film membrane, which supported the sensing junctions of the thermoelectric transducer, gave the system a temperature resolution of 0.125 mK, a low heat capacity of 1.2 nJ mK(-1), and a rapid (unfiltered) response time of 12 ms. The application of the system in ultra-low-volume cell-based assays could provide a rapid endogenous screen. It offers important additional advantages over existing methods in that it is generic in nature, it does not require the use of recombinant cell lines or of detailed assay development, and finally, it can enable the use of primary cell lines or tissue biopsies.

Glucocorticoid receptor ligand binding in monocytic cells using a microplate assay.

PubMed

Jansen, J; Uitdehaag, B; Koper, J W; van Den Berg, T K

1999-01-01

Glucocorticoids have profound effects on macrophage function and are widely used as anti-inflammatory drugs. Glucocorticoids receptor (GR) ligand binding capacity is a major determinant of cellular glucocorticoid sensitivity. The number and affinity of GR can be measured in a whole cell binding assay using (3)H-dexamethasone. Here, we describe a rapid and simple microplate assay for GR measurement using the human promonocytic cell line THP-1. Copyright 2000 S. Karger AG, Basel.

DNA damage evaluation of hydroxyapatite on fibroblast cell L929 using the single cell gel electrophoresis assay.

PubMed

Rajab, N F; Yaakob, T A; Ong, B Y; Hamid, M; Ali, A M; Annuar, B O; Inayat-Hussain, S H

2004-05-01

Hydroxyapatite is the main component of the bone which is a potential biomaterial substance that can be applied in orthopaedics. In this study, the biocompatibility of this biomaterial was assessed using an in vitro technique. The cytotoxicity and genotoxicity effect of HA2 and HA3 against L929 fibroblast cell was evaluated using the MTT Assay and Alkaline Comet Assay respectively. Both HA2 and HA3 compound showed low cytotoxicity effect as determined using MTT Assay. Cells viability following 72 hours incubation at maximum concentration of both HA2 and HA3 (200 mg/ml) were 75.3 +/- 8.8% and 86.7 +/- 13.1% respectively. However, the cytotoxicity effect of ZnSO4.7H2O as a positive control showed an IC50 values of 46 mg/ml (160 microM). On the other hand, both HA2 and HA3 compound showed a slight genotoxicity effect as determined using the Alkaline Comet Assay following incubation at the concentration 200 mg/ml for 72 hours. This assay has been widely used in genetic toxicology to detect DNA strand breaks and alkali-labile site. The percentage of the cells with DNA damage for both substance was 27.7 +/- 1.3% and 15.6 +/- 1.0% for HA2 and HA3 respectively. Incubation of the cells for 24 hours with 38 microg/ml (IC25) of positive control showed an increase in percentage of cells with DNA damage (67.5 +/- 0.7%). In conclusion, our study indicated that both hydroxyapatite compounds showed a good biocompatibility in fibroblast cells.

Capture of circulating tumor cells using photoacoustic flowmetry and two phase flow

NASA Astrophysics Data System (ADS)

O'Brien, Christine M.; Rood, Kyle D.; Bhattacharyya, Kiran; DeSouza, Thiago; Sengupta, Shramik; Gupta, Sagar K.; Mosley, Jeffrey D.; Goldschmidt, Benjamin S.; Sharma, Nikhilesh; Viator, John A.

2012-06-01

Melanoma is the deadliest form of skin cancer, yet current diagnostic methods are unable to detect early onset of metastatic disease. Patients must wait until macroscopic secondary tumors form before malignancy can be diagnosed and treatment prescribed. Detection of cells that have broken off the original tumor and travel through the blood or lymph system can provide data for diagnosing and monitoring metastatic disease. By irradiating enriched blood samples spiked with cultured melanoma cells with nanosecond duration laser light, we induced photoacoustic responses in the pigmented cells. Thus, we can detect and enumerate melanoma cells in blood samples to demonstrate a paradigm for a photoacoustic flow cytometer. Furthermore, we capture the melanoma cells using microfluidic two phase flow, a technique that separates a continuous flow into alternating microslugs of air and blood cell suspension. Each slug of blood cells is tested for the presence of melanoma. Slugs that are positive for melanoma, indicated by photoacoustic waves, are separated from the cytometer for further purification and isolation of the melanoma cell. In this paper, we evaluate the two phase photoacoustic flow cytometer for its ability to detect and capture metastastic melanoma cells in blood.

A microfluidic device for label-free, physical capture of circulating tumor cell-clusters

PubMed Central

Sarioglu, A. Fatih; Aceto, Nicola; Kojic, Nikola; Donaldson, Maria C.; Zeinali, Mahnaz; Hamza, Bashar; Engstrom, Amanda; Zhu, Huili; Sundaresan, Tilak K.; Miyamoto, David T.; Luo, Xi; Bardia, Aditya; Wittner, Ben S.; Ramaswamy, Sridhar; Shioda, Toshi; Ting, David T.; Stott, Shannon L.; Kapur, Ravi; Maheswaran, Shyamala; Haber, Daniel A.; Toner, Mehmet

2015-01-01

Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC-clusters). Existing technologies for CTC enrichment are designed primarily to isolate single CTCs, and while CTC-clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here, we developed a microchip technology (Cluster-Chip) specifically designed to capture CTC-clusters independent of tumor-specific markers from unprocessed blood. CTC-clusters are isolated through specialized bifurcating traps under low shear-stress conditions that preserve their integrity and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identify CTC-clusters in 30–40% of patients with metastatic cancers of the breast, prostate and melanoma. RNA sequencing of CTC-clusters confirms their tumor origin and identifies leukocytes within the clusters as tissue-derived macrophages. Together, the development of a device for efficient capture of CTC-clusters will enable detailed characterization of their biological properties and role in cancer metastasis. PMID:25984697

A smart core-sheath nanofiber that captures and releases red blood cells from the blood.

PubMed

Shi, Q; Hou, J; Zhao, C; Xin, Z; Jin, J; Li, C; Wong, S-C; Yin, J

2016-01-28

A smart core-sheath nanofiber for non-adherent cell capture and release is demonstrated. The nanofibers are fabricated by single-spinneret electrospinning of poly(N-isopropylacrylamide) (PNIPAAm), polycaprolactone (PCL) and nattokinase (NK) solution blends. The self-assembly of PNIPAAm and PCL blends during the electrospinning generates the core-sheath PCL/PNIPAAm nanofibers with PNIPAAm as the sheath. The PNIPAAm-based core-sheath nanofibers are switchable between hydrophobicity and hydrophilicity with temperature change and enhance stability in the blood. When the nanofibers come in contact with blood, the NK is released from the nanofibers to resist platelet adhesion on the nanofiber surface, facilitating the direct capture and isolation of red blood cells (RBCs) from the blood above phase-transition temperature of PNIPAAm. Meanwhile, the captured RBCs are readily released from the nanofibers with temperature stimuli in an undamaged manner. The release efficiency of up to 100% is obtained while maintaining cellular integrity and function. This work presents promising nanofibers to effectively capture non-adherent cells and release for subsequent molecular analysis and diagnosis of single cells.

Technique for obtaining highly enriched, quiescent immature Langerhans cells suitable for ex vivo assays.

PubMed

Tchou, Isabelle; Sabido, Odile; Lambert, Claude; Misery, Laurent; Garraud, Olivier; Genin, Christian

2003-03-03

Epidermis and surface epithelium-dendritic cells comprise of immature cells termed Langerhans cells (LCs), which express characteristically the Birbeck granules, along with surface markers such as CD1a. These cells can capture a pathogen and then migrate and differentiate to a more mature stage. During this maturation process, dentritic cells express surface markers differentially. In physio-pathological models of infection where LCs are involved, it is critically important to ensure that the LCs tested in vitro are still immature and are not artefactually matured-dentritic cells. For experimental purposes, LCs were isolated from skin epidermis obtained from patients undergoing plastic surgery. This work thus aimed at collecting fresh LCs ex vivo and at testing the cells for phenotypic and functional characteristics of the immature stage. After mechanic disruption of the epidermis and proceeding for single cell suspension obtaining, two methods for purification were tested in parallel: (a) a positive immuno-magnetic separation by anti-CD1a-coated beads and (b) a purely mechanic purification system based on a three-step Ficoll floatation process. Both systems were equally efficient in terms of purification and yield. By using flow cytometry phenotyping, we have demonstrated that the use of magnetic beads led to some degree of maturation of CD1a(+) LCs, contrary to the repeated Ficoll floatation. This work calls attention for the use of certain monoclonal antibodies such as anti-CD1a to purify immature dendritic cells as they pre-activate these cells. Pre-activation would render a number of assays on the early events of LC physiology invalid, contrary to the purification of fresh skin epidermis LCs by means of a repeated Ficoll floatation.

A NEW SENSITIVE ASSAY FOR ANTIBODY AGAINST CELL SURFACE ANTIGENS BASED ON INHIBITION OF CELL-DEPENDENT ANTIBODY-MEDIATED CYTOTOXICITY

PubMed Central

Halloran, Phil; Schirrmacher, Volker; Festenstein, Hilliard

1974-01-01

Inhibition of cell-dependent antibody-mediated cytotoxicity has been investigated as a new assay for antibody against cell surface antigens. The cytotoxicity system consisted of effector cells (normal mouse spleen cells), target cells (61Cr-labeled chicken erythrocytes), and antitarget cell antibody. Addition of antibody against cell surface antigens in the effector cell population regularly inhibited the cytotoxicity measured in this system. This cytotoxicity inhibition assay (CIA) detected antibody with a variety of specificities: anti-H-2, anti-Thy 1.2, anti-immunoglobulin, and antimouse bone marrow-derived lymphocyte antigen. When the inhibition by anti-H-2 sera was analyzed using effector cells from congenic mice, the activity was found to be directed against specificities mapping in the H-2K, H-2D, and I regions of the H-2 complex, correlating well with the specificities characterized by complement-dependent assays. A comparison between the sensitivity of the CIA and complement-dependent lysis revealed that the CIA was 2–11 times more sensitive for anti-H-2 antisera and 20–780 times more sensitive for certain antisera against subpopulations of the spleen cells (i.e., T cells or B cells). The CIA proved to be precise, sensitive, and reliable. It may become a very useful antibody assay in various species including man. PMID:4547657

Using a medium-throughput comet assay to evaluate the global DNA methylation status of single cells

PubMed Central

Lewies, Angélique; Van Dyk, Etresia; Wentzel, Johannes F.; Pretorius, Pieter J.

2014-01-01

The comet assay is a simple and cost effective technique, commonly used to analyze and quantify DNA damage in individual cells. The versatility of the comet assay allows introduction of various modifications to the basic technique. The difference in the methylation sensitivity of the isoschizomeric restriction enzymes HpaII and MspI are used to demonstrate the ability of the comet assay to measure the global DNA methylation level of individual cells when using cell cultures. In the experiments described here, a medium-throughput comet assay and methylation sensitive comet assay are combined to produce a methylation sensitive medium-throughput comet assay to measure changes in the global DNA methylation pattern in individual cells under various growth conditions. PMID:25071840

Semi-microdroplet assay for cell adhesion molecules. M.S. Thesis

NASA Technical Reports Server (NTRS)

Tawa, Lawrence Shinzo

1988-01-01

A new cell-to-cell adhesion assay was devised. Using dissociated embryos of the sea urchin, this procedure involves rotating a 0.100 ml suspension of single cells with 0.100 ml of the solution to be tested in the bulb portion of a transfer pipet with the tip removed. After 1 hour of rotation at 60 rpm at 15 C, the contents of each bulb were transferred into individual wells of a 96 well flat bottom plate. After the plate was incubated for 1 hour at 15 C, black and white photographs were taken with a 35 mm camera attached to an inverted photomicroscope. Examining a proof sheet of the negatives directly allowed a rapid evaluation of suspected cell adhesion promoting factors. A ranking system was used to evaluate all samples. The assay was tested by examining the effect of specific solutions on the aggregation of single cells obtained from dissociated 23 hour embryos.

Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

NASA Astrophysics Data System (ADS)

Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si; Wang, Jin; Huang, Nan

2016-11-01

Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses.

PubMed

Chang, G J; Trent, D W; Vorndam, A V; Vergne, E; Kinney, R M; Mitchell, C J

1994-02-01

We previously described a reverse transcriptase-PCR using flavivirus genus-conserved and virus species-specific amplimers (D. W. Trent and G. J. Chang, p. 355-371, in Y. Becker and C. Darai; ed., Frontiers of Virology, vol. 1, 1992). Target amplification was improved by redesigning the amplimers, and a sensitive enzyme-linked immunosorbent assay (ELISA) technique has been developed to detect amplified digoxigenin (DIG)-modified DNA. A single biotin motif and multiple DIG motifs were incorporated into each amplicon, which permitted amplicon capture by a biotin-streptavidin interaction and detection with DIG-specific antiserum in a colorimetric ELISA. We evaluated the utility of this assay for detecting St. Louis encephalitis (SLE) viral RNA in infected mosquitoes and dengue viral RNA in human serum specimens. The reverse transcriptase-PCR-ELISA was as sensitive as isolation of SLE virus by cell culture in detecting SLE viral RNA in infected mosquitoes. The test was 89% specific and 95 to 100% sensitive for identification of dengue viral RNA in serum specimens compared with isolation of virus by Aedes albopictus C6/36 cell culture and identification by the indirect immunofluorescence assay.

An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses.

PubMed Central

Chang, G J; Trent, D W; Vorndam, A V; Vergne, E; Kinney, R M; Mitchell, C J

1994-01-01

We previously described a reverse transcriptase-PCR using flavivirus genus-conserved and virus species-specific amplimers (D. W. Trent and G. J. Chang, p. 355-371, in Y. Becker and C. Darai; ed., Frontiers of Virology, vol. 1, 1992). Target amplification was improved by redesigning the amplimers, and a sensitive enzyme-linked immunosorbent assay (ELISA) technique has been developed to detect amplified digoxigenin (DIG)-modified DNA. A single biotin motif and multiple DIG motifs were incorporated into each amplicon, which permitted amplicon capture by a biotin-streptavidin interaction and detection with DIG-specific antiserum in a colorimetric ELISA. We evaluated the utility of this assay for detecting St. Louis encephalitis (SLE) viral RNA in infected mosquitoes and dengue viral RNA in human serum specimens. The reverse transcriptase-PCR-ELISA was as sensitive as isolation of SLE virus by cell culture in detecting SLE viral RNA in infected mosquitoes. The test was 89% specific and 95 to 100% sensitive for identification of dengue viral RNA in serum specimens compared with isolation of virus by Aedes albopictus C6/36 cell culture and identification by the indirect immunofluorescence assay. PMID:7512096

A smart core-sheath nanofiber that captures and releases red blood cells from the blood

NASA Astrophysics Data System (ADS)

Shi, Q.; Hou, J.; Zhao, C.; Xin, Z.; Jin, J.; Li, C.; Wong, S.-C.; Yin, J.

2016-01-01

A smart core-sheath nanofiber for non-adherent cell capture and release is demonstrated. The nanofibers are fabricated by single-spinneret electrospinning of poly(N-isopropylacrylamide) (PNIPAAm), polycaprolactone (PCL) and nattokinase (NK) solution blends. The self-assembly of PNIPAAm and PCL blends during the electrospinning generates the core-sheath PCL/PNIPAAm nanofibers with PNIPAAm as the sheath. The PNIPAAm-based core-sheath nanofibers are switchable between hydrophobicity and hydrophilicity with temperature change and enhance stability in the blood. When the nanofibers come in contact with blood, the NK is released from the nanofibers to resist platelet adhesion on the nanofiber surface, facilitating the direct capture and isolation of red blood cells (RBCs) from the blood above phase-transition temperature of PNIPAAm. Meanwhile, the captured RBCs are readily released from the nanofibers with temperature stimuli in an undamaged manner. The release efficiency of up to 100% is obtained while maintaining cellular integrity and function. This work presents promising nanofibers to effectively capture non-adherent cells and release for subsequent molecular analysis and diagnosis of single cells.A smart core-sheath nanofiber for non-adherent cell capture and release is demonstrated. The nanofibers are fabricated by single-spinneret electrospinning of poly(N-isopropylacrylamide) (PNIPAAm), polycaprolactone (PCL) and nattokinase (NK) solution blends. The self-assembly of PNIPAAm and PCL blends during the electrospinning generates the core-sheath PCL/PNIPAAm nanofibers with PNIPAAm as the sheath. The PNIPAAm-based core-sheath nanofibers are switchable between hydrophobicity and hydrophilicity with temperature change and enhance stability in the blood. When the nanofibers come in contact with blood, the NK is released from the nanofibers to resist platelet adhesion on the nanofiber surface, facilitating the direct capture and isolation of red blood cells (RBCs) from

Detection and capture of breast cancer cells with photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Bhattacharyya, Kiran; Goldschmidt, Benjamin S.; Viator, John A.

2016-08-01

According to the Centers for Disease Control and Prevention, breast cancer is the most common cancer and the second leading cause of cancer related deaths among women. Metastasis-the presence of secondary tumors caused by the spread of cancer cells via the circulatory or lymphatic systems-significantly worsens the prognosis of any breast cancer patient. A technique is developed to detect circulating breast cancer cells in human blood using a photoacoustic flow cytometry method. A Q-switched laser is used to interrogate thousands of blood cells with one pulse as they flow through the beam path. Cells that are optically absorbing, either naturally or artificially, emit an ultrasound wave as a result of the photoacoustic (PA) effect. Breast cancer cells are targeted with chromophores through immunochemistry in order to enhance optical absorption. After which, the PA cytometry device is calibrated to demonstrate the ability to detect single cells. Cultured breast cancer cells are added to whole blood to reach a biologically relevant concentration of about 25 to 45 breast cancer cells per 1 mL of blood. An in vitro PA flow cytometer is used to detect and isolate these cells followed by capture with the use of a micromanipulator. This method can not only be used to determine the disease state of the patient and the response to therapy but also it can be used for genetic testing and in vitro drug trials since the circulating cell can be captured and studied.

A Cell Programmable Assay (CPA) chip.

PubMed

Ju, Jongil; Warrick, Jay; Beebe, David J

2010-08-21

This article describes two kinds of "Cell Programmable Assay" (CPA) chips that utilize passive pumping for the culture and autonomous staining of cells to simply common protocols. One is a single timer channel CPA (sCPA) chip that has one timer channel and one main channel containing a cell culture chamber. The sCPA is used to culture and stain cells using Hoechst nuclear staining dye (a 2 step staining process). The other is a dual timer channel CPA (dCPA) chip that has two timer channels and one main channel with a chamber for cell culture. The dCPA is used here to culture, fix, permeablize, and stain cells using DAPI. The additional timer channel of the dCPA chip allows for automation of 3 steps. The CPA chips were successfully evaluated using HEK 293 cells. In addition, we provide a simplified equation for tuning or redesigning CPA chips to meet the needs of a variety of protocols that may require different timings. The equation is easy to use as it only depends upon the dimensions of microchannel and the volume of the reagent drops. The sCPA and dCPA chips can be readily modified to apply to a wide variety of common cell culture methods and procedures.

Ethanol-dispersed and antibody-conjugated polymer nanofibers for the selective capture and 3-dimensional culture of EpCAM-positive cells.

PubMed

Yoon, Junghyo; Yoon, Hee-Sook; Shin, Yoojin; Kim, Sanghyun; Ju, Youngjun; Kim, Jungbae; Chung, Seok

2017-07-01

Electrospun and ethanol-dispersed polystyrene-poly(styrene-co-maleic anhydride) (PS-PSMA) nanofibers (NFs) were used as a platform for the selective capture and three-dimensional culture of EpCAM-positive cells in cell culture medium and whole blood. The NFs were treated with streptavidin to facilitate bond formation between the amino groups of streptavidin and the maleic anhydride groups of the NFs. A biotinylated anti-EpCAM monoclonal antibody (mAb) was attached to the streptavidin-conjugated NFs via the selective binding of streptavidin and biotin. Upon simple mixing and shaking with EpCAM-positive cancer cells in a wide concentration range from 10 to 1000,000 cells per 10mL, the mAb-attached NFs (mAb-NFs) captured the Ep-CAM positive cells in an efficiency of 59%-67% depending on initial cell concentrations, with minor mechanical capture of 14%-36%. Captured cells were directly cultured, forming cell aggregates, in the NF matrix, which ensures the cell proliferation and follow-up analysis. Furthermore, the capture capacity of mAb-NFs was assessed in the presence of whole blood and blood lysates, indicating cluster formation that captured target cells. It is anticipated that the antibody-attached NFs can be employed for the capture and analysis of very rare EpCAM positive circulating cancer cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Capture and On-chip analysis of Melanoma Cells Using Tunable Surface Shear forces

NASA Astrophysics Data System (ADS)

Tsao, Simon Chang-Hao; Vaidyanathan, Ramanathan; Dey, Shuvashis; Carrascosa, Laura G.; Christophi, Christopher; Cebon, Jonathan; Shiddiky, Muhammad J. A.; Behren, Andreas; Trau, Matt

2016-01-01

With new systemic therapies becoming available for metastatic melanoma such as BRAF and PD-1 inhibitors, there is an increasing demand for methods to assist with treatment selection and response monitoring. Quantification and characterisation of circulating melanoma cells (CMCs) has been regarded as an excellent non-invasive candidate but a sensitive and efficient tool to do these is lacking. Herein we demonstrate a microfluidic approach for melanoma cell capture and subsequent on-chip evaluation of BRAF mutation status. Our approach utilizes a recently discovered alternating current electrohydrodynamic (AC-EHD)-induced surface shear forces, referred to as nanoshearing. A key feature of nanoshearing is the ability to agitate fluid to encourage contact with surface-bound antibody for the cell capture whilst removing nonspecific cells from the surface. By adjusting the AC-EHD force to match the binding affinity of antibodies against the melanoma-associated chondroitin sulphate proteoglycan (MCSP), a commonly expressed melanoma antigen, this platform achieved an average recovery of 84.7% from biological samples. Subsequent staining with anti-BRAFV600E specific antibody enabled on-chip evaluation of BRAFV600E mutation status in melanoma cells. We believe that the ability of nanoshearing-based capture to enumerate melanoma cells and subsequent on-chip characterisation has the potential as a rapid screening tool while making treatment decisions.

Mapping mitochondrial heteroplasmy in a Leydig tumor by laser capture micro-dissection and cycling temperature capillary electrophoresis.

PubMed

Refinetti, Paulo; Arstad, Christian; Thilly, William G; Morgenthaler, Stephan; Ekstrøm, Per Olaf

2017-01-01

The growth of tumor cells is accompanied by mutations in nuclear and mitochondrial genomes creating marked genetic heterogeneity. Tumors also contain non-tumor cells of various origins. An observed somatic mitochondrial mutation would have occurred in a founding cell and spread through cell division. Micro-anatomical dissection of a tumor coupled with assays for mitochondrial point mutations permits new insights into this growth process. More generally, the ability to detect and trace, at a histological level, somatic mitochondrial mutations in human tissues and tumors, makes these mutations into markers for lineage tracing. A tumor was first sampled by a large punch biopsy and scanned for any significant degree of heteroplasmy in a set of sequences containing known mutational hotspots of the mitochondrial genome. A heteroplasmic tumor was sliced at a 12 μm thickness and placed on membranes. Laser capture micro-dissection was used to take 25000 μm 2 subsamples or spots. After DNA amplification, cycling temperature capillary electrophoresis (CTCE) was used on the laser captured samples to quantify mitochondrial mutant fractions. Of six testicular tumors studied, one, a Leydig tumor, was discovered to carry a detectable degree of heteroplasmy for two separate point mutations: a C → T mutation at bp 64 and a T → C mutation found at bp 152. From this tumor, 381 spots were sampled with laser capture micro-dissection. The ordered distribution of spots exhibited a wide range of fractions of the mutant sequences from 0 to 100% mutant copies. The two mutations co-distributed in the growing tumor indicating they were present on the same genome copies in the founding cell. Laser capture microdissection of sliced tumor samples coupled with CTCE-based point mutation assays provides an effective and practical means to obtain maps of mitochondrial mutational heteroplasmy within human tumors.

Detection of an ALK Fusion in Colorectal Carcinoma by Hybrid Capture-Based Assay of Circulating Tumor DNA.

PubMed

Lai, Andrea Z; Schrock, Alexa B; Erlich, Rachel L; Ross, Jeffrey S; Miller, Vincent A; Yakirevich, Evgeny; Ali, Siraj M; Braiteh, Fadi

2017-07-01

ALK rearrangements have been observed in 0.05%-2.5% of patients with colorectal cancers (CRCs) and are predicted to be oncogenic drivers largely mutually exclusive of KRAS, NRAS, or BRAF alterations. Here we present the case of a patient with metastatic CRC who was treatment naïve at the time of molecular testing. Initial ALK immunohistochemistry (IHC) staining was negative, but parallel genomic profiling of both circulating tumor DNA (ctDNA) and tissue using similar hybrid capture-based assays each identified an identical STRN-ALK fusion. Subsequent ALK IHC staining of the same specimens was positive, suggesting that the initial result was a false negative. This report is the first instance of an ALK fusion in CRC detected using a ctDNA assay. Current guidelines for colorectal cancer (CRC) only recommend genomic assessment of KRAS, NRAS, BRAF, and microsatellite instability (MSI) status. ALK rearrangements are rare in CRC, but patients with activating ALK fusions have responded to targeted therapies ALK rearrangements can be detected by genomic profiling of ctDNA from blood or tissue, and this methodology may be informative in cases where immunohistochemistry (IHC) or other standard testing is negative. © AlphaMed Press 2017.

Highly efficient capture and harvest of circulating tumor cells on a microfluidic chip integrated with herringbone and micropost arrays.

PubMed

Xue, Peng; Wu, Yafeng; Guo, Jinhong; Kang, Yuejun

2015-04-01

Circulating tumor cells (CTCs), which are derived from primary tumor site and transported to distant organs, are considered as the major cause of metastasis. So far, various techniques have been applied for CTC isolation and enumeration. However, there exists great demand to improve the sensitivity of CTC capture, and it remains challenging to elute the cells efficiently from device for further biomolecular and cellular analyses. In this study, we fabricate a dual functional chip integrated with herringbone structure and micropost array to achieve CTC capture and elution through EpCAM-based immunoreaction. Hep3B tumor cell line is selected as the model of CTCs for processing using this device. The results demonstrate that the capture limit of Hep3B cells can reach up to 10 cells (per mL of sample volume) with capture efficiency of 80% on average. Moreover, the elution rate of the captured Hep3B cells can reach up to 69.4% on average for cell number ranging from 1 to 100. These results demonstrate that this device exhibits dual functions with considerably high capture rate and elution rate, indicating its promising capability for cancer diagnosis and therapeutics.

A homogeneous cell-based assay for measurement of endogenous paraoxonase 1 activity.

PubMed

Ahmad, Syed; Carter, Jade J; Scott, John E

2010-05-01

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme that plays an important role in organophosphate detoxification and prevention of atherosclerosis. Thus, there is significant interest in identifying nutritional and pharmacological enhancers of PON1 activity. To identify such compounds, we developed a rapid homogeneous assay to detect endogenous cell-associated PON1 activity. PON1 activity was measured by the simple addition of fluorigenic PON1 substrate DEPFMU to live Huh7 cells in medium and monitoring change in fluorescence. A specific PON1 inhibitor, 2-hydroxyquinoline, was used to confirm that the observed activity was due to PON1. The assay was optimized and characterized with regard to time course, substrate and sodium chloride concentration, number of cells, and tolerance to dimethyl sulfoxide and serum. Aspirin, quercetin, and simvastatin are compounds reported to increase PON1 expression. Consistent with the literature and Western blot data, these compounds enhanced PON1 activity in this assay with comparable efficacies and potencies. A known toxic compound did not increase assay signal. This assay method also detected PON1 activity in normal hepatocytes. Thus, a novel homogeneous assay for detection of endogenous PON1 expression has been developed and is amenable to high-throughput screening for the identification of small molecules that enhance PON1 expression. 2010 Elsevier Inc. All rights reserved.

Implementation and Challenges of Direct Acoustic Dosing into Cell-Based Assays.

PubMed

Roberts, Karen; Callis, Rowena; Ikeda, Tim; Paunovic, Amalia; Simpson, Carly; Tang, Eric; Turton, Nick; Walker, Graeme

2016-02-01

Since the adoption of Labcyte Echo Acoustic Droplet Ejection (ADE) technology by AstraZeneca in 2005, ADE has become the preferred method for compound dosing into both biochemical and cell-based assays across AstraZeneca research and development globally. The initial implementation of Echos and the direct dosing workflow provided AstraZeneca with a unique set of challenges. In this article, we outline how direct Echo dosing has evolved over the past decade in AstraZeneca. We describe the practical challenges of applying ADE technology to 96-well, 384-well, and 1536-well assays and how AstraZeneca developed and applied software and robotic solutions to generate fully automated and effective cell-based assay workflows. © 2015 Society for Laboratory Automation and Screening.

Telomere maintenance in laser capture microdissection-purified Barrett's adenocarcinoma cells and effect of telomerase inhibition in vivo.

PubMed

Shammas, Masood A; Qazi, Aamer; Batchu, Ramesh B; Bertheau, Robert C; Wong, Jason Y Y; Rao, Manjula Y; Prasad, Madhu; Chanda, Diptiman; Ponnazhagan, Selvarangan; Anderson, Kenneth C; Steffes, Christopher P; Munshi, Nikhil C; De Vivo, Immaculata; Beer, David G; Gryaznov, Sergei; Weaver, Donald W; Goyal, Raj K

2008-08-01

The aims of this study were to investigate telomere function in normal and Barrett's esophageal adenocarcinoma (BEAC) cells purified by laser capture microdissection and to evaluate the effect of telomerase inhibition in cancer cells in vitro and in vivo. Epithelial cells were purified from surgically resected esophagi. Telomerase activity was measured by modified telomeric repeat amplification protocol and telomere length was determined by real-time PCR assay. To evaluate the effect of telomerase inhibition, adenocarcinoma cell lines were continuously treated with a specific telomerase inhibitor (GRN163L) and live cell number was determined weekly. Apoptosis was evaluated by Annexin labeling and senescence by beta-galactosidase staining. For in vivo studies, severe combined immunodeficient mice were s.c. inoculated with adenocarcinoma cells and following appearance of palpable tumors, injected i.p. with saline or GRN163L. Telomerase activity was significantly elevated whereas telomeres were shorter in BEAC cells relative to normal esophageal epithelial cells. The treatment of adenocarcinoma cells with telomerase inhibitor, GRN163L, led to loss of telomerase activity, reduction in telomere length, and growth arrest through induction of both the senescence and apoptosis. GRN163L-induced cell death could also be expedited by addition of the chemotherapeutic agents doxorubicin and ritonavir. Finally, the treatment with GRN163L led to a significant reduction in tumor volume in a subcutaneous tumor model. We show that telomerase activity is significantly elevated whereas telomeres are shorter in BEAC and suppression of telomerase inhibits proliferation of adenocarcinoma cells both in vitro and in vivo.

A Combined Negative and Positive Enrichment Assay for Cancer Cells Isolation and Purification.

PubMed

Cheng, Boran; Wang, Shuyi; Chen, Yuanyuan; Fang, Yuan; Chen, Fangfang; Wang, Zhenmeng; Xiong, Bin

2016-02-01

Cancer cells that detach from solid tumor and circulate in the peripheral blood (CTCs) have been considered as a new "biomarker" for the detection and characterization of cancers. However, isolating and detecting cancer cells from the cancer patient peripheral blood have been technically challenging, owing to the small sub-population of CTCs (a few to hundreds per milliliter). Here we demonstrate a simple and efficient cancer cells isolation and purification method. A biocompatible and surface roughness controllable TiO2 nanofilm was deposited onto a glass slide to achieve enhanced topographic interactions with nanoscale cellular surface components, again, anti-CD45 (a leukocyte common antigen) and anti-EpCAM (epithelial cell adhesion molecule) were then coated onto the surface of the nanofilm for advance depletion of white blood cells (WBCs) and specific isolation of CTCs, respectively. Comparing to the conventional positive enrichment technology, this method exhibited excellent biocompatibility and equally high capture efficiency. Moreover, the maximum number of background cells (WBCs) was removed, and viable and functional cancer cells were isolated with high purity. Utilizing the horizontally packed TiO2 nanofilm improved pure CTC-capture through combining cell-capture-agent and cancer cell-preferred nanoscale topography, which represented a new method capable of obtaining biologically functional CTCs for subsequent molecular analysis. © The Author(s) 2014.

Polyethylene Glycol-Functionalized Magnetic Fe₃O₄/P(MMA-AA) Composite Nanoparticles Enhancing Efficient Capture of Circulating Tumor Cells.

PubMed

Ma, Shaohua; Zhan, Xiaohui; Yang, Minggang; Lan, Fang; Wu, Yao; Gu, Zhongwei

2018-04-01

Circulating tumor cells (CTCs) played a significant role in early diagnosis and prognosis of carcinomas, and efficient capture of CTCs was highly desired to provide important and reliable evidence for clinical diagnosis. In present work, we successfully synthesized functional magnetic Fe3O4/P(MMA-AA) composite nanoparticles (FCNPs) inspired by a counterbalance concept for recognition and capture of CTCs. This counterbalance, composed of polyethylene glycol (PEG) suppressing cell adhesion and anti-epithelial-cell-adhesion-molecule (anti-EpCAM) antibody targeting tumor cells, could both enhance the specific capture of tumor cells and reduce unspecific adhesion of normal cells. The study showed that the PEG density on the surface of the FCNPs affected the specificity of the materials, and a density of ca. 15% was efficient for reducing the unspecific adhesion. After incubation with the mixture of HepG2 cells and Jurkat T cells, the FCNPs reached a capture efficiency as high as about 86.5% of the cancer cells, suggesting great potential on detection of CTCs in the diagnoses and prognoses of cancer metastasis.

OpenComet: An automated tool for comet assay image analysis

PubMed Central

Gyori, Benjamin M.; Venkatachalam, Gireedhar; Thiagarajan, P.S.; Hsu, David; Clement, Marie-Veronique

2014-01-01

Reactive species such as free radicals are constantly generated in vivo and DNA is the most important target of oxidative stress. Oxidative DNA damage is used as a predictive biomarker to monitor the risk of development of many diseases. The comet assay is widely used for measuring oxidative DNA damage at a single cell level. The analysis of comet assay output images, however, poses considerable challenges. Commercial software is costly and restrictive, while free software generally requires laborious manual tagging of cells. This paper presents OpenComet, an open-source software tool providing automated analysis of comet assay images. It uses a novel and robust method for finding comets based on geometric shape attributes and segmenting the comet heads through image intensity profile analysis. Due to automation, OpenComet is more accurate, less prone to human bias, and faster than manual analysis. A live analysis functionality also allows users to analyze images captured directly from a microscope. We have validated OpenComet on both alkaline and neutral comet assay images as well as sample images from existing software packages. Our results show that OpenComet achieves high accuracy with significantly reduced analysis time. PMID:24624335

OpenComet: an automated tool for comet assay image analysis.

PubMed

Gyori, Benjamin M; Venkatachalam, Gireedhar; Thiagarajan, P S; Hsu, David; Clement, Marie-Veronique

2014-01-01

Reactive species such as free radicals are constantly generated in vivo and DNA is the most important target of oxidative stress. Oxidative DNA damage is used as a predictive biomarker to monitor the risk of development of many diseases. The comet assay is widely used for measuring oxidative DNA damage at a single cell level. The analysis of comet assay output images, however, poses considerable challenges. Commercial software is costly and restrictive, while free software generally requires laborious manual tagging of cells. This paper presents OpenComet, an open-source software tool providing automated analysis of comet assay images. It uses a novel and robust method for finding comets based on geometric shape attributes and segmenting the comet heads through image intensity profile analysis. Due to automation, OpenComet is more accurate, less prone to human bias, and faster than manual analysis. A live analysis functionality also allows users to analyze images captured directly from a microscope. We have validated OpenComet on both alkaline and neutral comet assay images as well as sample images from existing software packages. Our results show that OpenComet achieves high accuracy with significantly reduced analysis time.

Quantum Dot Nanotoxicity Investigations Using Human Lung Cells and TOXOR Electrochemical Enzyme Assay Methodology.

PubMed

O'Hara, Tony; Seddon, Brian; O'Connor, Andrew; McClean, Siobhán; Singh, Baljit; Iwuoha, Emmanuel; Fuku, Xolile; Dempsey, Eithne

2017-01-27

Recent studies have suggested that certain nanomaterials can interfere with optically based cytotoxicity assays resulting in underestimations of nanomaterial toxicity. As a result there has been growing interest in the use of whole cell electrochemical biosensors for nanotoxicity applications. Herein we report application of an electrochemical cytotoxicity assay developed in house (TOXOR) in the evaluation of toxic effects of mercaptosuccinic acid capped cadmium telluride quantum dots (MSA capped CdTe QDs), toward mammalian cells. MSA capped CdTe QDs were synthesized, characterized, and their cytotoxicity toward A549 human lung epithelial cells investigated. The internalization of QDs within cells was scrutinized via confocal microscopy. The cytotoxicity assay is based on the measurement of changes in cellular enzyme acid phosphatase upon 24 h exposure to QDs. Acid phosphatase catalyzes dephosphorylation of 2-naphthyl phosphate to 2-naphthol (determined by chronocoulometry) and is indicative of metabolic activity in cells. The 24 h IC50 (concentration resulting in 50% reduction in acid phosphatase activity) value for MSA capped CdTe QDs was found to be 118 ± 49 μg/mL using the TOXOR assay and was in agreement with the MTT assay (157 ± 31 μg/mL). Potential uses of this electrochemical assay include the screening of nanomaterials, environmental toxins, in addition to applications in the pharmaceutical, food, and health sectors.

Human adipose-derived stem cells (ADSC) and human periodontal ligament stem cells (PDLSC) as cellular substrates of a toxicity prediction assay.

PubMed

Corrêa, Natássia Caroline Resende; Kuligovski, Crisciele; Paschoal, Ariane Caroline Campos; Abud, Ana Paula Ressetti; Rebelatto, Carmen Lucia Kuniyoshi; Leite, Lidiane Maria Boldrini; Senegaglia, Alexandra Cristina; Dallagiovanna, Bruno; Aguiar, Alessandra Melo de

2018-02-01

With the increasing need to develop in vitro assays to replace animal use, human stem cell-derived methods are emerging and showing outstanding contributions to the toxicological screening of substances. Adult human stem cells such as adipose-derived stem cells (ADSC) and periodontal ligament stem cells (PDLSC) were used as cell substrates for a cytotoxicity assay and toxicity prediction using the neutral red uptake (NRU) assay. First, primary cell cultures from three independent donors, from each tissue source, were characterized as mesenchymal stem cells (MSC) by plastic adherence and appropriate immunophenotype for MSC markers (positive for CD90, CD73, and CD105 and negative for CD11b, CD34, CD45, HLADR, and CD19). Furthermore, ADSC and PDLSC were able to differentiate into adipocytes and osteoblasts when maintained under the same culture conditions previously established for the NRU assay. NRU assays for three reference test substances were performed. R 2 was higher than 0.85 for all conditions, showing the feasibility to calculate IC 50 values. The IC 50 values were then used to predict the LD 50 of the test substances, which were comparable to previous results and the ICCVAM standard test report. Primary ADSC and PDLSC showed the potential to be considered as additional models for use in cytotoxicity assays. Copyright © 2017 Elsevier Inc. All rights reserved.

Comprehensive Profiling of Radiosensitive Human Cell Lines with DNA Damage Response Assays Identifies the Neutral Comet Assay as a Potential Surrogate for Clonogenic Survival

PubMed Central

Nahas, Shareef A.; Davies, Robert; Fike, Francesca; Nakamura, Kotoka; Du, Liutao; Kayali, Refik; Martin, Nathan T.; Concannon, Patrick; Gatti, Richard A.

2015-01-01

In an effort to explore the possible causes of human radiosensitivity and identify more rapid assays for cellular radiosensitivity, we interrogated a set of assays that evaluate cellular functions involved in recognition and repair of DNA double-strand breaks: (1) neutral comet assay, (2) radiation-induced γ-H2AX focus formation, (3) the temporal kinetics of structural maintenance of chromosomes 1 phosphorylation, (4) intra-S-phase checkpoint integrity, and (5) mitochondrial respiration. We characterized a unique panel of 19 “radiosensitive” human lymphoblastoid cell lines from individuals with undiagnosed diseases suggestive of a DNA repair disorder. Radiosensitivity was defined by reduced cellular survival using a clonogenic survival assay. Each assay identified cell lines with defects in DNA damage response functions. The highest concordance rate observed, 89% (17/19), was between an abnormal neutral comet assay and reduced survival by the colony survival assay. Our data also suggested that the neutral comet assay would be a more rapid surrogate for analyzing DNA repair/processing disorders. PMID:21962002

Serial interactome capture of the human cell nucleus.

PubMed

Conrad, Thomas; Albrecht, Anne-Susann; de Melo Costa, Veronica Rodrigues; Sauer, Sascha; Meierhofer, David; Ørom, Ulf Andersson

2016-04-04

Novel RNA-guided cellular functions are paralleled by an increasing number of RNA-binding proteins (RBPs). Here we present 'serial RNA interactome capture' (serIC), a multiple purification procedure of ultraviolet-crosslinked poly(A)-RNA-protein complexes that enables global RBP detection with high specificity. We apply serIC to the nuclei of proliferating K562 cells to obtain the first human nuclear RNA interactome. The domain composition of the 382 identified nuclear RBPs markedly differs from previous IC experiments, including few factors without known RNA-binding domains that are in good agreement with computationally predicted RNA binding. serIC extends the number of DNA-RNA-binding proteins (DRBPs), and reveals a network of RBPs involved in p53 signalling and double-strand break repair. serIC is an effective tool to couple global RBP capture with additional selection or labelling steps for specific detection of highly purified RBPs.

Assessment of the predictive capacity of the optimized in vitro comet assay using HepG2 cells.

PubMed

Hong, Yoon-Hee; Jeon, Hye Lyun; Ko, Kyung Yuk; Kim, Joohwan; Yi, Jung-Sun; Ahn, Ilyoung; Kim, Tae Sung; Lee, Jong Kwon

2018-03-01

Evaluation of DNA damage is critical during the development of new drugs because it is closely associated with genotoxicity and carcinogenicity. The in vivo comet assay to assess DNA damage is globally harmonized as OECD TG 489. However, a comet test guideline that evaluates DNA damage without sacrificing animals does not yet exist. The goal of this study was to select an appropriate cell line for optimization of the in vitro comet assay to assess DNA damage. We then evaluated the predictivity of the in vitro comet assay using the selected cell line. In addition, the effect of adding S9 was evaluated using 12 test chemicals. For cell line selection, HepG2, Chinese hamster lung (CHL/IU), and TK6 cell lines were evaluated. We employed a method for the in vitro comet assay based on that for the in vivo comet assay. The most appropriate cell line was determined by% tail DNA increase after performing in vitro comet assays with 6 test chemicals. The predictivity of the in vitro comet assay using the selected cell line was measured with 10 test chemicals (8 genotoxins and 2 non-genotoxic chemicals). The HepG2 cell line was found to be the most appropriate, and in vitro comet assays using HepG2 cells exhibited a high accuracy of 90% (9/10). This study suggests that HepG2 is an optimal cell line for the in vitro comet assay to assess DNA damage. Copyright © 2018 Elsevier B.V. All rights reserved.

Stem Cell-Like Differentiation Potentials of Endometrial Side Population Cells as Revealed by a Newly Developed In Vivo Endometrial Stem Cell Assay

PubMed Central

Miyazaki, Kaoru; Maruyama, Tetsuo; Masuda, Hirotaka; Yamasaki, Akiko; Uchida, Sayaka; Oda, Hideyuki; Uchida, Hiroshi; Yoshimura, Yasunori

2012-01-01

Background Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a woman's reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP), but not endometrial main population cells (EMP), exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche) to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay. Methodology/Principal Findings ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom), a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells. Conclusions/Significance We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo endometrial tissue

Human iPSC-Derived Endothelial Cell Sprouting Assay in Synthetic Hydrogel Arrays

EPA Science Inventory

Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can rec...

EL4 cell-based colorimetric toxin neutralization activity assays for determination of neutralizing anti-ricin antibodies.

PubMed

Lindsey, Changhong Y; Brown, J Edward; Torabazar, Nahid R; Smith, Leonard A

2013-01-01

A recombinant ricin toxin A-chain 1-33/44-198 vaccine (RVEc), developed at the United States Army Medical Research Institute of Infectious Diseases as a vaccine candidate, is under investigation in a phase 1 clinical study. To effectively evaluate the immunogenicity of this ricin vaccine and to eliminate the use of radioactive material, an EL4 cell-based colorimetric toxin neutralization activity (TNA) assay using a CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent has been developed, optimized, and applied in the vaccine efficacy studies. The TNA assay measures the protective neutralizing anti-ricin antibodies in animal sera by determining the cell viability after ricin exposure in the assay system and comparing it to a purified mouse polyclonal antiricin IgG standard curve. The standard curve of the anti-ricin TNA assay closely fits a four-parameter logistic regression model. The unknown test sample concentration was expressed as microg/mL, but not the 50% effective concentration (EC50), which was determined by most TNA assays. The neutralizing endpoint titers, not the 50% effective dilution (ED50), of human specimens were measured with the TNA assay in support of the clinical study of the RVEc vaccine. The optimal amount of ricin toxin, EL4 cells, and concentration of standards used in the assay system was established to minimize false-negative and false-positive results of serum specimens from the nonclinical and clinical studies of RVEc. The testing conditions were adjusted to optimize assay performance. The colorimetric TNA assay replaced a radioactive TNA assay previously used in the ricin vaccine studies.

Luciferase reporter assay in Drosophila and mammalian tissue culture cells

PubMed Central

Yun, Chi

2014-01-01

Luciferase reporter gene assays are one of the most common methods for monitoring gene activity. Because of their sensitivity, dynamic range, and lack of endogenous activity, luciferase assays have been particularly useful for functional genomics in cell-based assays, such as RNAi screening. This unit describes delivery of two luciferase reporters with other nucleic acids (siRNA /dsRNA), measurement of the dual luciferase activities, and analysis of data generated. The systematic query of gene function (RNAi) combined with the advances in luminescent technology have made it possible to design powerful whole genome screens to address diverse and significant biological questions. PMID:24652620

Automated reagent-dispensing system for microfluidic cell biology assays.

PubMed

Ly, Jimmy; Masterman-Smith, Michael; Ramakrishnan, Ravichandran; Sun, Jing; Kokubun, Brent; van Dam, R Michael

2013-12-01

Microscale systems that enable measurements of oncological phenomena at the single-cell level have a great capacity to improve therapeutic strategies and diagnostics. Such measurements can reveal unprecedented insights into cellular heterogeneity and its implications into the progression and treatment of complicated cellular disease processes such as those found in cancer. We describe a novel fluid-delivery platform to interface with low-cost microfluidic chips containing arrays of microchambers. Using multiple pairs of needles to aspirate and dispense reagents, the platform enables automated coating of chambers, loading of cells, and treatment with growth media or other agents (e.g., drugs, fixatives, membrane permeabilizers, washes, stains, etc.). The chips can be quantitatively assayed using standard fluorescence-based immunocytochemistry, microscopy, and image analysis tools, to determine, for example, drug response based on differences in protein expression and/or activation of cellular targets on an individual-cell level. In general, automation of fluid and cell handling increases repeatability, eliminates human error, and enables increased throughput, especially for sophisticated, multistep assays such as multiparameter quantitative immunocytochemistry. We report the design of the automated platform and compare several aspects of its performance to manually-loaded microfluidic chips.

Microfluidic assay of circulating endothelial cells in coronary artery disease patients with angina pectoris

PubMed Central

Chen, Shuiyu; Sun, Yukun; Neoh, Kuang Hong; Chen, Anqi; Li, Weiju; Yang, Xiaorui

2017-01-01

Background Circulating endothelial cells (CECs) are widely reported as a promising biomarker of endothelial damage/dysfunction in coronary artery disease (CAD). The two popular methods of CEC quantification include the use of immunomagnetic beads separation (IB) and flow cytometry analysis (FC); however, they suffer from two main shortcomings that affect their diagnostic and prognostic responses: non-specific bindings of magnetic beads to non-target cells and a high degree of variability in rare cell identification, respectively. We designed a microfluidic chip with spatially staggered micropillars for the efficient harvesting of CECs with intact cellular morphology in an attempt to revisit the diagnostic goal of CEC counts in CAD patients with angina pectoris. Methods A label-free microfluidic assay that involved an in-situ enumeration and immunofluorescent identification (DAPI+/CD146+/VEGFR1+/CD45-) of CECs was carried out to assess the CEC count in human peripheral blood samples. A total of 55 CAD patients with angina pectoris [16 with chronic stable angina (CSA) and 39 with unstable angina (UA)], together with 15 heathy controls (HCs) were enrolled in the study. Results CEC counts are significantly higher in both CSA and UA groups compared to the HC group [respective medians of 6.9, 10.0 and 1.5 cells/ml (p < 0.01)]. Further, a significant elevation of CEC count was observed in the three UA subgroups [low risk (5.3) vs. intermediate risk (10.8) vs. high risk (18.0) cells/ml, p < 0.001) classified in accordance to the TIMI NSTEMI/UA risk score system. From the receiver-operating characteristic curve analysis, the AUCs for distinguishing CSA and UA from HC were 0.867 and 0.938, respectively. The corresponding sensitivities were 87.5% and 84.6% and the specificities were 66.7% and 86.7%, respectively. Conclusions Our microfluidic assay system is efficient and stable for CEC capture and enumeration. The results showed that the CEC count has the potential to be a

Quality Assurance in the Polio Laboratory. Cell Sensitivity and Cell Authentication Assays.

PubMed

Dunn, Glynis

2016-01-01

The accuracy of poliovirus surveillance is largely dependent on the quality of the cell lines used for virus isolation, which is the foundation of poliovirus diagnostic work. Many cell lines are available for the isolation of enteroviruses, whilst genetically modified L20B cells can be used as a diagnostic tool for the identification of polioviruses. To be confident that cells can consistently isolate the virus of interest, it is necessary to have a quality assurance system in place, which will ensure that the cells in use are not contaminated with other cell lines or microorganisms and that they remain sensitive to the viruses being studied.The sensitivity of cell lines can be assessed by the regular testing of a virus standard of known titer in the cell lines used for virus isolation. The titers obtained are compared to previously obtained titers in the same assay, so that any loss of sensitivity can be detected.However, the detection of cell line cross contamination is more difficult. DNA bar coding is a technique that uses a short DNA sequence from a standardized position in the genome as a molecular diagnostic assay for species-level identification. For almost all groups of higher animals, the cytochrome c oxidase subunit 1 of mitochondrial DNA (CO1) is emerging as the standard barcode region. This region is 648 nucleotide base pairs long in most phylogenetic groups and is flanked by regions of conserved sequences, making it relatively easy to isolate and analyze. DNA barcodes vary among individuals of the same species to a very minor degree (generally less than 1-2 %), and a growing number of studies have shown that the COI sequences of even closely related species differ by several per cent, making it possible to identify different species with high confidence.

Cell-based cytotoxicity assays for engineered nanomaterials safety screening: exposure of adipose derived stromal cells to titanium dioxide nanoparticles.

PubMed

Xu, Yan; Hadjiargyrou, M; Rafailovich, Miriam; Mironava, Tatsiana

2017-07-11

Increasing production of nanomaterials requires fast and proper assessment of its potential toxicity. Therefore, there is a need to develop new assays that can be performed in vitro, be cost effective, and allow faster screening of engineered nanomaterials (ENMs). Herein, we report that titanium dioxide (TiO 2 ) nanoparticles (NPs) can induce damage to adipose derived stromal cells (ADSCs) at concentrations which are rated as safe by standard assays such as measuring proliferation, reactive oxygen species (ROS), and lactate dehydrogenase (LDH) levels. Specifically, we demonstrated that low concentrations of TiO 2 NPs, at which cellular LDH, ROS, or proliferation profiles were not affected, induced changes in the ADSCs secretory function and differentiation capability. These two functions are essential for ADSCs in wound healing, energy expenditure, and metabolism with serious health implications in vivo. We demonstrated that cytotoxicity assays based on specialized cell functions exhibit greater sensitivity and reveal damage induced by ENMs that was not otherwise detected by traditional ROS, LDH, and proliferation assays. For proper toxicological assessment of ENMs standard ROS, LDH, and proliferation assays should be combined with assays that investigate cellular functions relevant to the specific cell type.

Quantifying the effect of experimental design choices for in vitro scratch assays.

PubMed

Johnston, Stuart T; Ross, Joshua V; Binder, Benjamin J; Sean McElwain, D L; Haridas, Parvathi; Simpson, Matthew J

2016-07-07

Scratch assays are often used to investigate potential drug treatments for chronic wounds and cancer. Interpreting these experiments with a mathematical model allows us to estimate the cell diffusivity, D, and the cell proliferation rate, λ. However, the influence of the experimental design on the estimates of D and λ is unclear. Here we apply an approximate Bayesian computation (ABC) parameter inference method, which produces a posterior distribution of D and λ, to new sets of synthetic data, generated from an idealised mathematical model, and experimental data for a non-adhesive mesenchymal population of fibroblast cells. The posterior distribution allows us to quantify the amount of information obtained about D and λ. We investigate two types of scratch assay, as well as varying the number and timing of the experimental observations captured. Our results show that a scrape assay, involving one cell front, provides more precise estimates of D and λ, and is more computationally efficient to interpret than a wound assay, with two opposingly directed cell fronts. We find that recording two observations, after making the initial observation, is sufficient to estimate D and λ, and that the final observation time should correspond to the time taken for the cell front to move across the field of view. These results provide guidance for estimating D and λ, while simultaneously minimising the time and cost associated with performing and interpreting the experiment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Immuno capture PCR for rapid and sensitive identification of pathogenic Bacillus anthracis.

PubMed

Makam, Shivakiran S; Majumder, Saugata; Kingston, Joseph J; Urs, Radhika M; Tuteja, Urmil; Sripathi, Murali H; Batra, Harsh V

2013-12-01

Immuno capture PCR (IPCR) is a technique capable of detecting the pathogens with high specificity and sensitivity. Rapid and accurate detection of Bacillus anthracis was achieved using anti-EA1 antibodies to capture the cells and two primer sets targeting the virulence factors of the pathogen i.e., protective antigen (pag) and capsule (cap) in an IPCR format. Monoclonal antibodies specific to B. anthracis were generated against extractable antigen 1 protein and used as capture antibody onto 96 well polystyrene plates. Following the binding of the pathogen, the DNA extraction was carried out in the well itself and further processed for PCR assay. We compared IPCR described here with conventional duplex PCR using the same primers and sandwich ELISA using the monoclonal antibodies developed in the present study. IPCR was capable of detecting as few as 10 and 100 cfu ml⁻¹ of bacterial cells and spores, respectively. IPCR was found to be 2-3 logs more sensitive than conventional duplex PCR and the sandwich ELISA. The effect of other bacteria and any organic materials on IPCR was also analyzed and found that this method was robust with little change in the sensitivity in the presence of interfering agents. Moreover, we could demonstrate a simple process of microwave treatment for spore disruption which otherwise are resistant to chemical treatments. Also, the IPCR could clearly distinguish the pathogenic and nonpathogenic strains of B. anthracis in the same assay. This can help in saving resources on unnecessary decontamination procedures during false alarms.

Dual-responsive surfaces modified with phenylboronic acid-containing polymer brush to reversibly capture and release cancer cells.

PubMed

Liu, Hongliang; Li, Yingying; Sun, Kang; Fan, Junbing; Zhang, Pengchao; Meng, Jingxin; Wang, Shutao; Jiang, Lei

2013-05-22

Artificial stimuli-responsive surfaces that can mimic the dynamic function of living systems have attracted much attention. However, there exist few artificial systems capable of responding to dual- or multistimulation as the natural system does. Herein, we synthesize a pH and glucose dual-responsive surface by grafting poly(acrylamidophenylboronic acid) (polyAAPBA) brush from aligned silicon nanowire (SiNW) array. The as-prepared surface can reversibly capture and release targeted cancer cells by precisely controlling pH and glucose concentration, exhibiting dual-responsive AND logic. In the presence of 70 mM glucose, the surface is pH responsive, which can vary from a cell-adhesive state to a cell-repulsive state by changing the pH from 6.8 to 7.8. While keeping the pH at 7.8, the surface becomes glucose responsive--capturing cells in the absence of glucose and releasing cells by adding 70 mM glucose. Through simultaneously changing the pH and glucose concentration from pH 6.8/0 mM glucose to pH 7.8/70 mM glucose, the surface is dual responsive with the capability to switch between cell capture and release for at least 5 cycles. The cell capture and release process on this dual-responsive surface is noninvasive with cell viability higher than 95%. Moreover, topographical interaction between the aligned SiNW array and cell protrusions greatly amplifies the responsiveness and accelerates the response rate of the dual-responsive surface between cell capture and release. The responsive mechanism of the dual-responsive surface is systematically studied using a quartz crystal microbalance, which shows that the competitive binding between polyAAPBA/sialic acid and polyAAPBA/glucose contributes to the dual response. Such dual-responsive surface can significantly impact biomedical and biological applications including cell-based diagnostics, in vivo drug delivery, etc.

Development of an in vitro PIG-A gene mutation assay in human cells

PubMed Central

Rees, Benjamin J; Tate, Matthew; Lynch, Anthony M; Thornton, Catherine A; Jenkins, Gareth J; Walmsley, Richard M; Johnson, George E

2017-01-01

Abstract Mutagens can be carcinogens, and traditionally, they have been identified in vitro using the Salmonella ‘Ames’ reverse mutation assay. However, prokaryotic DNA packaging, replication and repair systems are mechanistically very different to those in the humans we inevitably seek to protect. Therefore, for many years, mammalian cell line genotoxicity assays that can detect eukaryotic mutagens as well as clastogens and aneugens have been used. The apparent lack of specificity in these largely rodent systems, due partly to their mutant p53 status, has contributed to the use of animal studies to resolve data conflicts. Recently, silencing mutations at the PIG-A locus have been demonstrated to prevent glycophosphatidylinositol (GPI) anchor synthesis and consequentially result in loss of GPI-anchored proteins from the cell’s extracellular surface. The successful exploitation of this mutant phenotype in animal studies has triggered interest in the development of an analogous in vitro PIG-A mutation screening assay. This article describes the development of a robust assay design using metabolically active human cells. The assay includes viability and cell membrane integrity assessment and conforms to the future ideas of the 21st-century toxicology testing. PMID:28057708

RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA), an Assay for Detecting RNA-Protein Complexes in Intact Cells.

PubMed

Roussis, Ioannis M; Guille, Matthew; Myers, Fiona A; Scarlett, Garry P

2016-01-01

Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein complexes as a consequence of the complexities associated with working with RNA. Here we present a method for the modification of the existing In Situ Hybridisation-Proximity Ligation Assay (ISH-PLA) protocol to adapt it to the study of RNA regulation (rISH-PLA). As proof of principle we used the well-characterised interaction of the Xenopus laevis Staufen RNA binding protein with Vg1 mRNA, the complex of which co-localises to the vegetal pole of Xenopus oocytes. The applicability of both the Stau1 antibody and the Locked Nucleic Acid probe (LNA) recognising Vg1 mRNA were independently validated by whole-mount Immunohistochemistry and whole-mount in situ hybridisation assays respectively prior to combining them in the rISH-PLA assay. The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogenous cell populations from which conventional RNA-protein interaction detection techniques suffer. This technique will be particularly usefully for studying the activity of RNA binding proteins (RBPs) in complex mixtures of cells, for example tissue sections or whole embryos.

High throughput RNAi assay optimization using adherent cell cytometry.

PubMed

Nabzdyk, Christoph S; Chun, Maggie; Pradhan, Leena; Logerfo, Frank W

2011-04-25

siRNA technology is a promising tool for gene therapy of vascular disease. Due to the multitude of reagents and cell types, RNAi experiment optimization can be time-consuming. In this study adherent cell cytometry was used to rapidly optimize siRNA transfection in human aortic vascular smooth muscle cells (AoSMC). AoSMC were seeded at a density of 3000-8000 cells/well of a 96 well plate. 24 hours later AoSMC were transfected with either non-targeting unlabeled siRNA (50 nM), or non-targeting labeled siRNA, siGLO Red (5 or 50 nM) using no transfection reagent, HiPerfect or Lipofectamine RNAiMax. For counting cells, Hoechst nuclei stain or Cell Tracker green were used. For data analysis an adherent cell cytometer, Celigo® was used. Data was normalized to the transfection reagent alone group and expressed as red pixel count/cell. After 24 hours, none of the transfection conditions led to cell loss. Red fluorescence counts were normalized to the AoSMC count. RNAiMax was more potent compared to HiPerfect or no transfection reagent at 5 nM siGLO Red (4.12 +/-1.04 vs. 0.70 +/-0.26 vs. 0.15 +/-0.13 red pixel/cell) and 50 nM siGLO Red (6.49 +/-1.81 vs. 2.52 +/-0.67 vs. 0.34 +/-0.19). Fluorescence expression results supported gene knockdown achieved by using MARCKS targeting siRNA in AoSMCs. This study underscores that RNAi delivery depends heavily on the choice of delivery method. Adherent cell cytometry can be used as a high throughput-screening tool for the optimization of RNAi assays. This technology can accelerate in vitro cell assays and thus save costs.

High throughput RNAi assay optimization using adherent cell cytometry

PubMed Central

2011-01-01

Background siRNA technology is a promising tool for gene therapy of vascular disease. Due to the multitude of reagents and cell types, RNAi experiment optimization can be time-consuming. In this study adherent cell cytometry was used to rapidly optimize siRNA transfection in human aortic vascular smooth muscle cells (AoSMC). Methods AoSMC were seeded at a density of 3000-8000 cells/well of a 96well plate. 24 hours later AoSMC were transfected with either non-targeting unlabeled siRNA (50 nM), or non-targeting labeled siRNA, siGLO Red (5 or 50 nM) using no transfection reagent, HiPerfect or Lipofectamine RNAiMax. For counting cells, Hoechst nuclei stain or Cell Tracker green were used. For data analysis an adherent cell cytometer, Celigo® was used. Data was normalized to the transfection reagent alone group and expressed as red pixel count/cell. Results After 24 hours, none of the transfection conditions led to cell loss. Red fluorescence counts were normalized to the AoSMC count. RNAiMax was more potent compared to HiPerfect or no transfection reagent at 5 nM siGLO Red (4.12 +/-1.04 vs. 0.70 +/-0.26 vs. 0.15 +/-0.13 red pixel/cell) and 50 nM siGLO Red (6.49 +/-1.81 vs. 2.52 +/-0.67 vs. 0.34 +/-0.19). Fluorescence expression results supported gene knockdown achieved by using MARCKS targeting siRNA in AoSMCs. Conclusion This study underscores that RNAi delivery depends heavily on the choice of delivery method. Adherent cell cytometry can be used as a high throughput-screening tool for the optimization of RNAi assays. This technology can accelerate in vitro cell assays and thus save costs. PMID:21518450

GFP-based fluorescence assay for CAG repeat instability in cultured human cells.

PubMed

Santillan, Beatriz A; Moye, Christopher; Mittelman, David; Wilson, John H

2014-01-01

Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries.

Measurement of DNA damage in rat urinary bladder transitional cells: improved selective harvest of transitional cells and detailed Comet assay protocols.

PubMed

Wang, Amy; Robertson, John L; Holladay, Steven D; Tennant, Alan H; Lengi, Andrea J; Ahmed, S Ansar; Huckle, William R; Kligerman, Andrew D

2007-12-01

Urinary bladder transitional epithelium is the main site of bladder cancer, and the use of transitional cells to study carcinogenesis/genotoxicity is recommended over the use of whole bladders. Because the transitional epithelium is only a small fraction of the whole bladder, the alkaline single cell gel electrophoresis assay (Comet assay), which requires only a small number of cells per sample, is especially suitable for measuring DNA damage in transitional cells. However, existed procedures of cell collection did not yield transitional cells with a high purity, and pooling of samples was needed for Comet assay. The goal of this study was to develop an optimized protocol to evaluate DNA damage in the urinary bladder transitional epithelium. This was achieved by an enzymatic stripping method (trypsin-EDTA incubation plus gentle scraping) to selectively harvest transitional cells from rat bladders, and the use of the alkaline Comet assay to detect DNA strand breaks, alkaline labile sites, and DNA-protein crosslinks. Step by step procedures are reported here. Cells collected from a single rat bladder were sufficient for multiple Comet assays. With this new protocol, increases in DNA damage were detected in transitional cells after in vitro exposure to the positive control agents, hydrogen peroxide or formaldehyde. Repair of the induced DNA damage occurred within 4h. This indicated the capacity for DNA repair was maintained in the harvested cells. The new protocol provides a simple and inexpensive method to detect various types of DNA damage and to measure DNA damage repair in urinary bladder transitional cells.

A two-magnet strategy for improved mixing and capture from biofluids

PubMed Central

Doyle, Andrew B.; Haselton, Frederick R.

2016-01-01

Magnetic beads are a popular method for concentrating biomolecules from solution and have been more recently used in multistep pre-arrayed microfluidic cartridges. Typical processing strategies rely on a single magnet, resulting in a tight cluster of beads and requiring long incubation times to achieve high capture efficiencies, especially in highly viscous patient samples. This report describes a two-magnet strategy to improve the interaction of the bead surface with the surrounding fluid inside of a pre-arrayed, self-contained assay-in-a-tube. In the two-magnet system, target biomarker capture occurs at a rate three times faster than the single-magnet system. In clinically relevant biomatrices, we find a 2.5-fold improvement in biomarker capture at lower sample viscosities with the two-magnet system. In addition, we observe a 20% increase in the amount of protein captured at high viscosity for the two-magnet configuration relative to the single magnet approach. The two-magnet approach offers a means to achieve higher biomolecule extraction yields and shorter assay times in magnetic capture assays and in self-contained processor designs. PMID:27158286

A Comparison of Real-Time and Endpoint Cell Viability Assays for Improved Synthetic Lethal Drug Validation.

PubMed

Single, Andrew; Beetham, Henry; Telford, Bryony J; Guilford, Parry; Chen, Augustine

2015-12-01

Cell viability assays fulfill a central role in drug discovery studies. It is therefore important to understand the advantages and disadvantages of the wide variety of available assay methodologies. In this study, we compared the performance of three endpoint assays (resazurin reduction, CellTiter-Glo, and nuclei enumeration) and two real-time systems (IncuCyte and xCELLigence). Of the endpoint approaches, both the resazurin reduction and CellTiter-Glo assays showed higher cell viabilities when compared directly to stained nuclei counts. The IncuCyte and xCELLigence real-time systems were comparable, and both were particularly effective at tracking the effects of drug treatment on cell proliferation at sub-confluent growth. However, the real-time systems failed to evaluate contrasting cell densities between drug-treated and control-treated cells at full growth confluency. Here, we showed that using real-time systems in combination with endpoint assays alleviates the disadvantages posed by each approach alone, providing a more effective means to evaluate drug toxicity in monolayer cell cultures. Such approaches were shown to be effective in elucidating the toxicity of synthetic lethal drugs in an isogenic pair of MCF10A breast cell lines. © 2015 Society for Laboratory Automation and Screening.

Micropatterned comet assay enables high throughput and sensitive DNA damage quantification

PubMed Central

Ge, Jing; Chow, Danielle N.; Fessler, Jessica L.; Weingeist, David M.; Wood, David K.; Engelward, Bevin P.

2015-01-01

The single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring many classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. However, limited throughput and difficulties with reproducibility have limited its utility, particularly for clinical and epidemiological studies. To address these limitations, we created a microarray comet assay. The use of a micrometer scale array of cells increases the number of analysable comets per square centimetre and enables automated imaging and analysis. In addition, the platform is compatible with standard 24- and 96-well plate formats. Here, we have assessed the consistency and sensitivity of the microarray comet assay. We showed that the linear detection range for H2O2-induced DNA damage in human lymphoblastoid cells is between 30 and 100 μM, and that within this range, inter-sample coefficient of variance was between 5 and 10%. Importantly, only 20 comets were required to detect a statistically significant induction of DNA damage for doses within the linear range. We also evaluated sample-to-sample and experiment-to-experiment variation and found that for both conditions, the coefficient of variation was lower than what has been reported for the traditional comet assay. Finally, we also show that the assay can be performed using a 4× objective (rather than the standard 10× objective for the traditional assay). This adjustment combined with the microarray format makes it possible to capture more than 50 analysable comets in a single image, which can then be automatically analysed using in-house software. Overall, throughput is increased more than 100-fold compared to the traditional assay. Together, the results presented here demonstrate key advances in comet assay technology that improve the throughput, sensitivity, and robustness, thus enabling larger scale clinical and epidemiological studies. PMID:25527723

Pertussis serology: assessment of IgG anti-PT ELISA for replacement of the CHO cell assay*

PubMed Central

DALBY, TINE; SØRENSEN, CHARLOTTE; PETERSEN, JESPER WESTPHAL; KROGFELT, KAREN ANGELIKI

2010-01-01

Dalby T, Sørensen C, Petersen JW, Krogfelt KA. Pertussis serology: assessment of IgG anti-PT ELISA for replacement of the CHO cell assay. APMIS 2010; 118: 968–72. Two types of serological assays are commonly used for the assessment of pertussis vaccine-induced antibodies; the Chinese hamster ovary cell (CHO cell) assay and the immunoglobulin G (IgG) anti pertussis toxin (PT) enzyme-linked immunosorbent assay (IgG anti-PT ELISA). Recently, both the techniques have been modified to improve performance with sera with interfering activity (CHO cell assay) or with heat-treated sera (IgG anti-PT ELISA). These two improved techniques were compared by the analysis of 100 individual serum samples from a previous clinical trial and 213 sera from a longitudinal serum collection from 20 Danish adults recently vaccinated with the Danish acellular pertussis vaccine. The comparison showed a significant linear correlation between the results of the two assays with a p-value of <0.0001 for the 100 individual samples. We, therefore, conclude that the improved IgG anti-PT ELISA can be used as a replacement for the often troublesome and time-consuming CHO cell assay for the measurement of vaccine-induced human antibodies to PT. PMID:21091778

A cluster pattern algorithm for the analysis of multiparametric cell assays.

PubMed

Kaufman, Menachem; Bloch, David; Zurgil, Naomi; Shafran, Yana; Deutsch, Mordechai

2005-09-01

The issue of multiparametric analysis of complex single cell assays of both static and flow cytometry (SC and FC, respectively) has become common in recent years. In such assays, the analysis of changes, applying common statistical parameters and tests, often fails to detect significant differences between the investigated samples. The cluster pattern similarity (CPS) measure between two sets of gated clusters is based on computing the difference between their density distribution functions' set points. The CPS was applied for the discrimination between two observations in a four-dimensional parameter space. The similarity coefficient (r) ranges between 0 (perfect similarity) to 1 (dissimilar). Three CPS validation tests were carried out: on the same stock samples of fluorescent beads, yielding very low r's (0, 0.066); and on two cell models: mitogenic stimulation of peripheral blood mononuclear cells (PBMC), and apoptosis induction in Jurkat T cell line by H2O2. In both latter cases, r indicated similarity (r < 0.23) within the same group, and dissimilarity (r > 0.48) otherwise. This classification and algorithm approach offers a measure of similarity between samples. It relies on the multidimensional pattern of the sample parameters. The algorithm compensates for environmental drifts in this apparatus and assay; it also may be applied to more than four dimensions.

Capture-related stressors impair immune system function in sablefish

USGS Publications Warehouse

Lupes, S.C.; Davis, M.W.; Olla, B.L.; Schreck, C.B.

2006-01-01

The sablefish Anoplopoma fimbria is a valuable North Pacific Ocean species that, when not targeted in various commercial fisheries, is often a part of discarded bycatch. Predictions of the survival of discarded fish are dependent on understanding how a fish responds to stressful conditions. Our objective was to describe the immunological health of sablefish exposed to capture stressors. In laboratory experiments designed to simulate the capture process, we subjected sablefish to various stressors that might influence survival: towing in a net, hooking, elevated seawater and air temperatures, and air exposure time. After stress was imposed, the in vitro mitogen-stimulated proliferation of sablefish leukocytes was used to evaluate the function of the immune system in an assay we validated for this species. The results demonstrated that regardless of fishing gear type, exposure to elevated seawater temperature, or time in air, the leukocytes from stressed sablefish exhibited significantly diminished proliferative responses to the T-cell mitogen, concanavalin A, or the B-cell mitogen, lipopolysaccharide. There was no difference in the immunological responses associated with seawater or air temperature. The duration and severity of the capture stressors applied in our study were harsh enough to induce significantly elevated levels of plasma cortisol and glucose, but there was no difference in the magnitude of levels among stressor treatments. These data suggest that immunological suppression occurs in sablefish subjected to capture-related stressors. The functional impairment of the immune system after capture presents a potential reason why delayed mortality is possible in discarded sablefish. Further studies are needed to determine whether delayed mortality in discarded sablefish can be caused by increased susceptibility to infectious agents resulting from stressor-mediated immunosuppression.

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells.

PubMed

Guillen-Ahlers, Hector; Rao, Prahlad K; Perumalla, Danu S; Montoya, Maria J; Jadhav, Avinash Y L; Shortreed, Michael R; Smith, Lloyd M; Olivier, Michael

2018-06-01

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

Assay Design Affects the Interpretation of T-Cell Receptor Gamma Gene Rearrangements

PubMed Central

Cushman-Vokoun, Allison M.; Connealy, Solomon; Greiner, Timothy C.

2010-01-01

Interpretation of capillary electrophoresis results derived from multiplexed fluorochrome-labeled primer sets can be complicated by small peaks, which may be incorrectly interpreted as clonal T-cell receptor-γ gene rearrangements. In this report, different assay designs were used to illustrate how design may adversely affect specificity. Ten clinical cases, with subclonal peaks containing one of the two infrequently used joining genes, were identified with a tri-color, one-tube assay. The DNA was amplified with the same NED fluorochrome on all three joining primers, first combined (one-color assay) and then amplified separately using a single NED-labeled joining primer. The single primer assay design shows how insignificant peaks could easily be wrongly interpreted as clonal T-cell receptor-γ gene rearrangements. Next, the performance of the one-tube assay was compared with the two-tube BIOMED-2-based TCRG Gene Clonality Assay in a series of 44 cases. Whereas sensitivity was similar between the two methods (92.9% vs. 96.4%; P = 0.55), specificity was significantly less in the BIOMED-2 assay (87.5% vs. 56.3%; P = 0.049) when a 2× ratio was used to define clonality. Specificity was improved to 81.3% by the use of a 5× peak height ratio (P = 0.626). These findings illustrate how extra caution is needed in interpreting a design with multiple, separate distributions, which is more difficult to interpret than a single distribution assay. PMID:20959612

ELISPOT Assays in 384-Well Format: Up to 30 Data Points with One Million Cells

PubMed Central

Hanson, Jodi; Sundararaman, Srividya; Caspell, Richard; Karacsony, Edith; Karulin, Alexey Y.; Lehmann, Paul V.

2015-01-01

Comprehensive immune monitoring requires that frequencies of T cells, producing different cytokines, are measured to establish the magnitude of Th1, Th2, and Th17 components of cell-mediated immunity. Antigen titration provides additional information about the affinity of T cell response. In tumor immunity, it is also advisable to account for determinant spreading by testing multiple epitopes. Efforts for comprehensive immune monitoring would require substantial numbers of PBMC to run the above tests systematically, which in most test cases is limiting. Immune monitoring with ELISPOT assays have been performed, thus far, in a 96-well format. In this study we show that one can increase cell utilization by performing the assay in 384-well plates whose membrane surface area is one third that of 96-well plates. Systematic testing of PBMC for antigen-specific T cell response in the two formats demonstrated that the 384-well assay corresponds to a one-in-three miniaturization of the 96-well assay. The lowest number of cells that can be used in the 384-well format, while allowing for sufficient contact with APC, is 33,000 PBMC/well. Therefore, with one million PBMC typically obtained from 1 mL of blood, a 30 well T cell ELISPOT assay can be performed in a 384-well format. PMID:25643292

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells.

PubMed

Fang, Mingzhu; Kang, Hwan-Goo; Park, Youngil; Estrella, Brian; Zarbl, Helmut

2017-09-28

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both

The loss-of-allele assay for ES cell screening and mouse genotyping.

PubMed

Frendewey, David; Chernomorsky, Rostislav; Esau, Lakeisha; Om, Jinsop; Xue, Yingzi; Murphy, Andrew J; Yancopoulos, George D; Valenzuela, David M

2010-01-01

Targeting vectors used to create directed mutations in mouse embryonic stem (ES) cells consist, in their simplest form, of a gene for drug selection flanked by mouse genomic sequences, the so-called homology arms that promote site-directed homologous recombination between the vector and the target gene. The VelociGene method for the creation of targeted mutations in ES cells employs targeting vectors, called BACVecs, that are based on bacterial artificial chromosomes. Compared with conventional short targeting vectors, BacVecs provide two major advantages: (1) their much larger homology arms promote high targeting efficiencies without the need for isogenicity or negative selection strategies; and (2) they enable deletions and insertions of up to 100kb in a single targeting event, making possible gene-ablating definitive null alleles and other large-scale genomic modifications. Because of their large arm sizes, however, BACVecs do not permit screening by conventional assays, such as long-range PCR or Southern blotting, that link the inserted targeting vector to the targeted locus. To exploit the advantages of BACVecs for gene targeting, we inverted the conventional screening logic in developing the loss-of-allele (LOA) assay, which quantifies the number of copies of the native locus to which the mutation was directed. In a correctly targeted ES cell clone, the LOA assay detects one of the two native alleles (for genes not on the X or Y chromosome), the other allele being disrupted by the targeted modification. We apply the same principle in reverse as a gain-of-allele assay to quantify the copy number of the inserted targeting vector. The LOA assay reveals a correctly targeted clone as having lost one copy of the native target gene and gained one copy of the drug resistance gene or other inserted marker. The combination of these quantitative assays makes LOA genotyping unequivocal and amenable to automated scoring. We use the quantitative polymerase chain reaction

Quantitative analyses reveal distinct sensitivities of the capture of HIV-1 primary viruses and pseudoviruses to broadly neutralizing antibodies.

PubMed

Kim, Jiae; Jobe, Ousman; Peachman, Kristina K; Michael, Nelson L; Robb, Merlin L; Rao, Mangala; Rao, Venigalla B

2017-08-01

Development of vaccines capable of eliciting broadly neutralizing antibodies (bNAbs) is a key goal to controlling the global AIDS epidemic. To be effective, bNAbs must block the capture of HIV-1 to prevent viral acquisition and establishment of reservoirs. However, the role of bNAbs, particularly during initial exposure of primary viruses to host cells, has not been fully examined. Using a sensitive, quantitative, and high-throughput qRT-PCR assay, we found that primary viruses were captured by host cells and converted into a trypsin-resistant form in less than five minutes. We discovered, unexpectedly, that bNAbs did not block primary virus capture, although they inhibited the capture of pseudoviruses/IMCs and production of progeny viruses at 48h. Further, viruses escaped bNAb inhibition unless the bNAbs were present in the initial minutes of exposure of virus to host cells. These findings will have important implications for HIV-1 vaccine design and determination of vaccine efficacy. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

ViroSpot microneutralization assay for antigenic characterization of human influenza viruses.

PubMed

van Baalen, Carel A; Jeeninga, Rienk E; Penders, Germaine H W M; van Gent, Brenda; van Beek, Ruud; Koopmans, Marion P G; Rimmelzwaan, Guus F

2017-01-03

The hemagglutination inhibition (HI) assay has been used for the antigenic characterization of influenza viruses for decades. However, the majority of recent seasonal influenza A viruses of the H3N2 subtype has lost the capacity to agglutinate erythrocytes of various species. The hemagglutination (HA) activity of other A(H3N2) strains is generally sensitive to the action of the neuraminidase inhibitor oseltamivir, which indicates that the neuraminidase and not the hemagglutinin is responsible for the HA activity. These findings complicate the antigenic characterization and selection of A(H3N2) vaccine strains, calling for alternative antigenic characterization assays. Here we describe the development and use of the ViroSpot microneutralization (MN) assay as a reliable and robust alternative for the HI assay. Serum neutralization of influenza A(H3N2) reference virus strains and epidemic isolates was determined by automated readout of immunostained cell monolayers, in a format designed to minimize the influence of infectious virus doses on serum neutralization titers. Neutralization of infection was largely independent from rates of viral replication and cell-to-cell transmission, facilitating the comparison of different virus isolates. Other advantages of the ViroSpot MN assay include its relative insensitivity to variation in test dose of infectious virus, automated capture and analyses of residual infection patterns, and compatibility with standardized large scale analyses. Using this assay, a number of epidemic influenza A(H3N2) strains that failed to agglutinate erythrocytes, were readily characterized antigenically. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

GFP-complementation assay to detect functional CPP and protein delivery into living cells

PubMed Central

Milech, Nadia; Longville, Brooke AC; Cunningham, Paula T; Scobie, Marie N; Bogdawa, Heique M; Winslow, Scott; Anastasas, Mark; Connor, Theresa; Ong, Ferrer; Stone, Shane R; Kerfoot, Maria; Heinrich, Tatjana; Kroeger, Karen M; Tan, Yew-Foon; Hoffmann, Katrin; Thomas, Wayne R; Watt, Paul M; Hopkins, Richard M

2015-01-01

Efficient cargo uptake is essential for cell-penetrating peptide (CPP) therapeutics, which deliver widely diverse cargoes by exploiting natural cell processes to penetrate the cell’s membranes. Yet most current CPP activity assays are hampered by limitations in assessing uptake, including confounding effects of conjugated fluorophores or ligands, indirect read-outs requiring secondary processing, and difficulty in discriminating internalization from endosomally trapped cargo. Split-complementation Endosomal Escape (SEE) provides the first direct assay visualizing true cytoplasmic-delivery of proteins at biologically relevant concentrations. The SEE assay has minimal background, is amenable to high-throughput processes, and adaptable to different transient and stable cell lines. This split-GFP-based platform can be useful to study transduction mechanisms, cellular imaging, and characterizing novel CPPs as pharmaceutical delivery agents in the treatment of disease. PMID:26671759

Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization.

PubMed

Fisher, Gregory W; Fuhrman, Margaret H; Adler, Sally A; Szent-Gyorgyi, Christopher; Waggoner, Alan S; Jarvik, Jonathan W

2014-09-01

G protein-coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describe new live-cell assays that use recombinant GPCRs to quantify two general features of GPCR cell biology-receptor desensitization and resensitization. The assays employ a fluorogen-activating protein (FAP) reporter that reversibly complexes with either of two soluble organic molecules (fluorogens) whose fluorescence is strongly enhanced when complexed with the FAP. Both assays require no wash or cleanup steps and are readily performed in microwell plates, making them adaptable to high-throughput drug discovery applications. © 2014 Society for Laboratory Automation and Screening.

The Effect of Primary Cancer Cell Culture Models on the Results of Drug Chemosensitivity Assays: The Application of Perfusion Microbioreactor System as Cell Culture Vessel

PubMed Central

Chen, Yi-Dao; Huang, Shiang-Fu; Wang, Hung-Ming

2015-01-01

To precisely and faithfully perform cell-based drug chemosensitivity assays, a well-defined and biologically relevant culture condition is required. For the former, a perfusion microbioreactor system capable of providing a stable culture condition was adopted. For the latter, however, little is known about the impact of culture models on the physiology and chemosensitivity assay results of primary oral cavity cancer cells. To address the issues, experiments were performed. Results showed that minor environmental pH change could significantly affect the metabolic activity of cells, demonstrating the importance of stable culture condition for such assays. Moreover, the culture models could also significantly influence the metabolic activity and proliferation of cells. Furthermore, the choice of culture models might lead to different outcomes of chemosensitivity assays. Compared with the similar test based on tumor-level assays, the spheroid model could overestimate the drug resistance of cells to cisplatin, whereas the 2D and 3D culture models might overestimate the chemosensitivity of cells to such anticancer drug. In this study, the 3D culture models with same cell density as that in tumor samples showed comparable chemosensitivity assay results as the tumor-level assays. Overall, this study has provided some fundamental information for establishing a precise and faithful drug chemosensitivity assay. PMID:25654105

Toxcast Profiling in a Human Stem Cell Assay for Developmental Toxicity (SOT)

EPA Science Inventory

We correlated the ToxCast library in a metabolic biomarker-based in vitro assay (Stemina devTOXqP) utilizing human embryonic stem (hES) cells (H9 line). This assay identifies the concentration of a chemical that disrupts cellular metabolism in a manner indicative of teratogenic...

Development of a Cell-Based Functional Assay for the Detection of Clostridium botulinum Neurotoxin Types A and E

PubMed Central

Basavanna, Uma; Muruvanda, Tim; Brown, Eric W.; Sharma, Shashi K.

2013-01-01

The standard procedure for definitive detection of BoNT-producing Clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (MBA). The mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. Cell-based assays provide an alternative to the MBA in screening for BoNT-producing Clostridia. Here, we describe a cell-based assay utilizing a fluorescence reporter construct expressed in a neuronal cell model to study toxin activity in situ. Our data indicates that the assay can detect as little as 100 pM BoNT/A activity within living cells, and the assay is currently being evaluated for the analysis of BoNT in food matrices. Among available in vitro assays, we believe that cell-based assays are widely applicable in high-throughput screenings and have the potential to at least reduce and refine animal assays if not replace it. PMID:23533420

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 (Pcell activity of approximately 18% was observed following 8 h of incubation at the 2:1 and 1:1 leucocyte to target cell ratios. Percent cytotoxic cell activity using calcein AM was similar to values reported for rainbow trout leucocytes using the 51Cr-release assay.

Capture, Release and Culture of Circulating Tumor Cells from Pancreatic Cancer Patients using an Enhanced Mixing Chip

PubMed Central

Sheng, Weian; Ogunwobi, Olorunseun O.; Chen, Tao; Zhang, Jinling; George, Thomas J.; Liu, Chen; Fan, Z. Hugh

2013-01-01

Circulating tumor cells (CTCs) from peripheral blood hold important information for cancer diagnosis and disease monitoring. Analysis of this “liquid biopsy” holds the promise to usher in a new era of personalized therapeutic treatments and real-time monitoring for cancer patients. But the extreme rarity of CTCs in blood makes their isolation and characterization technologically challenging. This paper reports the development of a geometrically enhanced mixing (GEM) chip for high-efficiency and high-purity tumor cell capture. We also successfully demonstrated the release and culture of the captured tumor cells, as well as the isolation of CTCs from cancer patients. The high-performance microchip is based on geometrically optimized micromixer structures, which enhance the transverse flow and flow folding, maximizing the interaction between CTCs and antibody-coated surfaces. With the optimized channel geometry and flow rate, the capture efficiency reached >90% with a purity of >84% when capturing spiked tumor cells in buffer. The system was further validated by isolating a wide range of spiked tumor cells (50–50,000) in 1 mL of lysed blood and whole blood. With the combination of trypsinization and high flow rate washing, captured tumor cells were efficiently released. The released cells were viable and able to proliferate, and showed no difference compared with intact cells that were not subjected to the capture and release process. Furthermore, we applied the device for detecting CTCs from metastatic pancreatic cancer patients’ blood; and CTCs were found from 17 out of 18 samples (>94%). We also tested the potential utility of the device in monitoring the response to anti-cancer drug treatment in pancreatic cancer patients, and the CTC numbers correlated with the clinical computed tomograms (CT scans) of tumors. The presented technology shows great promise for accurate CTC enumeration, biological studies of CTCs and cancer metastasis, as well as for cancer

Assaying Cellular Viability Using the Neutral Red Uptake Assay.

PubMed

Ates, Gamze; Vanhaecke, Tamara; Rogiers, Vera; Rodrigues, Robim M

2017-01-01

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

Novel Surface-Enhanced Raman Scattering-based Assays for Ultra-sensitive Detection of Human Pluripotent Stem Cells

PubMed Central

Han, Jingjia; Qian, Ximei; Wu, Qingling; Jha, Rajneesh; Duan, Jinshuai; Yang, Zhou; Maher, Kevin O.; Nie, Shuming; Xu, Chunhui

2017-01-01

Human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, but their derivatives need to be rigorously evaluated for residual stem cells to prevent teratoma formation. Here, we report the development of novel surface-enhanced Raman scattering (SERS)-based assays that can detect trace numbers of undifferentiated hPSCs in mixed cell populations in a highly specific, ultra-sensitive, and time-efficient manner. By targeting stem cell surface markers SSEA-5 and TRA-1-60 individually or simultaneously, these SERS assays were able to identify as few as 1 stem cell in 106 cells, a sensitivity (0.0001%) which was ~2,000 to 15,000-fold higher than that of flow cytometry assays. Using the SERS assay, we demonstrate that the aggregation of hPSC-based cardiomyocyte differentiation cultures into 3D spheres significantly reduced SSEA-5+ and TRA-1-60+ cells compared with parallel 2D cultures. Thus, SERS may provide a powerful new technology for quality control of hPSC-derived products for preclinical and clinical applications. PMID:27509304

Microfluidic immunocapture of circulating pancreatic cells using parallel EpCAM and MUC1 capture: characterization, optimization and downstream analysis.

PubMed

Thege, Fredrik I; Lannin, Timothy B; Saha, Trisha N; Tsai, Shannon; Kochman, Michael L; Hollingsworth, Michael A; Rhim, Andrew D; Kirby, Brian J

2014-05-21

We have developed and optimized a microfluidic device platform for the capture and analysis of circulating pancreatic cells (CPCs) and pancreatic circulating tumor cells (CTCs). Our platform uses parallel anti-EpCAM and cancer-specific mucin 1 (MUC1) immunocapture in a silicon microdevice. Using a combination of anti-EpCAM and anti-MUC1 capture in a single device, we are able to achieve efficient capture while extending immunocapture beyond single marker recognition. We also have detected a known oncogenic KRAS mutation in cells spiked in whole blood using immunocapture, RNA extraction, RT-PCR and Sanger sequencing. To allow for downstream single-cell genetic analysis, intact nuclei were released from captured cells by using targeted membrane lysis. We have developed a staining protocol for clinical samples, including standard CTC markers; DAPI, cytokeratin (CK) and CD45, and a novel marker of carcinogenesis in CPCs, mucin 4 (MUC4). We have also demonstrated a semi-automated approach to image analysis and CPC identification, suitable for clinical hypothesis generation. Initial results from immunocapture of a clinical pancreatic cancer patient sample show that parallel capture may capture more of the heterogeneity of the CPC population. With this platform, we aim to develop a diagnostic biomarker for early pancreatic carcinogenesis and patient risk stratification.

Establishment of a cell-based wound healing assay for bio-relevant testing of wound therapeutics.

PubMed

Planz, Viktoria; Wang, Jing; Windbergs, Maike

Predictive in vitro testing of novel wound therapeutics requires adequate cell-based bio-assays. Such assays represent an integral part during preclinical development as pre-step before entering in vivo studies. Simple "scratch tests" based on defected skin cell monolayers exist, however these can solely be used for testing liquids, as cell monolayer destruction and excessive hydration limit their applicability for (semi-)solid systems like wound dressings. In this context, a cell-based wound healing assay is introduced for rapid and predictive testing of wound therapeutics independent of their physical state in a bio-relevant environment. A novel wound healing assay was established for bio-relevant and predictive testing of (semi-) solid wound therapeutics. The assay allows for physiologically relevant hydration of the tested wound therapeutics at the air-liquid interface and their removal without cell monolayer disruption. In a proof-of-concept study, the applicability and discriminative power could be demonstrated by examining unloaded and drug-loaded wound dressings with two different established wound healing actives (dexpanthenol and metyrapone) and their effect on skin cell behavior. The influence of the released drug on the cells´ healing behavior could successfully be monitored over time. Wound size assessment after 96h resulted in an eight fold smaller wound area for drug treated models compared to the ones treated with unloaded fibers and non-treated wounds. This assay provides valuable first insights towards the establishment of a valid screening and evaluation tool for preclinical wound therapeutic development from liquid to (semi-)solid systems to improve predictability in a simple, yet standardized way. Copyright © 2017 Elsevier Inc. All rights reserved.

Specific capture, recovery and culture of cancer cells using oriented antibody-modified polystyrene chips coated with agarose film.

PubMed

Jeong, Jiyun; Lee, Yeolin; Yoo, Yeongeun; Lee, Myung Kyu

2018-02-01

Agarose gel can be used for three dimensional (3D) cell culture because it prevents cell attachment. The dried agarose film coated on a culture plate also protected cell attachment and allowed 3D growth of cancer cells. We developed an efficient method for agarose film coating on an oxygen-plasma treated micropost polystyrene chip prepared by an injection molding process. The agarose film was modified to maleimide or Ni-NTA groups for covalent or cleavable attachment of photoactivatable Fc-specific antibody binding proteins (PFcBPs) via their N-terminal cysteine residues or 6xHis tag, respectively. The antibodies photocrosslinked onto the PFcBP-modified chips specifically captured the target cells without nonspecific binding, and the captured cells grew 3D modes on the chips. The captured cells on the cleavable antibody-modified chips were easily recovered by treatment of commercial trypsin-EDTA solution. Under fluidic conditions using an antibody-modified micropost chip, the cells were mainly captured on the micropost walls of the chip rather than on the bottom of it. The presented method will also be applicable for immobilization of oriented antibodies on various microfluidic chips with different structures. Copyright © 2017 Elsevier B.V. All rights reserved.

Enzyme-linked immunosorbent assay using a recombinant baculovirus-expressed Bacillus anthracis protective antigen (PA): measurement of human anti-PA antibodies.

PubMed Central

Iacono-Connors, L C; Novak, J; Rossi, C; Mangiafico, J; Ksiazek, T

1994-01-01

We developed an antigen capture enzyme-linked immunosorbent assay (ELISA) which does not require purified protective antigen (PA) for detection of human antibodies to Bacillus anthracis PA. Lysates of Spodoptera frugiperda (Sf-9) cells infected with recombinant baculovirus containing the PA gene were used as the source of PA to develop the ELISA. Recombinant PA from crude Sf-9 cell lysates or PA purified from B. anthracis Sterne strain was captured by an anti-PA monoclonal antibody coated onto microtiter plates. We demonstrated that human serum antibody titers to PA were identical in the ELISA whether we used crude Sf-9 cell lysates containing recombinant baculovirus-expressed PA or purified Sterne PA. Finally, false-positive results observed in a direct ELISA were eliminated with this antigen capture ELISA. Thus, the antigen capture ELISA with crude preparations of baculovirus-expressed PA is reliable, safe, and inexpensive for determining anti-PA antibody levels in human sera. PMID:7496927

A microfluidics assay to study invasion of human placental trophoblast cells.

PubMed

Abbas, Yassen; Oefner, Carolin Melati; Polacheck, William J; Gardner, Lucy; Farrell, Lydia; Sharkey, Andrew; Kamm, Roger; Moffett, Ashley; Oyen, Michelle L

2017-05-01

Pre-eclampsia, fetal growth restriction and stillbirth are major pregnancy disorders throughout the world. The underlying pathogenesis of these diseases is defective placentation characterized by inadequate invasion of extravillous placental trophoblast cells into the uterine arteries. How trophoblast invasion is controlled remains an unanswered question but is influenced by maternal uterine immune cells called decidual natural killer cells. Here, we describe an in vitro microfluidic invasion assay to study the migration of primary human trophoblast cells. Each experiment can be performed with a small number of cells making it possible to conduct research on human samples despite the challenges of isolating primary trophoblast cells. Cells are exposed to a chemical gradient and tracked in a three-dimensional microenvironment using real-time high-resolution imaging, so that dynamic readouts on cell migration such as directionality, motility and velocity are obtained. The microfluidic system was validated using isolated trophoblast and a gradient of granulocyte-macrophage colony-stimulating factor, a cytokine produced by activated decidual natural killer cells. This microfluidic model provides detailed analysis of the dynamics of trophoblast migration compared to previous assays and can be modified in future to study in vitro how human trophoblast behaves during placentation. © 2017 The Authors.

Use of a Novel Fluidics Microbead Trap/Flow-cell Enhances Speed and Sensitivity of Bead-Based Bioassays

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

Ozanich, Rich M.; Antolick, Kathryn C.; Bruckner-Lea, Cindy J.

2007-09-15

Automated devices and methods for biological sample preparation often utilize surface functionalized microbeads (superparamagnetic or non-magnetic) to allow capture, purification and pre-concentration of trace amounts of proteins, cells, or nucleic acids (DNA/RNA) from complex samples. We have developed unique methods and hardware for trapping either magnetic or non-magnetic functionalized beads that allow samples and reagents to be efficiently perfused over a micro-column of beads. This approach yields enhanced mass transport and up to 5-fold improvements in assay sensitivity or speed, dramatically improving assay capability relative to assays conducted in more traditional “batch modes” (i.e., in tubes or microplate wells). Summarymore » results are given that highlight the analytical performance improvements obtained for automated microbead processing systems utilizing novel microbead trap/flow-cells for various applications, including: 1) simultaneous capture of multiple cytokines using an antibody-coupled polystyrene bead assay with subsequent flow cytometry detection; 2) capture of nucleic acids using oligonucleotide coupled polystyrene beads with flow cytometry detection; and 3) capture of Escherichia coli 0157:H7 (E. coli) from 50 mL sample volumes using antibody-coupled superparamagnetic microbeads with subsequent culturing to assess capture efficiency.« less

p38 Signaling and Receptor Recycling Events in a Microfluidic Endothelial Cell Adhesion Assay

PubMed Central

Vickers, Dwayne A. L.; Chory, Emma J.; Harless, Megan C.; Murthy, Shashi K.

2013-01-01

Adhesion-based microfluidic cell separation has proven to be very useful in applications ranging from cancer diagnostics to tissue engineering. This process involves functionalizing microchannel surfaces with a capture molecule. High specificity and purity capture can be achieved using this method. Despite these advances, little is known about the mechanisms that govern cell capture within these devices and their relationships to basic process parameters such as fluid shear stress and the presence of soluble factors. This work examines how the adhesion of human endothelial cells (ECs) is influenced by a soluble tetrapeptide, Arg-Glu-Asp-Val (REDV) and fluidic shear stress. The ability of these ECs to bind within microchannels coated with REDV is shown to be governed by shear- and soluble-factor mediated changes in p38 mitogen-activated protein kinase expression together with recycling of adhesion receptors from the endosome. PMID:23762436

GFP-Based Fluorescence Assay for CAG Repeat Instability in Cultured Human Cells

PubMed Central

Santillan, Beatriz A.; Moye, Christopher; Mittelman, David; Wilson, John H.

2014-01-01

Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries. PMID:25423602

Paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

PubMed

Yeow, Natasha; McLiesh, Heather; Guan, Liyun; Shen, Wei; Garnier, Gil

2016-07-01

A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. Graphical abstract A rapid and simple paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

Micro-abrasion package capture cell experiment on the trailing edge of LDEF: Impactor chemistry and whipple bumper shield efficiencies

NASA Technical Reports Server (NTRS)

Fitzgerald, Howard J.; Yano, Hajime

1995-01-01

Four of the eight available double layer microparticle capture cells, flown as the experiment A0023 on the trailing (West) face of LDEF, have been extensively studied. An investigation of the chemistry of impactors has been made using SEM/EDX techniques and the effectiveness of the capture cells as bumper shields has also been examined. Studies of these capture cells gave positive EDX results, with 53 percent of impact sites indicating the presence of some chemical residues, the predominant residue identified as being silicon in varying quantities.

A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

PubMed

Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

2014-07-01

The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis. Copyright © 2014 Elsevier B.V. All rights reserved.

Quantivirus® HPV E6/E7 RNA 3.0 assay (bDNA) is as sensitive, but less specific than Hybrid Capture 2 test.

PubMed

Shen, Yong; Gong, Jiaomei; He, Yanxia; Cheng, Guomei; Okunieff, Paul; Li, Xiaofu

2013-02-01

Human papillomavirus (HPV) infection is the primary cause of cervical cancer. The Quantivirus(®) HPV E6/E7 RNA 3.0 assay (DiaCarta, CA, USA) detects E6/E7 mRNA of 13 high risk subtypes and 6 low risk subtypes. Cervical specimens collected in PreservCyt were processed for HPV detection. Cervical biopsies were taken only from those women with abnormal colposcopy. 200 out of 272 (73.5%) cases were mRNA positive. The percentage of HPV E6/E7 mRNA positive samples increases with the severity of the cytological diagnosis, but not in histological diagnosis. In 146 patients with both tests, the E6/E7 mRNA assay had significant higher positivity rate than the Hybrid Capture 2 assay (75.3% versus 62.3%). The HPV mRNA assay and the HC2 assay had the same sensitivity of high grade cervical intraepithelial neoplasia (CIN 2+), 82.4% (14/17) (95% confidence interval [CI], 64.3, 100). However, the specificity of CIN 2+ for the HPV mRNA assay was significantly lower than HC2 assay. Receiver operating characteristic curve analysis was used to compare the diagnostic performance of the E6/E7 mRNA and HC2. E6/E7 mRNA achieved 58.8% sensitivity with 74.1% specificity, HC2, achieved 47.1% sensitivity with 70.7% specificity. The overall performance of HPV E6/E7 mRNA assay for detecting CIN 2+ was lower than HC2. This study does not support the use of this assay in screening for cervical cancer prevention alone. Copyright © 2012 Elsevier B.V. All rights reserved.

A quantitative evaluation of cell migration by the phagokinetic track motility assay.

PubMed

Nogalski, Maciej T; Chan, Gary C T; Stevenson, Emily V; Collins-McMillen, Donna K; Yurochko, Andrew D

2012-12-04

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles(1,2,3). Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread(4,5). Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell(6,7,8). However, many biological questions about cell migration remain unanswered. The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers. The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip(9,10). With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells(11,12), fibroblasts(9), neutrophils(13), skeletal muscle cells(14

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System.

PubMed

Kumaresan, Pappanaicken; Figliola, Mathew; Moyes, Judy S; Huls, M Helen; Tewari, Priti; Shpall, Elizabeth J; Champlin, Richard; Cooper, Laurence J N

2015-10-05

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-γ). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced

Sensing Cell-Culture Assays with Low-Cost Circuitry.

PubMed

Pérez, Pablo; Huertas, Gloria; Maldonado-Jacobi, Andrés; Martín, María; Serrano, Juan A; Olmo, Alberto; Daza, Paula; Yúfera, Alberto

2018-06-11

An alternative approach for cell-culture end-point protocols is proposed herein. This new technique is suitable for real-time remote sensing. It is based on Electrical Cell-substrate Impedance Spectroscopy (ECIS) and employs the Oscillation-Based Test (OBT) method. Simple and straightforward circuit blocks form the basis of the proposed measurement system. Oscillation parameters - frequency and amplitude - constitute the outcome, directly correlated with the culture status. A user can remotely track the evolution of cell cultures in real time over the complete experiment through a web tool continuously displaying the acquired data. Experiments carried out with commercial electrodes and a well-established cell line (AA8) are described, obtaining the cell number in real time from growth assays. The electrodes have been electrically characterized along the design flow in order to predict the system performance and the sensitivity curves. Curves for 1-week cell growth are reported. The obtained experimental results validate the proposed OBT for cell-culture characterization. Furthermore, the proposed electrode model provides a good approximation for the cell number and the time evolution of the studied cultures.

Radiobiological response of U251MG, CHO-K1 and V79 cell lines to accelerator-based boron neutron capture therapy

PubMed Central

Sato, Eisuke; Zaboronok, Alexander; Yamamoto, Tetsuya; Nakai, Kei; Taskaev, Sergey; Volkova, Olga; Mechetina, Ludmila; Taranin, Alexander; Kanygin, Vladimir; Isobe, Tomonori; Mathis, Bryan J; Matsumura, Akira

2018-01-01

Abstract In the current article, we provide in vitro efficacy evaluation of a unique accelerator-based neutron source, constructed at the Budker Institute of Nuclear Physics (Novosibirsk, Russian Federation), for boron neutron capture therapy (BNCT), which is particularly effective in the case of invasive cancers. U251MG, CHO-K1 and V79 cells were incubated and irradiated in various concentrations of boric acid with epithermal neutrons for 2–3 h in a plexiglass phantom, using 2.0 MeV proton energy and 1.5–3.0 mA proton current, resulting in a neutron fluence of 2.16 × 1012 cm−2. The survival curves of cells loaded with boron were normalized to those irradiated without boron (to exclude the influence of the fast neutron and gamma dose components) and fit to the linear–quadratic (LQ) model. Colony formation assays showed the following cell survival rates (means ± SDs): CHO-K1: 0.348 ± 0.069 (10 ppm), 0.058 ± 0.017 (20 ppm), 0.018 ± 0.005 (40 ppm); V79: 0.476 ± 0.160 (10 ppm), 0.346 ± 0.053 (20 ppm), 0.078 ± 0.015 (40 ppm); and U251MG: 0.311 ± 0.061 (10 ppm), 0.131 ± 0.022 (20 ppm), 0.020 ± 0.010 (40 ppm). The difference between treated cells and controls was significant in all cases (P < 0.01) and confirmed that the neutron source and irradiation regimen were sufficient for control over cell colony formation. We believe our study will serve as a model for ongoing in vitro experiments on neutron capture therapy to advance in this area for further development of accelerator-based BNCT into the clinical phase. PMID:29281044

CellAnimation: an open source MATLAB framework for microscopy assays.

PubMed

Georgescu, Walter; Wikswo, John P; Quaranta, Vito

2012-01-01

Advances in microscopy technology have led to the creation of high-throughput microscopes that are capable of generating several hundred gigabytes of images in a few days. Analyzing such wealth of data manually is nearly impossible and requires an automated approach. There are at present a number of open-source and commercial software packages that allow the user to apply algorithms of different degrees of sophistication to the images and extract desired metrics. However, the types of metrics that can be extracted are severely limited by the specific image processing algorithms that the application implements, and by the expertise of the user. In most commercial software, code unavailability prevents implementation by the end user of newly developed algorithms better suited for a particular type of imaging assay. While it is possible to implement new algorithms in open-source software, rewiring an image processing application requires a high degree of expertise. To obviate these limitations, we have developed an open-source high-throughput application that allows implementation of different biological assays such as cell tracking or ancestry recording, through the use of small, relatively simple image processing modules connected into sophisticated imaging pipelines. By connecting modules, non-expert users can apply the particular combination of well-established and novel algorithms developed by us and others that are best suited for each individual assay type. In addition, our data exploration and visualization modules make it easy to discover or select specific cell phenotypes from a heterogeneous population. CellAnimation is distributed under the Creative Commons Attribution-NonCommercial 3.0 Unported license (http://creativecommons.org/licenses/by-nc/3.0/). CellAnimationsource code and documentation may be downloaded from www.vanderbilt.edu/viibre/software/documents/CellAnimation.zip. Sample data are available at www

Evaluation of a rat tracheal epithelial cell culture assay system to identify respiratory carcinogens

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

Steele, V.E.; Arnold, J.T.; Arnold, J.V.

1989-01-01

To evaluate a short-term epithelial cell assay system to detect respiratory carcinogens, primary cultures of rat tracheal epithelial cells were exposed to a series of 17 compounds and scored for morphologically transformed cell colonies 28 days later. The test compounds included known carcinogens and noncarcinogens in volatile or liquid form. Tracheal epithelial cells were isolate from F344 rats, plated onto collagen-coated dishes, and exposed to the test compounds on day 1 for 24 hours. At day 30 the cultures were fixed, stained, and scored for colonies having a density greater than 1,300 cells/mm{sup 2}. With standardized protocols, such colonies aremore » very infrequent in media and solvent control cultures. Concentration levels for each chemical were chosen over a range from nontoxic to toxic levels. Highly positive compounds in this assay included benzo(a)pyrene, benzo(l)aceanthrylene, 3-methylcholanthrene, and formaldehyde. Compounds which were negative in this assay included pyrene, benzo(e)pyrene, and 4-nitroquinoline-N-oxide. Examining the concordance of in vitro results with whole animal carcinogenesis studies revealed an accuracy of 88% with one false-positive and one false-negative compound. The results of these studies indicate that the rat tracheal epithelial cell assay may be useful in identifying potential respiratory carcinogens in our environment.« less

A High-Content Live-Cell Viability Assay and Its Validation on a Diverse 12K Compound Screen.

PubMed

Chiaravalli, Jeanne; Glickman, J Fraser

2017-08-01

We have developed a new high-content cytotoxicity assay using live cells, called "ImageTOX." We used a high-throughput fluorescence microscope system, image segmentation software, and the combination of Hoechst 33342 and SYTO 17 to simultaneously score the relative size and the intensity of the nuclei, the nuclear membrane permeability, and the cell number in a 384-well microplate format. We then performed a screen of 12,668 diverse compounds and compared the results to a standard cytotoxicity assay. The ImageTOX assay identified similar sets of compounds to the standard cytotoxicity assay, while identifying more compounds having adverse effects on cell structure, earlier in treatment time. The ImageTOX assay uses inexpensive commercially available reagents and facilitates the use of live cells in toxicity screens. Furthermore, we show that we can measure the kinetic profile of compound toxicity in a high-content, high-throughput format, following the same set of cells over an extended period of time.

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.

Detection of human papillomavirus in pterygium and conjunctival papilloma by hybrid capture II and PCR assays.

PubMed

Takamura, Y; Kubo, E; Tsuzuki, S; Akagi, Y

2008-11-01

To elucidate the putative role of human papillomavirus (HPV) infection in pterygium and conjunctival papilloma. Hybrid capture II (HC-II) and polymerase chain reaction (PCR) assays were performed to detect HPV in pterygium (42 samples obtained from 40 patients) and conjunctival papilloma (8 samples from 6 patients). The amount of HPV DNA was evaluated by measurement of relative light units (RLUs) on a luminometer. All papilloma samples were positive for HPV DNA by PCR and HC-II. The RLU values for specimens of recurrent and re-recurrent papilloma were markedly higher than those for specimens of primary lesions. HPV was detected by PCR in 2 of 42 (4.8%) beta-globin-positive pterygium specimens, whereas HC-II showed that HPV was negative in all pterygium samples. Our results support the hypothesis that HPV DNA is associated with the pathogenesis of conjunctival papilloma, but not pterygium. RLU measurement by HC-II may serve as a marker for evaluating the activity of HPV in conjunctival tumours.

Gamma-ray imaging assay of cells 3-5 of the east cell line in the 235-F plutonium fuel form facility

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

Brand, A. D.; Aucott, T. J.; Diprete, D. P.

In August and September, 2016, scientists from the Savannah River National Laboratory (SRNL) took a series of gamma-ray imaging measurements through the cell windows in front of Cells 3-5 on the east line of the Plutonium Fuel Form (PuFF) Facility using an electrically cooled, high-purity germanium detector. A Germanium Gamma Ray Imager (GeGI) was utilized since it allowed for the location from which the radiation was being emitted to be identified by incoming gamma-ray energy. This measurement technique provided a tool which allowed for the relative concentration of Pu-238 to be mapped throughout each cell. The mapping and new assaymore » data were then used to update the model used in an assay discussed in a 2014 report (SRNL-STI-2014-00629) and to calculate a more accurate value for the holdup in each of the cells [1]. Note that the mapping and new assay data did not replace the previous assay data in the model. Rather, the mapping and new assay data provided additional details on source distribution, which supplemented the previous assay data.« less

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

PubMed Central

Gallerani, Giulia; Cocchi, Claudia; Bocchini, Martine; Piccinini, Filippo; Fabbri, Francesco

2017-01-01

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments. PMID:29286485

Novel surface-enhanced Raman scattering-based assays for ultra-sensitive detection of human pluripotent stem cells.

PubMed

Han, Jingjia; Qian, Ximei; Wu, Qingling; Jha, Rajneesh; Duan, Jinshuai; Yang, Zhou; Maher, Kevin O; Nie, Shuming; Xu, Chunhui

2016-10-01

Human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, but their derivatives need to be rigorously evaluated for residual stem cells to prevent teratoma formation. Here, we report the development of novel surface-enhanced Raman scattering (SERS)-based assays that can detect trace numbers of undifferentiated hPSCs in mixed cell populations in a highly specific, ultra-sensitive, and time-efficient manner. By targeting stem cell surface markers SSEA-5 and TRA-1-60 individually or simultaneously, these SERS assays were able to identify as few as 1 stem cell in 10(6) cells, a sensitivity (0.0001%) which was ∼2000 to 15,000-fold higher than that of flow cytometry assays. Using the SERS assay, we demonstrate that the aggregation of hPSC-based cardiomyocyte differentiation cultures into 3D spheres significantly reduced SSEA-5(+) and TRA-1-60(+) cells compared with parallel 2D cultures. Thus, SERS may provide a powerful new technology for quality control of hPSC-derived products for preclinical and clinical applications. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cell death and renewal during prey capture and digestion in the carnivorous sponge Asbestopluma hypogea (Porifera: Poecilosclerida).

PubMed

Martinand-Mari, Camille; Vacelet, Jean; Nickel, Michael; Wörheide, Gert; Mangeat, Paul; Baghdiguian, Stephen

2012-11-15

The sponge Asbestopluma hypogea is unusual among sponges due to its peculiar carnivorous feeding habit. During various stages of its nutrition cycle, the sponge is subjected to spectacular morphological modifications. Starved animals are characterized by many elongated filaments, which are crucial for the capture of prey. After capture, and during the digestion process, these filaments actively regress before being regenerated during a subsequent period of starvation. Here, we demonstrate that these morphological events rely on a highly dynamic cellular turnover, implying a coordinated sequence of programmed cell death (apoptosis and autophagy), cell proliferation and cell migration. A candidate niche for cell renewal by stem cell proliferation and differentiation was identified at the base of the sponge peduncle, characterized by higher levels of BrdU/EdU incorporation. Therefore, BrdU/EdU-positive cells of the peduncle base are candidate motile cells responsible for the regeneration of the prey-capturing main sponge body, i.e. the dynamic filaments. Altogether, our results demonstrate that dynamics of cell renewal in sponge appear to be regulated by cellular mechanisms as multiple and complex as those already identified in bilaterian metazoans.

Miniaturized and High-Throughput Assays for Analysis of T-Cell Immunity Specific for Opportunistic Pathogens and HIV

PubMed Central

Ivaldi, Federico; Starc, Nadia; Landi, Fabiola; Locatelli, Franco; Rutella, Sergio; Tripodi, Gino; Manca, Fabrizio

2014-01-01

Monitoring of antigen-specific T-cell responses is valuable in numerous conditions that include infectious diseases, vaccinations, and opportunistic infections associated with acquired or congenital immune defects. A variety of assays that make use of peripheral lymphocytes to test activation markers, T-cell receptor expression, or functional responses are currently available. The last group of assays calls for large numbers of functional lymphocytes. The number of cells increases with the number of antigens to be tested. Consequently, cells may be the limiting factor, particularly in lymphopenic subjects and in children, the groups that more often require immune monitoring. We have developed immunochemical assays that measure secreted cytokines in the same wells in which peripheral blood mononuclear cells (PBMC) are cultured. This procedure lent itself to miniaturization and automation. Lymphoproliferation and the enzyme-linked immunosorbent spot (ELISPOT) assay have been adapted to a miniaturized format. Here we provide examples of immune profiles and describe a comparison between miniaturized assays based on cytokine secretion or proliferation. We also demonstrate that these assays are convenient for use in testing antigen specificity in established T-cell lines, in addition to analysis of PBMC. In summary, the applicabilities of miniaturization to save cells and reagents and of automation to save time and increase accuracy were demonstrated in this study using different methodological approaches valuable in the clinical immunology laboratory. PMID:24477854

Cytotoxicity assays with fish cells as an alternative to the acute lethality test with fish.

PubMed

Segner, Helmut

2004-10-01

In ecotoxicology, in vitro assays with fish cells are currently applied for mechanistic studies, bioanalytical purposes and toxicity screening. This paper discusses the potential of cytotoxicity assays with fish cells to reduce, refine or replace acute lethality tests using fish. Basal cytotoxicity data obtained with fish cell lines or fish primary cell cultures show a reasonable to good correlation with lethality data from acute toxicity tests, with the exception of compounds that exert a specific mode of toxic action. Basal cytotoxicity data from fish cell lines also correlate well with cytotoxicity data from mammalian cell lines. However, both the piscine and mammalian in vitro assays are clearly less sensitive than the fish test. Therefore, in vivo LC50 values (concentrations of the test compounds that are lethal to 50% of the fish in the experiment within 96 hours) currently cannot be predicted from in vitro values. This in vitro-in vivo difference in sensitivity appears to be true for both fish cell lines and mammalian cell lines. Given the good in vitro-in vivo correlation in toxicity ranking, together with the clear-cut difference in sensitivity, the role of cytotoxicity assays in a tiered alternative testing strategy could be in priority setting in relation to toxic hazard and in the toxicity classification of chemicals and environmental samples.

Nanomaterial-based Microfluidic Chips for the Capture and Detection of Circulating Tumor Cells.

PubMed

Sun, Duanping; Chen, Zuanguang; Wu, Minhao; Zhang, Yuanqing

2017-01-01

Circulating tumor cells (CTCs), a type of cancer cells that spreads from primary or metastatic tumors into the bloodstream, can lead to a new fatal metastasis. As a new type of liquid biopsy, CTCs have become a hot pursuit and detection of CTCs offers the possibility for early diagnosis of cancers, earlier evaluation of chemotherapeutic efficacy and cancer recurrence, and choice of individual sensitive anti-cancer drugs. The fundamental challenges of capturing and characterizing CTCs are the extremely low number of CTCs in the blood and the intrinsic heterogeneity of CTCs. A series of microfluidic devices have been proposed for the analysis of CTCs with automation capability, precise flow behaviors, and significant advantages over the conventional larger scale systems. This review aims to provide in-depth insights into CTCs analysis, including various nanomaterial-based microfluidic chips for the capture and detection of CTCs based on the specific biochemical and physical properties of CTCs. The current developmental trends and promising research directions in the establishment of microfluidic chips for the capture and detection of CTCs are also discussed.

In-situ gamma-ray assay of the east cell line in the 235-F Plutonium fuel form facility

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

Diprete, D.

On September 17th -19th , 2013, scientists from SRNL took a series of in-situ gamma-ray measurements in the maintenance trench beneath Cells 1-5 on the east line of the PuFF facility using a well-collimated, high-purity germanium detector. The cell interiors were assayed along with the furnaces and storage coolers that protrude beneath the cells. The detector efficiency was estimated using a combination of MCNP simulations and empirical measurements. Data analysis was performed using three gamma-rays emitted by Pu-238 (99.85 keV, 152.7 keV, and 766.4 keV) providing three independent estimates of the mass of Pu-238 holdup in each of the cells.more » The weighted mean of these three results was used as the best estimate of Pu-238 holdup in the East Cell Line of PuFF. The results of the assay measurements are found in the table on the following page along with the results from the scoping assay performed in 2006. All uncertainties in this table (as well as the rest of the report) are reported at 1σ. Summing the assay results and treating MDAs as M 238Pu= 0 ± MDA, the total holdup in the East Cell Line was 240 ± 40 grams. This result is 100 grams lower than the previous estimate, a 0.55σ difference. The uncertainty in the Pu-238 holdup is also reduced substantially relative to the 2006 scoping assay. However, the current assay results are in agreement with the 2006 scoping assay results due to the large uncertainty associated with the 2006 scoping assays. The current assay results support the conclusion that the 2006 results bound the Pu-238 mass in Cells 1-5. These results should be considered preliminary since additional measurements of the East Cell line are scheduled for 2017 and 2018. Those measurements will provide detailed information about the distribution of Pu-238 in the cells to be used to refine the results of the current assay.« less

Transparent, biocompatible nanostructured surfaces for cancer cell capture and culture

PubMed Central

Cheng, Boran; He, Zhaobo; Zhao, Libo; Fang, Yuan; Chen, Yuanyuan; He, Rongxiang; Chen, Fangfang; Song, Haibin; Deng, Yuliang; Zhao, Xingzhong; Xiong, Bin

2014-01-01

Circulating tumor cells (CTCs) in the blood which have detached from both the primary tumor and any metastases may be considered as a “liquid biopsy” and are expected to replace tumor biopsies in the monitoring of treatment response and determining patient prognosis. Here, we introduce a facile and efficient CTC detection material made of hydroxyapatite/chitosan (HA/CTS), which is beneficial because of its transparency and excellent biological compatibility. Atomic force microscopy images show that the roughness of the HA/CTS nanofilm (HA/CTSNF) substrates can be controlled by changing the HA:CTS ratio. Enhanced local topographic interactions between nano-components on cancer cell membranes, and the antibody coated nanostructured substrate lead to improved CTC capture and separation. This remarkable nanostructured substrate has the potential for CTC culture in situ and merits further analysis. CTCs captured from artificial blood samples were observed in culture on HA/CTSNF substrates over a period of 14 days by using conventional staining methods (hematoxylin eosin and Wright’s stain). We conclude that these substrates are multifunctional materials capable of isolating and culturing CTCs for subsequent studies. PMID:24904216

High-throughput assays for DNA gyrase and other topoisomerases

PubMed Central

Maxwell, Anthony; Burton, Nicolas P.; O'Hagan, Natasha

2006-01-01

We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format. PMID:16936317

High-throughput assays for DNA gyrase and other topoisomerases.

PubMed

Maxwell, Anthony; Burton, Nicolas P; O'Hagan, Natasha

2006-01-01

We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format.

A versatile assay for RNA-binding proteins in living cells

PubMed Central

Strein, Claudia; Alleaume, Anne-Marie; Rothbauer, Ulrich; Hentze, Matthias W.; Castello, Alfredo

2014-01-01

RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not reflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their responses to stimuli. While electrophoresis mobility shift assays (EMSA) have been used for such determinations, their results serve at best as proxies for the RBP activities in living cells. Here, we describe a quantitative dual fluorescence method to analyze protein–mRNA interactions in vivo. Known or candidate RBPs are fused to fluorescent proteins (eGFP, YFP), expressed in cells, cross-linked in vivo to RNA by ultraviolet light irradiation, and immunoprecipitated, after lysis, with a single chain antibody fragment directed against eGFP (GFP-binding protein, GBP). Polyadenylated RNA-binding activity of fusion proteins is assessed by hybridization with an oligo(DT) probe coupled with a red fluorophore. Since UV light is directly applied to living cells, the assay can be used to monitor dynamic changes in RNA-binding activities in response to biological or pharmacological stimuli. Notably, immunoprecipitation and hybridization can also be performed with commercially available GBP-coupled 96-well plates (GFP-multiTrap), allowing highly parallel RNA-binding measurements in a single experiment. Therefore, this method creates the possibility to conduct in vivo high-throughput RNA-binding assays. We believe that this fast and simple radioactivity-free method will find many useful applications in RNA biology. PMID:24664470

Using a split luciferase assay (SLA) to measure the kinetics of cell-cell fusion mediated by herpes simplex virus glycoproteins.

PubMed

Saw, Wan Ting; Matsuda, Zene; Eisenberg, Roselyn J; Cohen, Gary H; Atanasiu, Doina

2015-11-15

Herpes simplex virus (HSV) entry and cell-cell fusion require the envelope proteins gD, gH/gL and gB. We propose that receptor-activated conformational changes to gD activate gH/gL, which then triggers gB (the fusogen) into an active form. To study this dynamic process, we have adapted a dual split protein assay originally developed to study the kinetics of human immunodeficiency virus (HIV) mediated fusion. This assay uses a chimera of split forms of renilla luciferase (RL) and green fluorescent protein (GFP). Effector cells are co-transfected with the glycoproteins and one of the split reporters. Receptor-bearing target cells are transfected with the second reporter. Co-culture results in fusion and restoration of RL, which can convert a membrane permeable substrate into a luminescent product, thereby enabling one to monitor initiation and extent of fusion in live cells in real time. Restoration of GFP can also be studied by fluorescence microscopy. Two sets of split reporters have been developed: the original one allows one to measure fusion kinetics over hours whereas the more recent version was designed to enhance the sensitivity of RL activity allowing one to monitor both initiation and rates of fusion in minutes. Here, we provide a detailed, step-by-step protocol for the optimization of the assay (which we call the SLA for split luciferase assay) using the HSV system. We also show several examples of the power of this assay to examine both the initiation and kinetics of cell-cell fusion by wild type forms of gD, gB, gH/gL of both serotypes of HSV as well as the effect of mutations and antibodies that alter the kinetics of fusion. The SLA can be applied to other viral systems that carry out membrane fusion. Copyright © 2015 Elsevier Inc. All rights reserved.

Using a Split Luciferase Assay (SLA) to measure the kinetics of cell-cell fusion mediated by herpes simplex virus glycoproteins

PubMed Central

Saw, Wan Ting; Matsuda, Zene; Eisenberg, Roselyn J; Cohen, Gary H; Atanasiu, Doina

2015-01-01

Herpes simplex virus (HSV) entry and cell-cell fusion require the envelope proteins gD, gH/gL and gB. We propose that receptor-activated conformational changes to gD activate gH/gL, which then triggers gB (the fusogen) into an active form. To study this dynamic process, we have adapted a dual split protein assay originally developed to study the kinetics of human immunodeficiency virus (HIV) mediated fusion. This assay uses a chimera of split forms of renilla luciferase (RL) and green fluorescent protein (GFP). Effector cells are co-transfected with the glycoproteins and one of the split reporters. Receptor-bearing target cells are transfected with the second reporter. Co-culture results in fusion and restoration of RL, which can convert a membrane permeable substrate into a luminescent product, thereby enabling one to monitor initiation and extent of fusion in live cells in real time. Restoration of GFP can also be studied by fluorescence microscopy. Two sets of split reporters have been developed: the original one allows one to measure fusion kinetics over hours whereas the more recent version was designed to enhance the sensitivity of RL activity allowing one to monitor both initiation and rates of fusion in minutes. Here, we provide a detailed, step-by-step protocol for the optimization of the assay (which we call the SLA for split luciferase assay) using the HSV system. We also show several examples of the power of this assay to examine both the initiation and kinetics of cell-cell fusion by wild type forms of gD, gB, gH/gL of both serotypes of HSV as well as the effect of mutations and antibodies that alter the kinetics of fusion. The SLA can be applied to other viral systems that carry out membrane fusion. PMID:26022509

The XTT Cell Proliferation Assay Applied to Cell Layers Embedded in Three-Dimensional Matrix

PubMed Central

Huyck, Lynn; Ampe, Christophe

2012-01-01

Abstract Cell proliferation, a main target in cancer therapy, is influenced by the surrounding three-dimensional (3D) extracellular matrix (ECM). In vitro drug screening is, thus, optimally performed under conditions in which cells are grown (embedded or trapped) in dense 3D matrices, as these most closely mimic the adhesive and mechanical properties of natural ECM. Measuring cell proliferation under these conditions is, however, technically more challenging compared with two-dimensional (2D) culture and other “3D culture conditions,” such as growth on top of a matrix (pseudo-3D) or in spongy scaffolds with large pore sizes. Consequently, such measurements are only slowly applied on a wider scale. To advance this, we report on the equal quality (dynamic range, background, linearity) of measuring the proliferation of cell layers embedded in dense 3D matrices (collagen, Matrigel) compared with cells in 2D culture using the easy (one-step) and in 2D well-validated, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)-assay. The comparison stresses the differences in proliferation kinetics and drug sensitivity of matrix-embedded cells versus 2D culture. Using the specific cell-layer-embedded 3D matrix setup, quantitative measurements of cell proliferation and cell invasion are shown to be possible in similar assay conditions, and cytostatic, cytotoxic, and anti-invasive drug effects can thus be reliably determined and compared in physiologically relevant settings. This approach in the 3D matrix holds promise for improving early-stage, high-throughput drug screening, targeting either highly invasive or highly proliferative subpopulations of cancers or both. PMID:22574651

Detection, isolation, and capture of circulating breast cancer cells with photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Bhattacharyya, Kiran; Njoroge, Martin; Goldschmidt, Benjamin S.; Gaffigan, Brian; Rood, Kyle; Viator, John A.

2013-03-01

According to the CDC, breast cancer is the most common cancer and the second leading cause of cancer related deaths among women. Metastasis, or the presence of secondary tumors caused by the spread of cancer cells via the circulatory or lymphatic systems, significantly worsens the prognosis of any breast cancer patient. In this study, a technique is developed to detect circulating breast cancer cells in human blood using a photoacoustic flow cytometry method. A Q-switched laser with a 5 ns pulse at 532 nm is used to interrogate thousands of cells with one pulse as they flow through the beam path. Cells which are pigmented, either naturally or artificially, emit an ultrasound wave as a result of the photoacoustic (PA) effect. Breast cancer cells are targeted with chromophores through immunochemistry in order to provide pigment. After which, the device is calibrated to demonstrate a single-cell detection limit. Cultured breast cancer cells are added to whole blood to reach a biologically relevant concentration of about 25-45 breast cancer cells per 1 mL of blood. An in vitro photoacoustic flow cytometer is used to detect and isolate these cells followed by capture with the use of a micromanipulator. This method can not only be used to determine the disease state of the patient and the response to therapy, it can also be used for genetic testing and in vitro drug trials since the circulating cell can be captured and studied.

Chemical-free lysis and fractionation of cells by use of surface acoustic waves for sensitive protein assays.

PubMed

Salehi-Reyhani, Ali; Gesellchen, Frank; Mampallil, Dileep; Wilson, Rab; Reboud, Julien; Ces, Oscar; Willison, Keith R; Cooper, Jonathan M; Klug, David R

2015-02-17

We exploit the mechanical action of surface acoustic waves (SAW) to differentially lyse human cancer cells in a chemical-free manner. The extent to which cells were disrupted is reported for a range of SAW parameters, and we show that the presence of 10 μm polystyrene beads is required to fully rupture cells and their nuclei. We show that SAW is capable of subcellular fractionation through the chemical-free isolation of nuclei from whole cells. The concentration of protein was assessed in lysates with a sensitive microfluidic antibody capture (MAC) chip. An antibody-based sandwich assay in a microfluidic microarray format was used to detect unlabeled human tumor suppressor protein p53 in crude lysates, without any purification step, with single-molecule resolution. The results are digital, enabling sensitive quantification of proteins with a dynamic range >4 orders of magnitude. For the conditions used, the efficiency of SAW-induced mechanical lysis was determined to be 12.9% ± 0.7% of that for conventional detergent-based lysis in yielding detectable protein. A range of possible loss mechanisms that could lead to the drop in protein yield are discussed. Our results show that the methods described here are amenable to an integrated point-of-care device for the assessment of tumor protein expression in fine needle aspirate biopsies.

Detection of E. coli O157:H7 in complex matrices under varying flow parameters with a robotic fluorometric assay system

NASA Astrophysics Data System (ADS)

Leskinen, Stephaney D.; Schlemmer, Sarah M.; Kearns, Elizabeth A.; Lim, Daniel V.

2009-02-01

The development of rapid assays for detection of microbial pathogens in complex matrices is needed to protect public health due to continued outbreaks of disease from contaminated foods and water. An Escherichia coli O157:H7 detection assay was designed using a robotic, fluorometric assay system. The system integrates optics, fluidics, robotics and software for the detection of foodborne pathogens or toxins in as many as four samples simultaneously. It utilizes disposable fiber optic waveguides coated with biotinylated antibodies for capture of target analytes from complex sample matrices. Computer-controlled rotation of sample cups allows complete contact between the sample and the waveguide. Detection occurs via binding of a fluorophore-labeled antibody to the captured target, which leads to an increase in the fluorescence signal. Assays are completed within twenty-five minutes. Sample matrices included buffer, retentate (material recovered from the filter of the Automated Concentration System (ACS) following hollow fiber ultrafiltration), spinach wash and ground beef. The matrices were spiked with E. coli O157:H7 (103-105 cells/ml) and the limits of detection were determined. The effect of sample rotation on assay sensitivity was also examined. Rotation parameters for each sample matrix included 10 ml with rotation, 5 ml with rotation and 0.1 ml without rotation. Detection occurred at 104 cells/ml in buffer and spinach wash and at 105 cells/ml in retentate and ground beef. Detection was greater for rotated samples in each matrix except ground beef. Enhanced detection of E. coli from large, rotated volumes of complex matrices was confirmed.

Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay.

PubMed

Speit, Günter; Schütz, Petra; Bausinger, Julia

2016-06-01

The comet assay in combination with the polymerase inhibitor aphidicolin (APC) has been used to measure DNA excision repair activity, DNA repair kinetics and individual DNA repair capacity. Since APC can enhance genotoxic effects of mutagens measured by the comet assay, this approach has been proposed for increasing the sensitivity of the comet assay in human biomonitoring. The APC-modified comet assay has mainly been performed with human blood and it was shown that it not only enhances the detection of DNA damage repaired by nucleotide excision repair (NER) but also damage typically repaired by base excision repair (BER). Recently, we reported that in contrast to blood leukocytes, A549 cells (a human lung adenocarcinoma cell line) seem to be insensitive towards the repair-inhibiting action of APC. To further elucidate the general usefulness of the APC-modified comet assay for studying repair in cultured mammalian cells, we comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15μM). APC was either added for 2h together with the mutagen or cells were pre-incubated for 30min with APC before the mutagen was added. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC. The actual cause for these differences is still unclear but potential molecular explanations are discussed. Irrespective of the underlying mechanism(s), our study revealed practical limitations of the use of the APC-modified comet assay. Copyright © 2016 Elsevier B.V. All rights reserved.

Inertial focusing and passive micro-mixing techniques for rare cells capturing microfluidic platform

NASA Astrophysics Data System (ADS)

Phadke, Manisha; Shaner, Sebastian; Shah, Shreyas; Rodriguez, Ygnacio; Wibowo, Denni; Whulanza, Yudan; Teriete, Peter; Allen, Jeff; Kassegne, Sam

2018-02-01

Isolation and capture of rare cells continues to be a daunting task that is still looking for an innovative and efficient method. While a variety of approaches have been suggested over the past several years, immunocapturing in a microfluidic platform carries a substantial promise as shown by recent published works. In this paper, we introduced a combination of inertial focusing and passive micro-mixing through 3D chevron-type features in a microchannel to induce chaotic mixing within antibody-coated microchannels and, ultimately, promote rare cell capture. The device introduced in this work contains curved microchannels that consist of a series of staggered chevron grooves. The curved channels enable inertial focusing while the chevron grooves allow for chaotic mixing. The microfluidics platform microfabricated through soft lithography has a polydimethylsiloxane (PDMS) foundation and was thinly coated with an alginate hydrogel derivatized with streptavidin. We submitted that our qualitative and quantitative results demonstrated the potentials in advancements in rare cell isolation through this integration of two techniques.

Evaluation of a recombinant yeast cell estrogen screening assay.

PubMed Central

Coldham, N G; Dave, M; Sivapathasundaram, S; McDonnell, D P; Connor, C; Sauer, M J

1997-01-01

A wide range of chemicals with diverse structures derived from plant and environmental origins are reported to have hormonal activity. The potential for appreciable exposure of humans to such substances prompts the need to develop sensitive screening methods to quantitate and evaluate the risk to the public. Yeast cells transformed with plasmids encoding the human estrogen receptor and an estrogen responsive promoter linked to a reporter gene were evaluated for screening compounds for estrogenic activity. Relative sensitivity to estrogens was evaluated by reference to 17 beta-estradiol (E2) calibration curves derived using the recombinant yeast cells, MCF-7 human breast cancer cells, and a prepubertal mouse uterotrophic bioassay. The recombinant yeast cell bioassay (RCBA) was approximately two and five orders of magnitude more sensitive to E2 than MCF-7 cells and the uterotrophic assay, respectively. The estrogenic potency of 53 chemicals, including steroid hormones, synthetic estrogens, environmental pollutants, and phytoestrogens, was measured using the RCBA. Potency values produced with the RCBA relative to E2 (100) included estrone (9.6), diethylstilbestrol (74.3), tamoxifen (0.0047), alpha-zearalanol (1.3), equol (0.085), 4-nonylphenol (0.005), and butylbenzyl phathalate (0.0004), which were similar to literature values but generally higher than those produced by the uterotrophic assay. Exquisite sensitivity, absence of test compound biotransformation, ease of use, and the possibility of measuring antiestrogenic activity are important attributes that argue for the suitability of the RCBA in screening for potential xenoestrogens to evaluate risk to humans, wildlife, and the environment. Images Figure 1. Figure 2. Figure 3. Figure 4. PMID:9294720

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds.

PubMed

Bracke, Marc E; Roman, Bart I; Stevens, Christian V; Mus, Liselot M; Parmar, Virinder S; De Wever, Olivier; Mareel, Marc M

2015-06-06

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account

Development of a surrogate angiogenic potency assay for clinical-grade stem cell production.

PubMed

Lehman, Nicholas; Cutrone, Rochelle; Raber, Amy; Perry, Robert; Van't Hof, Wouter; Deans, Robert; Ting, Anthony E; Woda, Juliana

2012-09-01

Clinical results from acute myocardial infarction (AMI) patients treated with MultiStem®, a large-scale expanded adherent multipotent progenitor cell population (MAPC), have demonstrated a strong safety and benefit profile for these cells. The mechanism of benefit with MAPC treatment is a result, in part, of its ability to induce neovascularization through trophic support. Production of clinical-grade stem cell products requires the development of lot-release criteria based on potency assays that directly reflect the fundamental mechanistic pathway underlying the therapeutic response to verify manufacturing process consistency and product potency. Using an in vitro endothelial tube formation assay, a potency assay has been developed that reflects MAPC pro-angiogenic activity. Serum-free conditioned media collected from MAPC culture induced endothelial tube formation. A proteomic survey of angiogenic factors produced by the cells in vitro revealed candidate factors linked to angiogenic potency. Three cytokines, chemokine (C-X-C motif) ligand 5 (CXCL5), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF), were required for this angiogenic activity. Depletion of any of these factors from the media prevented tube formation, while adding back increasing amounts of these cytokines into the depleted serum-free conditioned media established the lower limits of each of the cytokines required to induce angiogenesis. A necessary threshold of angiogenic factor expression was established using an in vitro angiogenesis assay. By correlating the levels of the cytokines required to induce tube formation in vitro with levels of the factors found in the spent media from manufacturing production runs, detection of these factors was identified as a surrogate potency assay with defined pass/fail criteria.

Cell-free NADPH oxidase activation assays: "in vitro veritas".

PubMed

Pick, Edgar

2014-01-01

The superoxide (O2 (∙-))-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22 (phox) ) and four cytosolic regulatory proteins, p47 (phox) , p67 (phox) , p40 (phox) , and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 (∙-) generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by specific signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome. A consequent conformational change in Nox2 initiates the electron "flow" along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O2 (∙-). Methodological difficulties in the dissection of this complex mechanism led to the design "cell-free" systems (also known as "broken cells" or in vitro systems). In these, membrane receptor stimulation and all or part of the signal transduction sequence are missing, the accent being placed on the actual process of "NADPH oxidase assembly," thus on the formation of the complex between cytochrome b 558 and the cytosolic components and the resulting O2 (∙-) generation. Cell-free assays consist of a mixture of the individual components of the NADPH oxidase complex, derived from resting phagocytes or in the form of purified recombinant proteins, exposed in vitro to an activating agent (distinct from and unrelated to whole cell stimulants), in the presence of NADPH and oxygen. Activation is commonly quantified by measuring the primary product of the reaction, O2 (∙-), trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of the linear rate of O2 (∙-) production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount

Comparison of Abbott RealTime High-Risk HPV and Hybrid Capture 2 Assays for Detection of HPV Infection.

PubMed

Ko, Kiwoong; Yu, Shinae; Lee, Eun Hee; Park, Hyosoon; Woo, Hee-Yeon; Kwon, Min-Jung

2016-09-01

Various assays for detecting high-risk human papillomavirus (HR HPV) have been introduced recently, including the Abbott RealTime High-Risk HPV assay. We sought to compare the performance of Abbott PCR to Hybrid Capture 2 for the detection of HR HPV. A total of 941 cervical swab specimens were obtained. We submitted all specimens for HR HPV detection with HC2 and Abbott PCR, and then additionally analyzed discordant and concordant positive results using restriction fragment mass polymorphism (RFMP) genotyping analysis. HC2 detected one of 13 HR HPV types in 12.3% (116/941) of cases, while Abbott PCR detected one of 14 detectable HR HPV types in 12.9% (121/941) of cases. The overall agreement rate was 97.3% with a kappa coefficient of 0.879. Discordant results between these two assays were observed in 25 cases. HC2 showed a sensitivity of 90.0% and specificity of 95.9%, while Abbott PCR showed a sensitivity of 98.0% and specificity of 96.8% when using RFMP results as the gold standard. For HPV 16/18 detection, Abbott PCR showed 95.8%/88.9% sensitivity and 99.2%/99.8% specificity, respectively. The overall coinfection rate between HPV 16, 18 and non-16/18 was 9.9% (12/121) in Abbott PCR analysis. Considering its high agreement rate with HC2, higher sensitivity/specificity compared to HC2, and ability to differentiate HPV 16/18 from other HPV types, Abbott PCR could be a reliable laboratory testing method for the screening of HPV infections. © 2016 by the Association of Clinical Scientists, Inc.

Micropatterned comet assay enables high throughput and sensitive DNA damage quantification.

PubMed

Ge, Jing; Chow, Danielle N; Fessler, Jessica L; Weingeist, David M; Wood, David K; Engelward, Bevin P

2015-01-01

The single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring many classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. However, limited throughput and difficulties with reproducibility have limited its utility, particularly for clinical and epidemiological studies. To address these limitations, we created a microarray comet assay. The use of a micrometer scale array of cells increases the number of analysable comets per square centimetre and enables automated imaging and analysis. In addition, the platform is compatible with standard 24- and 96-well plate formats. Here, we have assessed the consistency and sensitivity of the microarray comet assay. We showed that the linear detection range for H2O2-induced DNA damage in human lymphoblastoid cells is between 30 and 100 μM, and that within this range, inter-sample coefficient of variance was between 5 and 10%. Importantly, only 20 comets were required to detect a statistically significant induction of DNA damage for doses within the linear range. We also evaluated sample-to-sample and experiment-to-experiment variation and found that for both conditions, the coefficient of variation was lower than what has been reported for the traditional comet assay. Finally, we also show that the assay can be performed using a 4× objective (rather than the standard 10× objective for the traditional assay). This adjustment combined with the microarray format makes it possible to capture more than 50 analysable comets in a single image, which can then be automatically analysed using in-house software. Overall, throughput is increased more than 100-fold compared to the traditional assay. Together, the results presented here demonstrate key advances in comet assay technology that improve the throughput, sensitivity, and robustness, thus enabling larger scale clinical and epidemiological studies. © The Author 2014. Published by

Cell based assays for anti-Plasmodium activity evaluation.

PubMed

Mokgethi-Morule, Thabang; N'Da, David D

2016-03-10

Malaria remains one of the most common and deadly infectious diseases worldwide. The severity of this global public health challenge is reflected by the approximately 198 million people, who were reportedly infected in 2013 and by the more than 584,000 related deaths in that same year. The rising emergence of drug resistance towards the once effective artemisinin combination therapies (ACTs) has become a serious concern and warrants more robust drug development strategies, with the objective of eradicating malaria infections. The intricate biology and life cycle of Plasmodium parasites complicate the understanding of the disease in such a way that would enhance the development of more effective chemotherapies that would achieve radical clinical cure and that would prevent disease relapse. Phenotypic cell based assays have for long been a valuable approach and involve the screening and analysis of diverse compounds with regards to their activities towards whole Plasmodium parasites in vitro. To achieve the Millennium Development Goal (MDG) of malaria eradication by 2020, new generation drugs that are active against all parasite stages (erythrocytic (blood), exo-erythrocytic (liver stages and gametocytes)) are needed. Significant advances are being made in assay development to overcome some of the practical challenges of assessing drug efficacy, particularly in the liver and transmission stage Plasmodium models. This review discusses primary screening models and the fundamental progress being made in whole cell based efficacy screens of anti-malarial activity. Ongoing challenges and some opportunities for improvements in assay development that would assist in the discovery of effective, safe and affordable drugs for malaria treatments are also discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) and IgM capture ELISA for detection of antibodies to lipopolysaccharide in adult typhoid fever patients in Pakistan.

PubMed

Sippel, J; Bukhtiari, N; Awan, M B; Krieg, R; Duncan, J F; Karamat, K A; Malik, I A; Igbal, L M; Legters, L

1989-06-01

Sera from 339 adult febrile patients in Pakistan were tested for antibodies to Salmonella typhi lipopolysaccharide by indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assay (ELISA) and IgM capture ELISA. A total of 55 patients had S. typhi cultured from their blood, 20 had S. typhi cultured from their stool, 24 were blood or stool culture positive for S. paratyphi A, 41 were culture negative but clinically diagnosed as having enteric fever, 41 had gastrointestinal or urinary tract infections, 41 were clinically diagnosed as having malaria, 20 were smear-positive patients with malaria, 58 had respiratory infections, and the remaining 39 individuals were placed in a miscellaneous group who did not have Salmonella infection. The sensitivities of the indirect IgG ELISA, indirect IgM ELISA, and IgM capture ELISA determined with specimens obtained from the blood culture-positive patients with typhoid fever (positive controls) were 80, 64, and 62%, respectively. The specificities of the assays determined with sera from the patients with respiratory infections (negative controls) were 95, 95, and 97%, respectively. The percentage of smear-positive patients with malaria who were positive by these assays was lower than that in the negative control group. The percentages of individuals in the other patient categories who were positive by these tests were between those obtained with the positive and negative controls. Of the positive controls, 26 were positive by both IgM assays, 9 were IgM positive only by indirect ELISA, and 8 were IgM positive only by IgM capture ELISA. A total of 70% of the positive control patients who were tested for O agglutinins by the Widal tube agglutination assay were positive; however, 29% of the negative control patients were also positive. The indirect IgG ELISA was the single most effective test for the serodiagnosis of typhoid fever in this population.

Indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) and IgM capture ELISA for detection of antibodies to lipopolysaccharide in adult typhoid fever patients in Pakistan.

PubMed Central

Sippel, J; Bukhtiari, N; Awan, M B; Krieg, R; Duncan, J F; Karamat, K A; Malik, I A; Igbal, L M; Legters, L

1989-01-01

Sera from 339 adult febrile patients in Pakistan were tested for antibodies to Salmonella typhi lipopolysaccharide by indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assay (ELISA) and IgM capture ELISA. A total of 55 patients had S. typhi cultured from their blood, 20 had S. typhi cultured from their stool, 24 were blood or stool culture positive for S. paratyphi A, 41 were culture negative but clinically diagnosed as having enteric fever, 41 had gastrointestinal or urinary tract infections, 41 were clinically diagnosed as having malaria, 20 were smear-positive patients with malaria, 58 had respiratory infections, and the remaining 39 individuals were placed in a miscellaneous group who did not have Salmonella infection. The sensitivities of the indirect IgG ELISA, indirect IgM ELISA, and IgM capture ELISA determined with specimens obtained from the blood culture-positive patients with typhoid fever (positive controls) were 80, 64, and 62%, respectively. The specificities of the assays determined with sera from the patients with respiratory infections (negative controls) were 95, 95, and 97%, respectively. The percentage of smear-positive patients with malaria who were positive by these assays was lower than that in the negative control group. The percentages of individuals in the other patient categories who were positive by these tests were between those obtained with the positive and negative controls. Of the positive controls, 26 were positive by both IgM assays, 9 were IgM positive only by indirect ELISA, and 8 were IgM positive only by IgM capture ELISA. A total of 70% of the positive control patients who were tested for O agglutinins by the Widal tube agglutination assay were positive; however, 29% of the negative control patients were also positive. The indirect IgG ELISA was the single most effective test for the serodiagnosis of typhoid fever in this population. PMID:2754002

MTS dye based colorimetric CTLL-2 cell proliferation assay for product release and stability monitoring of interleukin-15: assay qualification, standardization and statistical analysis.

PubMed

Soman, Gopalan; Yang, Xiaoyi; Jiang, Hengguang; Giardina, Steve; Vyas, Vinay; Mitra, George; Yovandich, Jason; Creekmore, Stephen P; Waldmann, Thomas A; Quiñones, Octavio; Alvord, W Gregory

2009-08-31

A colorimetric cell proliferation assay using soluble tetrazolium salt [(CellTiter 96(R) Aqueous One Solution) cell proliferation reagent, containing the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and an electron coupling reagent phenazine ethosulfate], was optimized and qualified for quantitative determination of IL-15 dependent CTLL-2 cell proliferation activity. An in-house recombinant Human (rHu)IL-15 reference lot was standardized (IU/mg) against an international reference standard. Specificity of the assay for IL-15 was documented by illustrating the ability of neutralizing anti-IL-15 antibodies to block the product specific CTLL-2 cell proliferation and the lack of blocking effect with anti-IL-2 antibodies. Under the defined assay conditions, the linear dose-response concentration range was between 0.04 and 0.17ng/ml of the rHuIL-15 produced in-house and 0.5-3.0IU/ml for the international standard. Statistical analysis of the data was performed with the use of scripts written in the R Statistical Language and Environment utilizing a four-parameter logistic regression fit analysis procedure. The overall variation in the ED(50) values for the in-house reference standard from 55 independent estimates performed over the period of 1year was 12.3% of the average. Excellent intra-plate and within-day/inter-plate consistency was observed for all four parameter estimates in the model. Different preparations of rHuIL-15 showed excellent intra-plate consistency in the parameter estimates corresponding to the lower and upper asymptotes as well as to the 'slope' factor at the mid-point. The ED(50) values showed statistically significant differences for different lots and for control versus stressed samples. Three R-scripts improve data analysis capabilities allowing one to describe assay variations, to draw inferences between data sets from formal statistical tests, and to set up improved assay acceptance

Human Mucosal Mast Cells Capture HIV-1 and Mediate Viral trans-Infection of CD4+ T Cells.

PubMed

Jiang, Ai-Ping; Jiang, Jin-Feng; Wei, Ji-Fu; Guo, Ming-Gao; Qin, Yan; Guo, Qian-Qian; Ma, Li; Liu, Bao-Chi; Wang, Xiaolei; Veazey, Ronald S; Ding, Yong-Bing; Wang, Jian-Hua

2015-12-30

The gastrointestinal mucosa is the primary site where human immunodeficiency virus type 1 (HIV-1) invades, amplifies, and becomes persistently established, and cell-to-cell transmission of HIV-1 plays a pivotal role in mucosal viral dissemination. Mast cells are widely distributed in the gastrointestinal tract and are early targets for invasive pathogens, and they have been shown to have increased density in the genital mucosa in HIV-infected women. Intestinal mast cells express numerous pathogen-associated molecular patterns (PAMPs) and have been shown to combat various viral, parasitic, and bacterial infections. However, the role of mast cells in HIV-1 infection is poorly defined. In this study, we investigated their potential contributions to HIV-1 transmission. Mast cells isolated from gut mucosal tissues were found to express a variety of HIV-1 attachment factors (HAFs), such as DC-SIGN, heparan sulfate proteoglycan (HSPG), and α4β7 integrin, which mediate capture of HIV-1 on the cell surface. Intriguingly, following coculture with CD4(+) T cells, mast cell surface-bound viruses were efficiently transferred to target T cells. Prior blocking with anti-HAF antibody or mannan before coculture impaired viral trans-infection. Cell-cell conjunctions formed between mast cells and T cells, to which viral particles were recruited, and these were required for efficient cell-to-cell HIV-1 transmission. Our results reveal a potential function of gut mucosal mast cells in HIV-1 dissemination in tissues. Strategies aimed at preventing viral capture and transfer mediated by mast cells could be beneficial in combating primary HIV-1 infection. In this study, we demonstrate the role of human mast cells isolated from mucosal tissues in mediating HIV-1 trans-infection of CD4(+) T cells. This finding facilitates our understanding of HIV-1 mucosal infection and will benefit the development of strategies to combat primary HIV-1 dissemination. Copyright © 2016, American Society

Capturing and concentrating adenovirus using magnetic anionic nanobeads

PubMed Central

Sakudo, Akikazu; Baba, Koichi; Ikuta, Kazuyoshi

2016-01-01

We recently demonstrated how various enveloped viruses can be efficiently concentrated using magnetic beads coated with an anionic polymer, poly(methyl vinyl ether-maleic anhydrate). However, the exact mechanism of interaction between the virus particles and anionic beads remains unclear. To further investigate whether these magnetic anionic beads specifically bind to the viral envelope, we examined their potential interaction with a nonenveloped virus (adenovirus). The beads were incubated with either adenovirus-infected cell culture medium or nasal aspirates from adenovirus-infected individuals and then separated from the supernatant by applying a magnetic field. After thoroughly washing the beads, adsorption of adenovirus was confirmed by a variety of techniques, including immunochromatography, polymerase chain reaction, Western blotting, and cell culture infection assays. These detection methods positively identified the hexon and penton capsid proteins of adenovirus along with the viral genome on the magnetic beads. Furthermore, various types of adenovirus including Types 5, 6, 11, 19, and 41 were captured using the magnetic bead procedure. Our bead capture method was also found to increase the sensitivity of viral detection. Adenovirus below the detectable limit for immunochromatography was efficiently concentrated using the magnetic bead procedure, allowing the virus to be successfully detected using this methodology. Moreover, these findings clearly demonstrate that a viral envelope is not required for binding to the anionic magnetic beads. Taken together, our results show that this capture procedure increases the sensitivity of detection of adenovirus and would, therefore, be a valuable tool for analyzing both clinical and experimental samples. PMID:27274228

Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells

PubMed Central

Cirelli, Kimberly M.; Dan, Jennifer M.; Morou, Antigoni; Daigneault, Audrey; Brassard, Nathalie; Silvestri, Guido; Routy, Jean-Pierre; Havenar-Daughton, Colin; Crotty, Shane

2017-01-01

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells. PMID:29065175

Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells.

PubMed

Reiss, Samantha; Baxter, Amy E; Cirelli, Kimberly M; Dan, Jennifer M; Morou, Antigoni; Daigneault, Audrey; Brassard, Nathalie; Silvestri, Guido; Routy, Jean-Pierre; Havenar-Daughton, Colin; Crotty, Shane; Kaufmann, Daniel E

2017-01-01

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells.

Self-propelled carbon nanotube based microrockets for rapid capture and isolation of circulating tumor cells

NASA Astrophysics Data System (ADS)

Banerjee, Shashwat S.; Jalota-Badhwar, Archana; Zope, Khushbu R.; Todkar, Kiran J.; Mascarenhas, Russel R.; Chate, Govind P.; Khutale, Ganesh V.; Bharde, Atul; Calderon, Marcelo; Khandare, Jayant J.

2015-05-01

Here, we report a non-invasive strategy for isolating cancer cells by autonomously propelled carbon nanotube (CNT) microrockets. H2O2-driven oxygen (O2) bubble-propelled microrockets were synthesized using CNT and Fe3O4 nanoparticles in the inner surface and covalently conjugating transferrin on the outer surface. Results show that self-propellant microrockets can specifically capture cancer cells.Here, we report a non-invasive strategy for isolating cancer cells by autonomously propelled carbon nanotube (CNT) microrockets. H2O2-driven oxygen (O2) bubble-propelled microrockets were synthesized using CNT and Fe3O4 nanoparticles in the inner surface and covalently conjugating transferrin on the outer surface. Results show that self-propellant microrockets can specifically capture cancer cells. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr01797a

Cell-of-Origin in Diffuse Large B-Cell Lymphoma: Are the Assays Ready for the Clinic?

PubMed

Scott, David W

2015-01-01

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma worldwide and consists of a heterogeneous group of cancers classified together on the basis of shared morphology, immunophenotype, and aggressive clinical behavior. It is now recognized that this malignancy comprises at least two distinct molecular subtypes identified by gene expression profiling: the activated B-cell-like (ABC) and the germinal center B-cell-like (GCB) groups-the cell-of-origin (COO) classification. These two groups have different genetic mutation landscapes, pathobiology, and outcomes following treatment. Evidence is accumulating that novel agents have selective activity in one or the other COO group, making COO a predictive biomarker. Thus, there is now a pressing need for accurate and robust methods to assign COO, to support clinical trials, and ultimately guide treatment decisions for patients. The "gold standard" methods for COO are based on gene expression profiling (GEP) of RNA from fresh frozen tissue using microarray technology, which is an impractical solution when formalin-fixed paraffin-embedded tissue (FFPET) biopsies are the standard diagnostic material. This review outlines the history of the COO classification before examining the practical implementation of COO assays applicable to FFPET biopsies. The immunohistochemistry (IHC)-based algorithms and gene expression-based assays suitable for the highly degraded RNA from FFPET are discussed. Finally, the technical and practical challenges that still need to be addressed are outlined before robust gene expression-based assays are used in the routine management of patients with DLBCL.

Boron neutron capture therapy induces apoptosis of glioma cells through Bcl-2/Bax

PubMed Central

2010-01-01

Background Boron neutron capture therapy (BNCT) is an alternative treatment modality for patients with glioma. The aim of this study was to determine whether induction of apoptosis contributes to the main therapeutic efficacy of BNCT and to compare the relative biological effect (RBE) of BNCT, γ-ray and reactor neutron irradiation. Methods The neutron beam was obtained from the Xi'an Pulsed Reactor (XAPR) and γ-rays were obtained from [60Co] γ source of the Fourth Military Medical University (FMMU) in China. Human glioma cells (the U87, U251, and SHG44 cell lines) were irradiated by neutron beams at the XAPR or [60Co] γ-rays at the FMMU with different protocols: Group A included control nonirradiated cells; Group B included cells treated with 4 Gy of [60Co] γ-rays; Group C included cells treated with 8 Gy of [60Co] γ-rays; Group D included cells treated with 4 Gy BPA (p-borono-phenylalanine)-BNCT; Group E included cells treated with 8 Gy BPA-BNCT; Group F included cells irradiated in the reactor for the same treatment period as used for Group D; Group G included cells irradiated in the reactor for the same treatment period as used for Group E; Group H included cells irradiated with 4 Gy in the reactor; and Group I included cells irradiated with 8 Gy in the reactor. Cell survival was determined using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium (MTT) cytotoxicity assay. The morphology of cells was detected by Hoechst33342 staining and transmission electron microscope (TEM). The apoptosis rate was detected by flow cytometer (FCM). The level of Bcl-2 and Bax protein was measured by western blot analysis. Results Proliferation of U87, U251, and SHG44 cells was much more strongly inhibited by BPA-BNCT than by irradiation with [60Co] γ-rays (P < 0.01). Nuclear condensation was determined using both a fluorescence technique and electron microscopy in all cell lines treated with BPA-BNCT. Furthermore, the cellular apoptotic rates in Group D and Group E

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.

Interspecific in vitro assay for the chimera-forming ability of human pluripotent stem cells.

PubMed

Masaki, Hideki; Kato-Itoh, Megumi; Umino, Ayumi; Sato, Hideyuki; Hamanaka, Sanae; Kobayashi, Toshihiro; Yamaguchi, Tomoyuki; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Nakauchi, Hiromitsu

2015-09-15

Functional assay limitations are an emerging issue in characterizing human pluripotent stem cells (PSCs). With rodent PSCs, chimera formation using pre-implantation embryos is the gold-standard assay of pluripotency (competence of progeny to differentiate into all three germ layers). In human PSCs (hPSCs), however, this can only be monitored via teratoma formation or in vitro differentiation, as ethical concerns preclude generation of human-human or human-animal chimeras. To circumvent this issue, we developed a functional assay utilizing interspecific blastocyst injection and in vitro culture (interspecies in vitro chimera assay) that enables the development and observation of embryos up to headfold stage. The assay uses mouse pre-implantation embryos and rat, monkey and human PSCs to create interspecies chimeras cultured in vitro to the early egg-cylinder stage. Intra- and interspecific chimera assays with rodent PSC lines were performed to confirm the consistency of results in vitro and in vivo. The behavior of chimeras developed in vitro appeared to recapitulate that of chimeras developed in vivo; that is, PSC-derived cells survived and were integrated into the epiblast of egg-cylinder-stage embryos. This indicates that the interspecific in vitro chimera assay is useful in evaluating the chimera-forming ability of rodent PSCs. However, when human induced PSCs (both conventional and naïve-like types) were injected into mouse embryos and cultured, some human cells survived but were segregated; unlike epiblast-stage rodent PSCs, they never integrated into the epiblast of egg-cylinder-stage embryos. These data suggest that the mouse-human interspecies in vitro chimera assay does not accurately reflect the early developmental potential/process of hPSCs. The use of evolutionarily more closely related species as host embryos might be necessary to evaluate the developmental potency of hPSCs. © 2015. Published by The Company of Biologists Ltd.

Alternative Methods for the Detection of Emerging Marine Toxins: Biosensors, Biochemical Assays and Cell-Based Assays

PubMed Central

Reverté, Laia; Soliño, Lucía; Carnicer, Olga; Diogène, Jorge; Campàs, Mònica

2014-01-01

The emergence of marine toxins in water and seafood may have a considerable impact on public health. Although the tendency in Europe is to consolidate, when possible, official reference methods based on instrumental analysis, the development of alternative or complementary methods providing functional or toxicological information may provide advantages in terms of risk identification, but also low cost, simplicity, ease of use and high-throughput analysis. This article gives an overview of the immunoassays, cell-based assays, receptor-binding assays and biosensors that have been developed for the screening and quantification of emerging marine toxins: palytoxins, ciguatoxins, cyclic imines and tetrodotoxins. Their advantages and limitations are discussed, as well as their possible integration in research and monitoring programs. PMID:25431968

Endothelial cell differentiation into capillary-like structures in response to tumour cell conditioned medium: a modified chemotaxis chamber assay.

PubMed

Garrido, T; Riese, H H; Aracil, M; Pérez-Aranda, A

1995-04-01

We have developed a modified chemotaxis chamber assay in which bovine aortic endothelial (BAE) cells degrade Matrigel basement membrane and migrate and form capillary-like structures on type I collagen. This capillary formation occurs in the presence of conditioned media from highly metastatic tumour cell lines, such as B16F10 murine melanoma or MDA-MD-231 human breast adenocarcinoma, but not in the presence of conditioned medium (CM) from the less invasive B16F0 cell line. Replacement of tumour cell CM by 10 ng ml-1 basic fibroblast growth factor (bFGF) also results in capillary-like structure formation by BAE cells. An anti-bFGF antibody blocks this effect, showing that bFGF is one of the factors responsible for the angiogenic response induced by B16F10 CM in our assay. Addition of an anti-laminin antibody reduces significantly the formation of capillary-like structures, probably by blocking the attachment of BAE cells to laminin present in Matrigel. The anti-angiogenic compound suramin inhibits in a dose-dependent manner (complete inhibition with 100 microM suramin) the migration and differentiation of BAE cells on type I collagen in response to B16F10 CM. This assay represents a new model system to study tumour-induced angiogenesis in vitro.

Development of a F actin-based live-cell fluorimetric microplate assay for diarrhetic shellfish toxins.

PubMed

Leira, F; Alvarez, C; Cabado, A G; Vieites, J M; Vieytes, M R; Botana, L M

2003-06-15

A new cytotoxicity assay for detection and quantitation of diarrhetic shellfish toxins (DSP) is presented. This assay is based upon fluorimetric determination of F-actin depolymerization induced by okadaic acid (OA)-class compounds in the BE(2)-M17 neuroblastoma cell line. No interferences were observed with other marine toxins such as saxitoxin, domoic acid, or yessotoxin, thus indicating a good specificity of the assay as expected by the direct relationship between protein phosphatase inhibition and cytoskeletal changes. The proposed method is rapid (<2h) and shows a linear response in the range of 50-300 nM OA. The detection limit of the assay for crude methanolic extracts of bivalves lies between 0.2 and 1.0 microg OA per gram of digestive glands, depending on the type of samples (fresh or canned), thus being similar to that of the mouse bioassay. The performance of this assay has been evaluated by comparative analysis of 32 toxic mussel samples by the F-actin assay, mouse bioassay, HPLC and PP2A inhibition assay. Results obtained by the F-actin method showed no differences with HPLC and significant correlation with PP2A inhibition assay (r(2)=0.71). No false negative results were obtained with this new cell assay, which also showed optimum reproducibility.

Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes.

PubMed

Treder, Krzysztof; Chołuj, Joanna; Zacharzewska, Bogumiła; Babujee, Lavanya; Mielczarek, Mateusz; Burzyński, Adam; Rakotondrafara, Aurélie M

2018-02-01

Potato virus Y (PVY) infection has been a global challenge for potato production and the leading cause of downgrading and rejection of seed crops for certification. Accurate and timely diagnosis is a key for effective disease control. Here, we have optimized a reverse transcription loop-mediated amplification (RT-LAMP) assay to differentiate the PVY O and N serotypes. The RT-LAMP assay is based on isothermal autocyclic strand displacement during DNA synthesis. The high specificity of this method relies heavily on the primer sets designed for the amplification of the targeted regions. We designed specific primer sets targeting a region within the coat protein gene that contains nucleotide signatures typical for O and N coat protein types, and these primers differ in their annealing temperature. Combining this assay with total RNA extraction by magnetic capture, we have established a highly sensitive, simplified and shortened RT-LAMP procedure as an alternative to conventional nucleic acid assays for diagnosis. This optimized procedure for virus detection may be used as a preliminary test for identifying the viral serotype prior to investing time and effort in multiplex RT-PCR tests when a specific strain is needed.

A facile, branched DNA assay to quantitatively measure glucocorticoid receptor auto-regulation in T-cell acute lymphoblastic leukemia

PubMed Central

Schwartz, Jason R.; Sarvaiya, Purvaba J.; Leiva, Lily E.; Velez, Maria C.; Singleton, Tammuella C.; Yu, Lolie C.; Vedeckis, Wayne V.

2012-01-01

Glucocorticoid (GC) steroid hormones are used to treat acute lymphoblastic leukemia (ALL) because of their pro-apoptotic effects in hematopoietic cells. However, not all leukemia cells are sensitive to GC, and no assay to stratify patients is available. In the GC-sensitive T-cell ALL cell line CEM-C7, auto-up-regulation of RNA transcripts for the glucocorticoid receptor (GR) correlates with increased apoptotic response. This study aimed to determine if a facile assay of GR transcript levels might be promising for stratifying ALL patients into hormone-sensitive and hormone-resistant populations. The GR transcript profiles of various lymphoid cell lines and 4 bone marrow samples from patients with T-cell ALL were analyzed using both an optimized branched DNA (bDNA) assay and a real-time quantitative reverse transcription-polymerase chain reaction assay. There were significant correlations between both assay platforms when measuring total GR (exon 5/6) transcripts in various cell lines and patient samples, but not for a probe set that detects a specific, low abundance GR transcript (exon 1A3). Our results suggest that the bDNA platform is reproducible and precise when measuring total GR transcripts and, with further development, may ultimately offer a simple clinical assay to aid in the prediction of GC-sensitivity in ALL patients. PMID:22739263

Microfluidic device with integrated microfilter of conical-shaped holes for high efficiency and high purity capture of circulating tumor cells

NASA Astrophysics Data System (ADS)

Tang, Yadong; Shi, Jian; Li, Sisi; Wang, Li; Cayre, Yvon E.; Chen, Yong

2014-08-01

Capture of circulating tumor cells (CTCs) from peripheral blood of cancer patients has major implications for metastatic detection and therapy analyses. Here we demonstrated a microfluidic device for high efficiency and high purity capture of CTCs. The key novelty of this approach lies on the integration of a microfilter with conical-shaped holes and a micro-injector with cross-flow components for size dependent capture of tumor cells without significant retention of non-tumor cells. Under conditions of constant flow rate, tumor cells spiked into phosphate buffered saline could be recovered and then cultured for further analyses. When tumor cells were spiked in blood of healthy donors, they could also be recovered at high efficiency and high clearance efficiency of white blood cells. When the same device was used for clinical validation, CTCs could be detected in blood samples of cancer patients but not in that of healthy donors. Finally, the capture efficiency of tumor cells is cell-type dependent but the hole size of the filter should be more closely correlated to the nuclei size of the tumor cells. Together with the advantage of easy operation, low-cost and high potential of integration, this approach offers unprecedented opportunities for metastatic detection and cancer treatment monitoring.

Critical elements in the development of cell therapy potency assays for ischemic conditions.

PubMed

Porat, Yael; Abraham, Eytan; Karnieli, Ohad; Nahum, Sagi; Woda, Juliana; Zylberberg, Claudia

2015-07-01

A successful potency assay for a cell therapy product (CTP) used in the treatment of ischemic conditions should quantitatively measure relevant biological properties that predict therapeutic activity. This is especially challenging because of numerous degrees of complexity stemming from factors that include a multifactorial complex mechanism of action, cell source, inherent cell characteristics, culture method, administration mode and the in vivo conditions to which the cells are exposed. The expected biological function of a CTP encompasses complex interactions that range from a biochemical, metabolic or immunological activity to structural replacement of damaged tissue or organ. Therefore, the requirements for full characterization of the active substance with respect to biological function could be taxing. Moreover, the specific mechanism of action is often difficult to pinpoint to a specific molecular entity; rather, it is more dependent on the functionality of the cellular components acting in a in a multifactorial fashion. In the case of ischemic conditions, the cell therapy mechanism of action can vary from angiogenesis, vasculogenesis and arteriogenesis that may activate different pathways and clinical outcomes. The CTP cellular attributes with relation to the suggested mechanism of action can be used for the development of quantitative and reproducible analytical potency assays. CTPs selected and released on the basis of such potency assays should have the highest probability of providing meaningful clinical benefit for patients. This White Paper will discuss and give examples for key elements in the development of a potency assay for treatment of ischemic disorders treated by the use of CTPs. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Anionic polymer, poly(methyl vinyl ether-maleic anhydride)-coated beads-based capture of human influenza A and B virus.

PubMed

Sakudo, Akikazu; Baba, Koichi; Tsukamoto, Megumi; Sugimoto, Atsuko; Okada, Takashi; Kobayashi, Takanori; Kawashita, Norihito; Takagi, Tatsuya; Ikuta, Kazuyoshi

2009-01-15

An anionic magnetic beads-based method was developed for the capture of human influenza A and B viruses from nasal aspirates, allantoic fluid and culture medium. A polymer, poly(methyl vinyl ether-maleic anhydride) [poly(MVE-MA)], was used to endow magnetic beads with a negative charge and bioadhesive properties. After incubation with samples containing human influenza virus, the beads were separated from supernatants by applying a magnetic field. The adsorption [corrected] of the virus by the beads was confirmed by hemagglutinin assay, immunochromatography, Western blotting, egg infection, and cell infection. Successful capture was proved using 5 H1N1 influenza A viruses, 10 H3N2 influenza A viruses, and 6 influenza B viruses. Furthermore, the infectivity in chicken embryonated eggs and Madin-Darby canine kidney (MDCK) cells of the captured human influenza virus was similar to that of the total viral quantity of starting materials. Therefore, this method of capture using magnetic beads coated with poly(MVE-MA) can be broadly used for the recovery of infectious human influenza viruses.

Clinical Applications of NanoVelcro Rare-Cell Assays for Detection and Characterization of Circulating Tumor Cells

PubMed Central

Chen, Jie-Fu; Zhu, Yazhen; Lu, Yi-Tsung; Hodara, Elisabeth; Hou, Shuang; Agopian, Vatche G.; Tomlinson, James S.; Posadas, Edwin M.; Tseng, Hsian-Rong

2016-01-01

Liquid biopsy of tumor through isolation of circulating tumor cells (CTCs) allows non-invasive, repetitive, and systemic sampling of disease. Although detecting and enumerating CTCs is of prognostic significance in metastatic cancer, it is conceivable that performing molecular and functional characterization on CTCs will reveal unprecedented insight into the pathogenic mechanisms driving lethal disease. Nanomaterial-embedded cancer diagnostic platforms, i.e., NanoVelcro CTC Assays represent a unique rare-cell sorting method that enables detection isolation, and characterization of CTCs in peripheral blood, providing an opportunity to noninvasively monitor disease progression in individual cancer patients. Over the past decade, a series of NanoVelcro CTC Assays has been demonstrated for exploring the full potential of CTCs as a clinical biomarker, including CTC enumeration, phenotyping, genotyping and expression profiling. In this review article, the authors will briefly introduce the development of three generations of NanoVelcro CTC Assays, and highlight the clinical applications of each generation for various types of solid cancers, including prostate cancer, pancreatic cancer, lung cancer, and melanoma. PMID:27375790

Capture and release of cancer cells using electrospun etchable MnO2 nanofibers integrated in microchannels

NASA Astrophysics Data System (ADS)

Liu, Hui-qin; Yu, Xiao-lei; Cai, Bo; You, Su-jian; He, Zhao-bo; Huang, Qin-qin; Rao, Lang; Li, Sha-sha; Liu, Chang; Sun, Wei-wei; Liu, Wei; Guo, Shi-shang; Zhao, Xing-zhong

2015-03-01

This paper introduces a cancer cell capture/release microchip based on the self-sacrificed MnO2 nanofibers. Through electrospinning, lift-off and soft-lithography procedures, MnO2 nanofibers are tactfully fabricated in microchannels to implement enrichment and release of cancer cells in liquid samples. The MnO2 nanofiber net which mimics the extra cellular matrix can lead to high capture ability with the help of a cancer cell-specific antibody bio-conjugation. Subsequently, an effective and friendly release method is carried out by using low concentration of oxalic acid to dissolve the MnO2 nanofiber substrate while keeping high viability of those released cancer cells at the same time. It is conceivable that our microchip may have potentials in realizing biomedical analysis of circulating tumor cells for biological and clinical researches in oncology.

An Electrochemical Cell for Selective Lithium Capture from Seawater.

PubMed

Kim, Joo-Seong; Lee, Yong-Hee; Choi, Seungyeon; Shin, Jaeho; Dinh, Hung-Cuong; Choi, Jang Wook

2015-08-18

Lithium (Li) is a core element of Li-ion batteries (LIBs). Recent developments in mobile electronics such as smartphones and tablet PCs as well as advent of large-scale LIB applications including electrical vehicles and grid-level energy storage systems have led to an increase in demand for LIBs, giving rise to a concern on the availability and market price of Li resources. However, the current Lime-Soda process that is responsible for greater than 80% of worldwide Li resource supply is applicable only in certain regions on earth where the Li concentrations are sufficiently high (salt lakes or salt pans). Moreover, not only is the process time-consuming (12-18 months), but post-treatments are also required for the purification of Li. Here, we have devised a location-independent electrochemical system for Li capture, which can operate within a short time period (a few hours to days). By engaging olivine LiFePO4 active electrode that improves interfacial properties via polydopamine coating, the electrochemical cell achieves 4330 times amplification in Li/Na ion selectivity (Li/Na molar ratio of initial solution = 0.01 and Li/Na molar ratio of final electrode = 43.3). In addition, the electrochemical system engages an I(-)/I3(-) redox couple in the other electrode for balancing of the redox states on both electrode sides and sustainable operations of the entire cell. Based on the electrochemical results, key material and interfacial properties that affect the selectivity in Li capture are identified.

Paramagnetic capture mode magnetophoretic microseparator for high efficiency blood cell separations.

PubMed

Han, Ki-Ho; Frazier, A Bruno

2006-02-01

This paper presents the characterization of continuous single-stage and three-stage cascade paramagnetic capture (PMC) mode magnetophoretic microseparators for high efficiency separation of red and white blood cells from diluted whole blood based on their native magnetic properties. The separation mechanism for both PMC microseparators is based on a high gradient magnetic separation (HGMS) method. This approach enables separation of blood cells without the use of additives such as magnetic beads. Experimental results for the single-stage PMC microseparator show that 91.1% of red blood cells were continuously separated from the sample at a volumetric flow rate of 5 microl h-1. In addition, the three-stage cascade PMC microseparator continuously separated 93.5% of red blood cells and 97.4% of white blood cells from whole blood at a volumetric flow rate of 5 microl h-1.

Comparison of the analytical and clinical performances of Abbott RealTime High Risk HPV, Hybrid Capture 2, and DNA Chip assays in gynecology patients.

PubMed

Park, Seungman; Kang, Youjin; Kim, Dong Geun; Kim, Eui-Chong; Park, Sung Sup; Seong, Moon-Woo

2013-08-01

The detection of high-risk (HR) HPV in cervical cancer screening is important for early diagnosis of cervical cancer or pre-cancerous lesions. We evaluated the analytical and clinical performances of 3 HR HPV assays in Gynecology patients. A total of 991 specimens were included in this study: 787 specimens for use with a Hybrid Capture 2 (HC2) and 204 specimens for a HPV DNA microarray (DNA Chip). All specimens were tested using an Abbott RealTime High Risk HPV assay (Real-time HR), PGMY PCR, and sequence analysis. Clinical sensitivities for severe abnormal cytology (severe than high-grade squamous intraepithelial lesion) were 81.8% for Real-time HR, 77.3% for HC2, and 66.7% for DNA Chip, and clinical sensitivities for severe abnormal histology (cervical intraepithelial neoplasia grade 2+) were 91.7% for HC2, 87.5% for Real-time HR, and 73.3% for DNA Chip. As compared to results of the sequence analysis, HC2, Real-time HR, and DNA Chip showed concordance rates of 94.3% (115/122), 90.0% (117/130), and 61.5% (16/26), respectively. The HC2 assay and Real-time HR assay showed comparable results to each other in both clinical and analytical performances, while the DNA Chip assay showed poor clinical and analytical performances. The Real-time HR assay can be a good alternative option for HR HPV testing with advantages of allowing full automation and simultaneous genotyping of HR types 16 and 18. Copyright © 2013 Elsevier Inc. All rights reserved.

Screening ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay

EPA Science Inventory

An Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to screen the ToxCast Phase I chemical library for effects on cellular differentiation and cell number. The U.S. Environmental Protection Agency (EPA) established the ...

Mussel micronucleus cytome assay.

PubMed

Bolognesi, Claudia; Fenech, Michael

2012-05-17

The micronucleus (MN) assay is one of the most widely used genotoxicity biomarkers in aquatic organisms, providing an efficient measure of chromosomal DNA damage occurring as a result of either chromosome breakage or chromosome mis-segregation during mitosis. The MN assay is today applied in laboratory and field studies using hemocytes and gill cells from bivalves, mainly from the genera Mytilus. These represent 'sentinel' organisms because of their ability to survive under polluted conditions and to accumulate both organic and inorganic pollutants. Because the mussel MN assay also includes scoring of different cell types, including necrotic and apoptotic cells and other nuclear anomalies, it is in effect an MN cytome assay. The mussel MN cytome (MUMNcyt) assay protocol we describe here reports the recommended experimental design, sample size, cell preparation, cell fixation and staining methods. The protocol also includes criteria and photomicrographs for identifying different cell types and scoring criteria for micronuclei (MNi) and nuclear buds. The complete procedure requires approximately 10 h for each experimental point/sampling station (ten animals).

Liquid biopsy in cancer patients: advances in capturing viable CTCs for functional studies using the EPISPOT assay.

PubMed

Alix-Panabières, Catherine; Pantel, Klaus

2015-01-01

Circulating tumor cells (CTCs) in the blood of cancer patients have received increasing attention as new diagnostic tool enabling 'liquid biopsies'. In contrast to the wealth of descriptive studies demonstrating the clinical relevance of CTCs as biomarkers, the extremely low concentration of CTCs in the peripheral blood of most cancer patients challenges further functional studies. This article discusses the current possibilities to enrich and, in particular, detect viable CTCs with emphasis on the EPithelial ImmunoSPOT technology. This functional assay detects viable CTCs at the single-cell level and has been used on hundreds of patients with different tumor types including epithelial tumors (breast, prostate and colon cancer) and melanomas. Moreover, the article summarizes recent advances in the in vitro and in vivo expansion of CTCs from cancer patients. These functional analyses will contribute to identifying the biological properties of metastatic cells and reveal new therapeutic targets against disseminating cancer cells.

Cell-based assays in GPCR drug discovery.

PubMed

Siehler, Sandra

2008-04-01

G protein-coupled receptors (GPCRs) transmit extracellular signals into the intracellular space, and play key roles in the physiological regulation of virtually every cell and tissue. Characteristic for the GPCR superfamily of cell surface receptors are their seven transmembrane-spanning alpha-helices, an extracellular N terminus and intracellular C-terminal tail. Besides transmission of extracellular signals, their activity is modulated by cellular signals in an auto- or transregulatory fashion. The molecular complexity of GPCRs and their regulated signaling networks triggered the interest in academic research groups to explore them further, and their drugability and role in pathophysiology triggers pharmaceutical research towards small molecular weight ligands and therapeutic antibodies. About 30% of marketed drugs target GPCRs, which underlines the importance of this target class. This review describes current and emerging cellular assays for the ligand discovery of GPCRs.

Albumin-coated monodisperse magnetic poly(glycidyl methacrylate) microspheres with immobilized antibodies: application to the capture of epithelial cancer cells.

PubMed

Horák, Daniel; Svobodová, Zuzana; Autebert, Julien; Coudert, Benoit; Plichta, Zdeněk; Královec, Karel; Bílková, Zuzana; Viovy, Jean-Louis

2013-01-01

Monodisperse (4 μm) macroporous crosslinked poly(glycidyl methacrylate) (PGMA) microspheres for use in microfluidic immunomagnetic cell sorting, with a specific application to the capture of circulating tumor cells (CTCs), were prepared by multistep swelling polymerization in the presence of cyclohexyl acetate porogen and hydrolyzed and ammonolyzed. Iron oxide was then precipitated in the microspheres to render them magnetic. Repeated precipitation made possible to raise the iron oxide content to more than 30 wt %. To minimize nonspecific adsorption of the microspheres in a microchannel and of cells on the microspheres, they were coated with albumin crosslinked with glutaraldehyde. Antibodies of epithelial cell adhesion molecule (anti-EpCAM) were then immobilized on the albumin-coated magnetic microspheres using the carbodiimide method. Capture of breast cancer MCF7 cells as a model of CTCs by the microspheres with immobilized anti-EpCAM IgG was performed in a batch experiment. Finally, MCF7 cells were captured by the anti-EpCAM-immobilized albumin-coated magnetic microspheres in an Ephesia chip. A very good rejection of lymphocytes was achieved. Thus, albumin-coated monodisperse magnetic PGMA microspheres with immobilized anti-EpCAM seem to be promising for capture of CTCs in a microfluidic device. Copyright © 2012 Wiley Periodicals, Inc.

Human papillomavirus (HPV) DNA triage of women with atypical squamous cells of undetermined significance with Amplicor HPV and Hybrid Capture 2 assays for detection of high-grade lesions of the uterine cervix.

PubMed

Dufresne, Simon; Sauthier, Philippe; Mayrand, Marie-Hélène; Petignat, Patrick; Provencher, Diane; Drouin, Pierre; Gauthier, Philippe; Dupuis, Marie-Josée; Michon, Bertrand; Ouellet, Stéphan; Hadjeres, Rachid; Ferenczy, Alex; Franco, Eduardo L; Coutlée, François

2011-01-01

Up to 20% of women having a cytology smear showing atypical squamous cells of undetermined significance (ASC-US) and infected with high-risk human papillomavirus (HR HPV) have high-grade cervical intraepithelial neoplasia (CIN 2/3). Results obtained with the Amplicor HPV and Hybrid Capture 2 (HC-2) assays for HR HPV DNA detection in women referred to colposcopy for an ASC-US smear were compared. Cervical samples in PreservCyt were tested for the presence of 13 HR HPV types with HC-2, with Amplicor at three cutoffs for positivity (0.2, 1.0, and 1.5 optical density units), and for 36 genotypes with the Linear Array (LA). Of 396 eligible women, 316 did not have CIN, 47 had CIN 1, 29 had CIN 2/3, and 4 had CIN of unknown grade. HR HPV was detected in 129 (32.6%) and 164 (41.4%) samples with HC-2 and Amplicor HPV (cutoff, 0.2), respectively (P = 0.01). Overall, 112 specimens were positive and 215 were negative with the HC-2 and Amplicor HPV assays (agreement of 82.6%; 95% confidence interval [CI], 78.5 to 86.0). The clinical sensitivity and specificity of Amplicor HPV at cutoffs of 0.2, 1.0 and 1.5 and of HC-2 for detection of CIN 2/3 were 89.7% (95% CI, 72.8 to 97.2) and 62.5% (95% CI, 57.5 to 52.4), 89.7% (95% CI, 72.8 to 97.2) and 64.5% (95% CI, 59.4 to 69.2), 89.7% (95% CI, 72.8 to 97.2) and 64.7% (95% CI, 59.7 to 69.5), and 93.1% (95% CI, 77.0 to 99.2) and 72.2% (95% CI, 67.4 to 76.5), respectively. Both HR HPV detection tests identified women with ASC-US who would benefit the most from colposcopy. Women with persistent HR HPV infection need further investigation despite a first normal colposcopy.

A Real Time Chemotaxis Assay Unveils Unique Migratory Profiles amongst Different Primary Murine Macrophages

PubMed Central

Iqbal, Asif J.; Regan-Komito, Daniel; Christou, Ivy; White, Gemma E.; McNeill, Eileen; Kenyon, Amy; Taylor, Lewis; Kapellos, Theodore S.; Fisher, Edward A.; Channon, Keith M.; Greaves, David R.

2013-01-01

Chemotaxis assays are an invaluable tool for studying the biological activity of inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of the data captured given that the assays are analysed at a single time-point. We report the optimisation and validation of a label-free, real-time cell migration assay based on electrical cell impedance to measure chemotaxis of different primary murine macrophage populations in response to a range of CC chemokines and other chemoattractant signalling molecules. We clearly demonstrate key differences in the migratory behavior of different murine macrophage populations and show that this dynamic system measures true macrophage chemotaxis rather than chemokinesis or fugetaxis. We highlight an absolute requirement for Gαi signaling and actin cytoskeletal rearrangement as demonstrated by Pertussis toxin and cytochalasin D inhibition. We also studied the chemotaxis of CD14+ human monocytes and demonstrate distinct chemotactic profiles amongst different monocyte donors to CCL2. This real-time chemotaxis assay will allow a detailed analysis of factors that regulate macrophage responses to chemoattractant cytokines and inflammatory mediators. PMID:23516549

A novel cell-based assay for inhibitory anti-muscarinic type 3 receptor antibodies in primary Sjögren's syndrome.

PubMed

Bastian, Isabell; Gordon, Tom P; Jackson, Michael W

2015-12-01

Inhibitory autoantibodies acting at the muscarinic acetylcholine receptor type 3 (M3R) are postulated to mediate autonomic dysfunction, including decreased salivary and lacrimal gland output and extra-glandular manifestations, in patients with primary Sjögren's syndrome. However, the contention that anti-M3R antibodies are pathogenic in patients remains untested, due to a lack of assays both sophisticated enough to detect inhibitory anti-M3R antibodies yet suitable for screening large patient cohorts. In the current study, we have established a cell-based bioassay of M3R activity, based on dual transfection of the M3R and a luciferase reporter gene. The bioassay is capable of capturing real-time agonist-mediated signalling of the M3R, which is inhibited specifically by patient IgG that have previously been demonstrated to have anti-M3R activity. The assay can be run in multi-well culture plates, and analysed using simple luminescence readers. As such, the new bioassay incorporating M3R-mediated luciferase transduction is the first assay adaptable to common diagnostic platforms that is capable of determining the presence in patient serum of functionally active anti-M3R autoantibodies. The new bioassay should prove useful for large cohort screening studies aiming to correlate the presence in patients of inhibitory anti-M3R antibodies with symptoms of both glandular and extra-glandular autonomic dysfunction. Copyright © 2015 Elsevier B.V. All rights reserved.

An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity.

PubMed

Barabas, Sascha; Spindler, Theresa; Kiener, Richard; Tonar, Charlotte; Lugner, Tamara; Batzilla, Julia; Bendfeldt, Hanna; Rascle, Anne; Asbach, Benedikt; Wagner, Ralf; Deml, Ludwig

2017-03-07

In healthy individuals, Cytomegalovirus (CMV) infection is efficiently controlled by CMV-specific cell-mediated immunity (CMI). Functional impairment of CMI in immunocompromized individuals however can lead to uncontrolled CMV replication and severe clinical complications. Close monitoring of CMV-specific CMI is therefore clinically relevant and might allow a reliable prognosis of CMV disease as well as assist personalized therapeutic decisions. Objective of this work was the optimization and technical validation of an IFN-γ ELISpot assay for a standardized, sensitive and reliable quantification of CMV-reactive effector cells. T-activated® immunodominant CMV IE-1 and pp65 proteins were used as stimulants. All basic assay parameters and reagents were tested and optimized to establish a user-friendly protocol and maximize the signal-to-noise ratio of the ELISpot assay. Optimized and standardized ELISpot revealed low intra-assay, inter-assay and inter-operator variability (coefficient of variation CV below 22%) and CV inter-site was lower than 40%. Good assay linearity was obtained between 6 × 10 4 and 2 × 10 5 PBMC per well upon stimulation with T-activated® IE-1 (R 2  = 0.97) and pp65 (R 2  = 0.99) antigens. Remarkably, stimulation of peripheral blood mononuclear cells (PBMC) with T-activated® IE-1 and pp65 proteins resulted in the activation of a broad range of CMV-reactive effector cells, including CD3 + CD4 + (Th), CD3 + CD8 + (CTL), CD3 - CD56 + (NK) and CD3 + CD56 + (NKT-like) cells. Accordingly, the optimized IFN-γ ELISpot assay revealed very high sensitivity (97%) in a cohort of 45 healthy donors, of which 32 were CMV IgG-seropositive. The combined use of T-activated® IE-1 and pp65 proteins for the stimulation of PBMC with the optimized IFN-γ ELISpot assay represents a highly standardized, valuable tool to monitor the functionality of CMV-specific CMI with great sensitivity and reliability.

Radiosensitivity of pimonidazole-unlabelled intratumour quiescent cell population to γ-rays, accelerated carbon ion beams and boron neutron capture reaction.

PubMed

Masunaga, S; Sakurai, Y; Tanaka, H; Hirayama, R; Matsumoto, Y; Uzawa, A; Suzuki, M; Kondo, N; Narabayashi, M; Maruhashi, A; Ono, K

2013-01-01

To detect the radiosensitivity of intratumour quiescent (Q) cells unlabelled with pimonidazole to accelerated carbon ion beams and the boron neutron capture reaction (BNCR). EL4 tumour-bearing C57BL/J mice received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all intratumour proliferating (P) cells. After the administration of pimonidazole, tumours were irradiated with γ-rays, accelerated carbon ion beams or reactor neutron beams with the prior administration of a (10)B-carrier. Responses of intratumour Q and total (P+Q) cell populations were assessed based on frequencies of micronucleation and apoptosis using immunofluorescence staining for BrdU. The response of pimonidazole-unlabelled tumour cells was assessed by means of apoptosis frequency using immunofluorescence staining for pimonidazole. Following γ-ray irradiation, the pimonidazole-unlabelled tumour cell fraction showed significantly enhanced radiosensitivity compared with the whole tumour cell fraction, more remarkably in the Q than total cell populations. However, a significantly greater decrease in radiosensitivity in the pimonidazole-unlabelled cell fraction, evaluated using a delayed assay or a decrease in radiation dose rate, was more clearly observed among the Q than total cells. These changes in radiosensitivity were suppressed following carbon ion beam and neutron beam-only irradiaton. In the BNCR, the use of a (10)B-carrier, especially L-para-boronophenylalanine-(10)B, enhanced the sensitivity of the pimonidazole-unlabelled cells more clearly in the Q than total cells. The radiosensitivity of the pimonidazole-unlabelled cell fraction depends on the quality of radiation delivered and characteristics of the (10)B-carrier used in the BNCR. The pimonidazole-unlabelled subfraction of Q tumour cells may be a critical target in tumour control.

A high-throughput in vitro ring assay for vasoactivity using magnetic 3D bioprinting

PubMed Central