flow cytometry chip: Topics by Science.gov

Sample records for flow cytometry chip

Mesenchymal Stem Cells for Vascular Target Discovery in Breast Cancer-Associated Angiogenesis

DTIC Science & Technology

2005-09-01

demonstrating this marker as demonstrated by flow cytometry . These GFP+ MSCs were subsequently analyzed for expression of commonly reported markers of...phenotypically and genotypically analyzed by flow cytometry and gene chip analysis, respectively. We have also shown that MSCs can then be stimulated to...positive MSCs retrieved by collagenase digestion of the Matrigel plug and sorted by flow cytometry . Sorting of these retrieved cells based on co-expression
Microfluidics and photonics for Bio-System-on-a-Chip: a review of advancements in technology towards a microfluidic flow cytometry chip.

PubMed

Godin, Jessica; Chen, Chun-Hao; Cho, Sung Hwan; Qiao, Wen; Tsai, Frank; Lo, Yu-Hwa

2008-10-01

Microfluidics and photonics come together to form a field commonly referred to as 'optofluidics'. Flow cytometry provides the field with a technology base from which both microfluidic and photonic components be developed and integrated into a useful device. This article reviews some of the more recent developments to familiarize a reader with the current state of the technologies and also highlights the requirements of the device and how researchers are working to meet these needs.
An optofluidic approach for gold nanoprobes based-cancer theranostics

NASA Astrophysics Data System (ADS)

Panwar, Nishtha; Song, Peiyi; Yang, Chengbin; Yong, Ken-Tye; Tjin, Swee Chuan

2017-02-01

Suppression of overexpressed gene mutations in cancer cells through RNA interference (RNAi) technique is a therapeutically effective modality for oncogene silencing. In general, transfection agent is needed for siRNA delivery. Also, it is a tedious and time consuming process to analyze the gene transfection using current conventional flow cytometry systems and commercially available transfection kits. Therefore, there are two urgent challenges that we need to address for understanding and real time monitoring the delivery of siRNA to cancer cells more effectively. One, nontoxic, biocompatible and stable non-viral transfection agents need to be developed and investigated for gene delivery in cancer cells. Two, new, portable optofluidic methods need to be engineered for determining the transfection efficiency of the nanoformulation in real time. First, we demonstrate the feasibility of using gold nanorods (AuNRs) as nanoprobes for the delivery of Interleukin-8 (IL-8) siRNA in a pancreatic cancer cell line- MiaPaCa-2. An optimum ratio of 10:1 for the AuNRs-siRNA nanoformulation required for efficient loading has been experimentally determined. Promising transfection rates (≈88%) of the nanoprobe-assisted gene delivery are quantified by flow cytometry and fluorescence imaging, which are higher than the commercial control, Oligofectamine. The excellent gene knockdown performance (over 81%) of the proposed model support in vivo trials for RNAi-based cancer theranostics. In addition to cancer theranostics, our nanoprobe combination can be also applied for disease outbreak monitoring like MERS. Second, we present an optical fiber-integrated microfluidic chip that utilizes simple hydrodynamic and optical setups for miniaturized on-chip flow cytometry. The chip provides a powerful and convenient tool to quantitatively determine the siRNA transfection into cancer cells without using bulky flow cytometer. These studies outline the role of AuNRs as potential non-viral gene delivery vehicles, and their suitability for microfluidics-based lab-on-chip flow cytometry applications.
Real-time Image Processing for Microscopy-based Label-free Imaging Flow Cytometry in a Microfluidic Chip.

PubMed

Heo, Young Jin; Lee, Donghyeon; Kang, Junsu; Lee, Keondo; Chung, Wan Kyun

2017-09-14

Imaging flow cytometry (IFC) is an emerging technology that acquires single-cell images at high-throughput for analysis of a cell population. Rich information that comes from high sensitivity and spatial resolution of a single-cell microscopic image is beneficial for single-cell analysis in various biological applications. In this paper, we present a fast image-processing pipeline (R-MOD: Real-time Moving Object Detector) based on deep learning for high-throughput microscopy-based label-free IFC in a microfluidic chip. The R-MOD pipeline acquires all single-cell images of cells in flow, and identifies the acquired images as a real-time process with minimum hardware that consists of a microscope and a high-speed camera. Experiments show that R-MOD has the fast and reliable accuracy (500 fps and 93.3% mAP), and is expected to be used as a powerful tool for biomedical and clinical applications.
Opto-fluidics based microscopy and flow cytometry on a cell phone for blood analysis.

PubMed

Zhu, Hongying; Ozcan, Aydogan

2015-01-01

Blood analysis is one of the most important clinical tests for medical diagnosis. Flow cytometry and optical microscopy are widely used techniques to perform blood analysis and therefore cost-effective translation of these technologies to resource limited settings is critical for various global health as well as telemedicine applications. In this chapter, we review our recent progress on the integration of imaging flow cytometry and fluorescent microscopy on a cell phone using compact, light-weight and cost-effective opto-fluidic attachments integrated onto the camera module of a smartphone. In our cell-phone based opto-fluidic imaging cytometry design, fluorescently labeled cells are delivered into the imaging area using a disposable micro-fluidic chip that is positioned above the existing camera unit of the cell phone. Battery powered light-emitting diodes (LEDs) are butt-coupled to the sides of this micro-fluidic chip without any lenses, which effectively acts as a multimode slab waveguide, where the excitation light is guided to excite the fluorescent targets within the micro-fluidic chip. Since the excitation light propagates perpendicular to the detection path, an inexpensive plastic absorption filter is able to reject most of the scattered light and create a decent dark-field background for fluorescent imaging. With this excitation geometry, the cell-phone camera can record fluorescent movies of the particles/cells as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the solution under test. With a similar opto-fluidic design, we have recently demonstrated imaging and automated counting of stationary blood cells (e.g., labeled white blood cells or unlabeled red blood cells) loaded within a disposable cell counting chamber. We tested the performance of this cell-phone based imaging cytometry and blood analysis platform by measuring the density of red and white blood cells as well as hemoglobin concentration in human blood samples, which showed a good match to our measurement results obtained using a commercially available hematology analyzer. Such a cell-phone enabled opto-fluidics microscopy, flow cytometry, and blood analysis platform could be especially useful for various telemedicine applications in remote and resource-limited settings.
Development of small and inexpensive digital data acquisition systems using a microcontroller-based approach†

PubMed Central

Naivar, Mark A.; Wilder, Mark E.; Habbersett, Robert C.; Woods, Travis A.; Sebba, David S.; Nolan, John P.; Graves, Steven W.

2014-01-01

Fully digital data acquisition systems for use in flow cytometry provide excellent flexibility and precision. Here, we demonstrate the development of a low cost, small, and low power digital flow cytometry data acquisition system using a single microcontroller chip with an integrated analog to digital converter (ADC). Our demonstration system uses a commercially available evaluation board making the system simple to integrate into a flow cytometer. We have evaluated this system using calibration microspheres analyzed on commercial, slow-flow, and CCD based flow cytometers. In our evaluations, our demonstration data system clearly resolves all eight peaks of a Rainbow microsphere set on both a slow-flow flow cytometer and a retrofitted BD FACScalibur, which indicates it has the sensitivity and resolution required for most flow cytometry applications. It is also capable of millisecond time resolution, full waveform collection, and selective triggering of data collection from a CCD camera. The capability of our demonstration system suggests that the use of microcontrollers for flow cytometry digital data-acquisition will be increasingly valuable for extending the life of older cytometers and provides a compelling data-system design approach for low-cost, portable flow cytometers. PMID:19852060
Development of small and inexpensive digital data acquisition systems using a microcontroller-based approach.

PubMed

Naivar, Mark A; Wilder, Mark E; Habbersett, Robert C; Woods, Travis A; Sebba, David S; Nolan, John P; Graves, Steven W

2009-12-01

Fully digital data acquisition systems for use in flow cytometry provide excellent flexibility and precision. Here, we demonstrate the development of a low cost, small, and low power digital flow cytometry data acquisition system using a single microcontroller chip with an integrated analog to digital converter (ADC). Our demonstration system uses a commercially available evaluation board making the system simple to integrate into a flow cytometer. We have evaluated this system using calibration microspheres analyzed on commercial, slow-flow, and CCD-based flow cytometers. In our evaluations, our demonstration data system clearly resolves all eight peaks of a Rainbow microsphere set on both a slow-flow flow cytometer and a retrofitted BD FACScalibur, which indicates it has the sensitivity and resolution required for most flow cytometry applications. It is also capable of millisecond time resolution, full waveform collection, and selective triggering of data collection from a CCD camera. The capability of our demonstration system suggests that the use of microcontrollers for flow cytometry digital data-acquisition will be increasingly valuable for extending the life of older cytometers and provides a compelling data-system design approach for low-cost, portable flow cytometers.
Flow cytometry as an improved method for the titration of Chlamydiaceae and other intracellular bacteria.

PubMed

Käser, T; Pasternak, J A; Hamonic, G; Rieder, M; Lai, K; Delgado-Ortega, M; Gerdts, V; Meurens, F

2016-05-01

Chlamydiaceae is a family of intracellular bacteria causing a range of diverse pathological outcomes. The most devastating human diseases are ocular infections with C. trachomatis leading to blindness and genital infections causing pelvic inflammatory disease with long-term sequelae including infertility and chronic pelvic pain. In order to enable the comparison of experiments between laboratories investigating host-chlamydia interactions, the infectious titer has to be determined. Titer determination of chlamydia is most commonly performed via microscopy of host cells infected with a serial dilution of chlamydia. However, other methods including fluorescent ELISpot (Fluorospot) and DNA Chip Scanning Technology have also been proposed to enumerate chlamydia-infected cells. For viruses, flow cytometry has been suggested as a superior alternative to standard titration methods. In this study we compared the use of flow cytometry with microscopy and Fluorospot for the titration of C. suis as a representative of other intracellular bacteria. Titer determination via Fluorospot was unreliable, while titration via microscopy led to a linear read-out range of 16 - 64 dilutions and moderate reproducibility with acceptable standard deviations within and between investigators. In contrast, flow cytometry had a vast linear read-out range of 1,024 dilutions and the lowest standard deviations given a basic training in these methods. In addition, flow cytometry was faster and material costs were lower compared to microscopy. Flow cytometry offers a fast, cheap, precise, and reproducible alternative for the titration of intracellular bacteria like C. suis. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.
Cytometer on a Chip

NASA Technical Reports Server (NTRS)

Fernandez, Salvador M.

2011-01-01

A cytometer now under development exploits spatial sorting of sampled cells on a microarray chip followed by use of grating-coupled surface-plasmon-resonance imaging (GCSPRI) to detect the sorted cells. This cytometer on a chip is a prototype of contemplated future miniature cytometers that would be suitable for rapidly identifying pathogens and other cells of interest in both field and laboratory applications and that would be attractive as alternatives to conventional flow cytometers. The basic principle of operation of a conventional flow cytometer requires fluorescent labeling of sampled cells, stringent optical alignment of a laser beam with a narrow orifice, and flow of the cells through the orifice, which is subject to clogging. In contrast, the principle of operation of the present cytometer on a chip does not require fluorescent labeling of cells, stringent optical alignment, or flow through a narrow orifice. The basic principle of operation of the cytometer on a chip also reduces the complexity, mass, and power of the associated laser and detection systems, relative to those needed in conventional flow cytometry. Instead of making cells flow in single file through a narrow flow orifice for sequential interrogation as in conventional flow cytometry, a liquid containing suspended sampled cells is made to flow over the front surface of a microarray chip on which there are many capture spots. Each capture spot is coated with a thin (approximately 50-nm) layer of gold that is, in turn, coated with antibodies that bind to cell-surface molecules characteristic of one the cell species of interest. The multiplicity of capture spots makes it possible to perform rapid, massively parallel analysis of a large cell population. The binding of cells to each capture spot gives rise to a minute change in the index of refraction at the surface of the chip. This change in the index of refraction is what is sensed in GCSPRI, as described briefly below. The identities of the various species in a sample of cells is spatially encoded in the chip by the pattern of capture spots. The number of cells of a particular species is determined from the magnitude of the GCSPRI signal from that spot. GCSPRI as used here can be summarized as follows: The cytometer chip is fabricated with a diffraction grating on its front surface. The chip is illuminated with a light emitting diode (LED) from the front. By proper choice of grating parameters and of the wavelength and the angle of incidence of a laser beam, laser light can be made to be coupled into an electromagnetic mode that resonates with surface plasmons and thus couples light into surface plasmons. Coupling of light into a surface plasmon at a given location reduces the amount of incident light reflected from that location. A change in the index of refraction at the surface of a capture spot gives rise to a change in the resonance condition. Depending on the specific design, the change in the index of refraction could manifest itself as a brightening or darkening, a change in the wavelength needed to excite the plasmon at a given angle of incidence, or a change in the angle of incidence needed to excite the plasmon at a given wavelength. Whereas a multiwavelength laser system with multichannel detection would be needed to detect multiple species in conventional flow cytometry, it suffices to use an LED and a single detector channel in the GCSPRI approach: this contributes significantly to reductions in cost, complexity, size, mass, and power. GCSPRI cytometer chips could be made of plastic and could be mass-produced cheaply by use of molding and other methods adopted from the manufacture of digital video disks. These methods are amenable to a high degree of miniaturization: such additional features as fluidic channels, reaction chambers, and fluid-coupling ports could readily be incorporated into the chips, without incurring substantial additional costs.
Cytometer on a Chip

NASA Technical Reports Server (NTRS)

Fernandez, Salvador M.

2011-01-01

A cytometer now under development exploits spatial sorting of sampled cells on a microarray chip followed by use of grating-coupled surface-plasmon-resonance imaging (GCSPRI) to detect the sorted cells. This cytometer on a chip is a prototype of contemplated future miniature cytometers that would be suitable for rapidly identifying pathogens and other cells of interest in both field and laboratory applications and that would be attractive as alternatives to conventional flow cytometers. The basic principle of operation of a conventional flow cytometer requires fluorescent labeling of sampled cells, stringent optical alignment of a laser beam with a narrow orifice, and flow of the cells through the orifice, which is subject to clogging. In contrast, the principle of operation of the present cytometer on a chip does not require fluorescent labeling of cells, stringent optical alignment, or flow through a narrow orifice. The basic principle of operation of the cytometer on a chip also reduces the complexity, mass, and power of the associated laser and detection systems, relative to those needed in conventional flow cytometry. Instead of making cells flow in single file through a narrow flow orifice for sequential interrogation as in conventional flow cytometry, a liquid containing suspended sampled cells is made to flow over the front surface of a microarray chip on which there are many capture spots. Each capture spot is coated with a thin (.50-nm) layer of gold that is, in turn, coated with antibodies that bind to cell-surface molecules characteristic of the cell species of interest. The multiplicity of capture spots makes it possible to perform rapid, massively parallel analysis of a large cell population. The binding of cells to each capture spot gives rise to a minute change in the index of refraction at the surface of the chip. This change in the index of refraction is what is sensed in GCSPRI, as described briefly below. The identities of the various species in a sample of cells is spatially encoded in the chip by the pattern of capture spots. The number of cells of a particular species is determined from the magnitude of the GCSPRI signal from that spot. GCSPRI as used here can be summarized as follows: The cytometer chip is fabricated with a diffraction grating on its front surface. The chip is illuminated with a light emitting diode (LED) from the front. By proper choice of grating parameters and of the wavelength and the angle of incidence of a laser beam, laser light can be made to be coupled into an electromagnetic mode that resonates with surface plasmons and thus couples light into surface plasmons. Coupling of light into a surface plasmon at a given location reduces the amount of incident light reflected from that location. A change in the index of refraction at the surface of a capture spot gives rise to a change in the resonance condition. Depending on the specific design, the change in the index of refraction could manifest itself as a brightening or darkening, a change in the wavelength needed to excite the plasmon at a given angle of incidence, or a change in the angle of incidence needed to excite the plasmon at a given wavelength. Whereas a multiwavelength laser system with multichannel detection would be needed to detect multiple species in conventional flow cytometry, it suffices to use an LED and a single detector channel in the GCSPRI approach: this contributes significantly to reductions in cost, complexity, size, mass, and power. GCSPRI cytometer chips could be made of plastic and could be mass-produced cheaply by use of molding and other methods adopted from the manufacture of digital video disks. These methods are amenable to a high degree of miniaturization: such additional features as fluidic channels, reaction chambers, and fluid-coupling ports could readily be incorporated into the chips, without incurring substantial additional costs.
Multi-channel imaging cytometry with a single detector

NASA Astrophysics Data System (ADS)

Locknar, Sarah; Barton, John; Entwistle, Mark; Carver, Gary; Johnson, Robert

2018-02-01

Multi-channel microscopy and multi-channel flow cytometry generate high bit data streams. Multiple channels (both spectral and spatial) are important in diagnosing diseased tissue and identifying individual cells. Omega Optical has developed techniques for mapping multiple channels into the time domain for detection by a single high gain, high bandwidth detector. This approach is based on pulsed laser excitation and a serial array of optical fibers coated with spectral reflectors such that up to 15 wavelength bins are sequentially detected by a single-element detector within 2.5 μs. Our multichannel microscopy system uses firmware running on dedicated DSP and FPGA chips to synchronize the laser, scanning mirrors, and sampling clock. The signals are digitized by an NI board into 14 bits at 60MHz - allowing for 232 by 174 pixel fields in up to 15 channels with 10x over sampling. Our multi-channel imaging cytometry design adds channels for forward scattering and back scattering to the fluorescence spectral channels. All channels are detected within the 2.5 μs - which is compatible with fast cytometry. Going forward, we plan to digitize at 16 bits with an A-toD chip attached to a custom board. Processing these digital signals in custom firmware would allow an on-board graphics processing unit to display imaging flow cytometry data over configurable scanning line lengths. The scatter channels can be used to trigger data buffering when a cell is present in the beam. This approach enables a low cost mechanically robust imaging cytometer.
Rise of the micromachines: microfluidics and the future of cytometry.

PubMed

Wlodkowic, Donald; Darzynkiewicz, Zbigniew

2011-01-01

The past decade has brought many innovations to the field of flow and image-based cytometry. These advancements can be seen in the current miniaturization trends and simplification of analytical components found in the conventional flow cytometers. On the other hand, the maturation of multispectral imaging cytometry in flow imaging and the slide-based laser scanning cytometers offers great hopes for improved data quality and throughput while proving new vistas for the multiparameter, real-time analysis of cells and tissues. Importantly, however, cytometry remains a viable and very dynamic field of modern engineering. Technological milestones and innovations made over the last couple of years are bringing the next generation of cytometers out of centralized core facilities while making it much more affordable and user friendly. In this context, the development of microfluidic, lab-on-a-chip (LOC) technologies is one of the most innovative and cost-effective approaches toward the advancement of cytometry. LOC devices promise new functionalities that can overcome current limitations while at the same time promise greatly reduced costs, increased sensitivity, and ultra high throughputs. We can expect that the current pace in the development of novel microfabricated cytometric systems will open up groundbreaking vistas for the field of cytometry, lead to the renaissance of cytometric techniques and most importantly greatly support the wider availability of these enabling bioanalytical technologies. Copyright © 2011 Elsevier Inc. All rights reserved.
Rise of the Micromachines: Microfluidics and the Future of Cytometry

PubMed Central

Wlodkowic, Donald; Darzynkiewicz, Zbigniew

2011-01-01

The past decade has brought many innovations to the field of flow and image-based cytometry. These advancements can be seen in the current miniaturization trends and simplification of analytical components found in the conventional flow cytometers. On the other hand, the maturation of multispectral imaging cytometry in flow imaging and the slide-based laser scanning cytometers offers great hopes for improved data quality and throughput while proving new vistas for the multiparameter, real-time analysis of cells and tissues. Importantly, however, cytometry remains a viable and very dynamic field of modern engineering. Technological milestones and innovations made over the last couple of years are bringing the next generation of cytometers out of centralized core facilities while making it much more affordable and user friendly. In this context, the development of microfluidic, lab-on-a-chip (LOC) technologies is one of the most innovative and cost-effective approaches toward the advancement of cytometry. LOC devices promise new functionalities that can overcome current limitations while at the same time promise greatly reduced costs, increased sensitivity, and ultra high throughputs. We can expect that the current pace in the development of novel microfabricated cytometric systems will open up groundbreaking vistas for the field of cytometry, lead to the renaissance of cytometric techniques and most importantly greatly support the wider availability of these enabling bioanalytical technologies. PMID:21704837
Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

PubMed Central

Zhu, Hongying; Ozcan, Aydogan

2013-01-01

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 μm over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water. PMID:23603893
Wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone.

PubMed

Zhu, Hongying; Ozcan, Aydogan

2013-04-11

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. - 10 μm over a very large field-of-view of - 81 mm(2). This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.
Femtosecond laser fabrication of fiber based optofluidic platform for flow cytometry applications

NASA Astrophysics Data System (ADS)

Serhatlioglu, Murat; Elbuken, Caglar; Ortac, Bulend; Solmaz, Mehmet E.

2017-02-01

Miniaturized optofluidic platforms play an important role in bio-analysis, detection and diagnostic applications. The advantages of such miniaturized devices are extremely low sample requirement, low cost development and rapid analysis capabilities. Fused silica is advantageous for optofluidic systems due to properties such as being chemically inert, mechanically stable, and optically transparent to a wide spectrum of light. As a three dimensional manufacturing method, femtosecond laser scanning followed by chemical etching shows great potential to fabricate glass based optofluidic chips. In this study, we demonstrate fabrication of all-fiber based, optofluidic flow cytometer in fused silica glass by femtosecond laser machining. 3D particle focusing was achieved through a straightforward planar chip design with two separately fabricated fused silica glass slides thermally bonded together. Bioparticles in a fluid stream encounter with optical interrogation region specifically designed to allocate 405nm single mode fiber laser source and two multi-mode collection fibers for forward scattering (FSC) and side scattering (SSC) signals detection. Detected signal data collected with oscilloscope and post processed with MATLAB script file. We were able to count number of events over 4000events/sec, and achieve size distribution for 5.95μm monodisperse polystyrene beads using FSC and SSC signals. Our platform shows promise for optical and fluidic miniaturization of flow cytometry systems.
Lab-on-Chip Cytometry Based on Magnetoresistive Sensors for Bacteria Detection in Milk

PubMed Central

Fernandes, Ana C.; Duarte, Carla M.; Cardoso, Filipe A.; Bexiga, Ricardo.; Cardoso, Susana.; Freitas, Paulo P.

2014-01-01

Flow cytometers have been optimized for use in portable platforms, where cell separation, identification and counting can be achieved in a compact and modular format. This feature can be combined with magnetic detection, where magnetoresistive sensors can be integrated within microfluidic channels to detect magnetically labelled cells. This work describes a platform for in-flow detection of magnetically labelled cells with a magneto-resistive based cell cytometer. In particular, we present an example for the validation of the platform as a magnetic counter that identifies and quantifies Streptococcus agalactiae in milk. PMID:25196163
Lab-on-chip cytometry based on magnetoresistive sensors for bacteria detection in milk.

PubMed

Fernandes, Ana C; Duarte, Carla M; Cardoso, Filipe A; Bexiga, Ricardo; Cardoso, Susana; Freitas, Paulo P

2014-08-21

Flow cytometers have been optimized for use in portable platforms, where cell separation, identification and counting can be achieved in a compact and modular format. This feature can be combined with magnetic detection, where magnetoresistive sensors can be integrated within microfluidic channels to detect magnetically labelled cells. This work describes a platform for in-flow detection of magnetically labelled cells with a magneto-resistive based cell cytometer. In particular, we present an example for the validation of the platform as a magnetic counter that identifies and quantifies Streptococcus agalactiae in milk.
Chip-Based Dynamic Real-Time Quantification of Drug-Induced Cytotoxicity in Human Tumor Cells

PubMed Central

Wlodkowic, Donald; Skommer, Joanna; McGuinness, Dagmara; Faley, Shannon; Kolch, Walter; Darzynkiewicz, Zbigniew; Cooper, Jonathan M.

2013-01-01

Cell cytotoxicity tests are among the most common bioassays using flow cytometry and fluorescence imaging analysis. The permeability of plasma membranes to charged fluorescent probes serves, in these assays, as a marker distinguishing live from dead cells. Since it is generally assumed that probes, such as propidium iodide (PI) or 7-amino-actinomycin D (7-AAD), are themselves cytotoxic, they are currently generally used only as the end-point markers of assays for live versus dead cells. In the current study, we provide novel insights into potential applications of these classical plasma membrane integrity markers in the dynamic tracking of drug-induced cytotoxicity. We show that treatment of a number of different human tumor cell lines in cultures for up to 72 h with the PI, 7-AAD, SYTOX Green (SY-G), SYTOX Red (SYR), TO-PRO, and YO-PRO had no effect on cell viability assessed by the integrity of plasma membrane, cell cycle progression, and rate of proliferation. We subsequently explore the potential of dynamic labeling with these markers in real-time analysis, by comparing results from both conventional cytometry and microfluidic chips. Considering the simplicity of the staining protocols and their low cost combined with the potential for real-time data collection, we show how that real-time fluorescent imaging and Lab-on-a-Chip platforms have the potential to be used for automated drug screening routines. PMID:19572560
Detection of sepsis in patient blood samples using CD64 expression in a microfluidic cell separation device.

PubMed

Zhang, Ye; Li, Wenjie; Zhou, Yun; Johnson, Amanda; Venable, Amanda; Hassan, Ahmed; Griswold, John; Pappas, Dimitri

2017-12-18

A microfluidic affinity separation device was developed for the detection of sepsis in critical care patients. An affinity capture method was developed to capture cells based on changes in CD64 expression in a single, simple microfluidic chip for sepsis detection. Both sepsis patient samples and a laboratory CD64+ expression model were used to validate the microfluidic assay. Flow cytometry analysis showed that the chip cell capture had a linear relationship with CD64 expression in laboratory models. The Sepsis Chip detected an increase in upregulated neutrophil-like cells when the upregulated cell population is as low as 10% of total cells spiked into commercially available aseptic blood samples. In a proof of concept study, blood samples obtained from sepsis patients within 24 hours of diagnosis were tested on the chip to further validate its performance. On-chip CD64+ cell capture from 10 patient samples (619 ± 340 cells per chip) was significantly different from control samples (32 ± 11 cells per chip) and healthy volunteer samples (228 ± 95 cells per chip). In addition, the on-chip cell capture has a linear relationship with CD64 expression indicating our approach can be used to measure CD64 expression based on total cell capture on Sepsis Chip. Our method has proven to be sensitive, accurate, rapid, and cost-effective. Therefore, this device is a promising detection platform for neutrophil activation and sepsis diagnosis.

Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA

PubMed Central

Tak For Yu, Zeta; Guan, Huijiao; Ki Cheung, Mei; McHugh, Walker M.; Cornell, Timothy T.; Shanley, Thomas P.; Kurabayashi, Katsuo; Fu, Jianping

2015-01-01

Immunoassays represent one of the most popular analytical methods for detection and quantification of biomolecules. However, conventional immunoassays such as ELISA and flow cytometry, even though providing high sensitivity and specificity and multiplexing capability, can be labor-intensive and prone to human error, making them unsuitable for standardized clinical diagnoses. Using a commercialized no-wash, homogeneous immunoassay technology (‘AlphaLISA’) in conjunction with integrated microfluidics, herein we developed a microfluidic immunoassay chip capable of rapid, automated, parallel immunoassays of microliter quantities of samples. Operation of the microfluidic immunoassay chip entailed rapid mixing and conjugation of AlphaLISA components with target analytes before quantitative imaging for analyte detections in up to eight samples simultaneously. Aspects such as fluid handling and operation, surface passivation, imaging uniformity, and detection sensitivity of the microfluidic immunoassay chip using AlphaLISA were investigated. The microfluidic immunoassay chip could detect one target analyte simultaneously for up to eight samples in 45 min with a limit of detection down to 10 pg mL−1. The microfluidic immunoassay chip was further utilized for functional immunophenotyping to examine cytokine secretion from human immune cells stimulated ex vivo. Together, the microfluidic immunoassay chip provides a promising high-throughput, high-content platform for rapid, automated, parallel quantitative immunosensing applications. PMID:26074253
Development of wide-angle 2D light scattering static cytometry

NASA Astrophysics Data System (ADS)

Xie, Linyan; Liu, Qiao; Shao, Changshun; Su, Xuantao

2016-10-01

We have recently developed a 2D light scattering static cytometer for cellular analysis in a label-free manner, which measures side scatter (SSC) light in the polar angular range from 79 to 101 degrees. Compared with conventional flow cytometry, our cytometric technique requires no fluorescent labeling of the cells, and static cytometry measurements can be performed without flow control. In this paper we present an improved label-free static cytometer that can obtain 2D light scattering patterns in a wider angular range. By illuminating the static microspheres on chip with a scanning optical fiber, wide-angle 2D light scattering patterns of single standard microspheres with a mean diameter of 3.87 μm are obtained. The 2D patterns of 3.87 μm microspheres contain both large-angle forward scatter (FSC) and SSC light in the polar angular range from 40 to 100 degrees, approximately. Experimental 2D patterns of 3.87 μm microspheres are in good agreement with Mie theory simulated ones. The wide-angle light scattering measurements may provide a better resolution for particle analysis as compared with the SSC measurements. Two dimensional light scattering patterns of HL-60 human acute leukemia cells are obtained by using our static cytometer. Compared with SSC 2D light scattering patterns, wide-angle 2D patterns contain richer information of the HL-60 cells. The obtaining of 2D light scattering patterns in a wide angular range could help to enhance the capabilities of our label-free static cytometry for cell analysis.
miRNA detection at single-cell resolution using microfluidic LNA flow-FISH

DOE PAGES

Wu, Meiye; Piccini, Matthew Ernest; Koh, Chung -Yan; ...

2014-08-20

Flow cytometry in combination with fluorescent in situ hybridization (flow-FISH) is a powerful technique that can be utilized to rapidly detect nucleic acids at single-cell resolution without the need for homogenization or nucleic acid extraction. Here, we describe a microfluidic-based method which enables the detection of microRNAs or miRNAs in single intact cells by flow-FISH using locked nucleic acid (LNA)-containing probes. Our method can be applied to all RNA species including mRNA and small noncoding RNA and is suitable for multiplexing with protein immunostaining in the same cell. For demonstration of our method, this chapter details the detection of miR155more » and CD69 protein in PMA and ionomycin-stimulated Jurkat cells. Here, we also include instructions on how to set up a microfluidic chip sample preparation station to prepare cells for imaging and analysis on a commercial flow cytometer or a custom-built micro-flow cytometer.« less
Microfluidic Impedance Flow Cytometry Enabling High-Throughput Single-Cell Electrical Property Characterization

PubMed Central

Chen, Jian; Xue, Chengcheng; Zhao, Yang; Chen, Deyong; Wu, Min-Hsien; Wang, Junbo

2015-01-01

This article reviews recent developments in microfluidic impedance flow cytometry for high-throughput electrical property characterization of single cells. Four major perspectives of microfluidic impedance flow cytometry for single-cell characterization are included in this review: (1) early developments of microfluidic impedance flow cytometry for single-cell electrical property characterization; (2) microfluidic impedance flow cytometry with enhanced sensitivity; (3) microfluidic impedance and optical flow cytometry for single-cell analysis and (4) integrated point of care system based on microfluidic impedance flow cytometry. We examine the advantages and limitations of each technique and discuss future research opportunities from the perspectives of both technical innovation and clinical applications. PMID:25938973
Coplanar electrode microfluidic chip enabling accurate sheathless impedance cytometry.

PubMed

De Ninno, Adele; Errico, Vito; Bertani, Francesca Romana; Businaro, Luca; Bisegna, Paolo; Caselli, Federica

2017-03-14

Microfluidic impedance cytometry offers a simple non-invasive method for single-cell analysis. Coplanar electrode chips are especially attractive due to ease of fabrication, yielding miniaturized, reproducible, and ultimately low-cost devices. However, their accuracy is challenged by the dependence of the measured signal on particle trajectory within the interrogation volume, that manifests itself as an error in the estimated particle size, unless any kind of focusing system is used. In this paper, we present an original five-electrode coplanar chip enabling accurate particle sizing without the need for focusing. The chip layout is designed to provide a peculiar signal shape from which a new metric correlating with particle trajectory can be extracted. This metric is exploited to correct the estimated size of polystyrene beads of 5.2, 6 and 7 μm nominal diameter, reaching coefficient of variations lower than the manufacturers' quoted values. The potential impact of the proposed device in the field of life sciences is demonstrated with an application to Saccharomyces cerevisiae yeast.
DOE Office of Scientific and Technical Information (OSTI.GOV)

Srivastava, Nimisha; Singh, Anup K

Microfluidic devices and methods for flow cytometry are described. In described examples, various sample handling and preparation steps may be carried out within a same microfluidic device as flow cytometry steps. A combination of imaging and flow cytometry is described. In some examples, spiral microchannels serve as incubation chambers. Examples of automated sample handling and flow cytometry are described.
Flow cytometry shows added value in diagnosing lymphoma in brain biopsies.

PubMed

van der Meulen, Matthijs; Bromberg, Jacoline E C; Lam, King H; Dammers, Ruben; Langerak, Anton W; Doorduijn, Jeanette K; Kros, Johan M; van den Bent, Martin J; van der Velden, Vincent H J

2018-05-10

To assess the sensitivity, specificity and turnaround time of flow cytometric analysis on brain biopsies compared to histology plus immunohistochemistry analysis in tumors with clinical suspicion of lymphoma. All brain biopsies performed between 2010 and 2015 at our institution and analyzed by both immunohistochemistry and flow cytometry were included in this retrospective study. Immunohistochemistry was considered the gold standard. In a total of 77 biopsies from 71 patients, 49 lymphomas were diagnosed by immunohistochemistry, flow cytometry results were concordant in 71 biopsies (92,2%). We found a specificity and sensitivity of flow cytometry of 100% and 87,8%, respectively. The time between the biopsy and reporting the result (turnaround time) was significantly shorter for flow cytometry, compared to immunohistochemistry (median: 1 versus 5 days). Flow cytometry has a high specificity and can confirm the diagnosis of a lymphoma significantly faster than immunohistochemistry. This allows for rapid initiation of treatment in this highly aggressive tumor. However, since its sensitivity is less than 100%, we recommend to perform histology plus immunohistochemistry in parallel to flow cytometry. This article is protected by copyright. All rights reserved. © 2018 International Clinical Cytometry Society.
Maintenance of head and neck tumor on-chip: gateway to personalized treatment?

PubMed Central

Bower, Ruth; Green, Victoria L; Kuvshinova, Elena; Kuvshinov, Dmitriy; Karsai, Laszlo; Crank, Stephen T; Stafford, Nicholas D; Greenman, John

2017-01-01

Aim: Head and neck squamous cell carcinomas (HNSCC) are solid tumors with low overall survival (40–60%). In a move toward personalized medicine, maintenance of tumor biopsies in microfluidic tissue culture devices is being developed. Methodology/results: HNSCC (n = 15) was dissected (5–10 mg) and either analyzed immediately or cultured in a microfluidic device (37°C) for 48 h. No difference was observed in morphology between pre- and postculture specimens. Dissociated samples were analyzed using trypan blue exclusion (viability), propidium iodide flow cytometry (death) and MTS assay (proliferation) with no significant difference observed highlighting tissue maintenance. Computational fluid dynamics showed laminar flow within the system. Conclusion: The microfluidic culture system successfully maintained HNSCC for 48 h, the culture system will allow testing of different treatment modalities with response monitoring. PMID:28670466
Impedance Flow Cytometry as a Tool to Analyze Microspore and Pollen Quality.

PubMed

Heidmann, Iris; Di Berardino, Marco

2017-01-01

Analyzing pollen quality in an efficient and reliable manner is of great importance to the industries involved in seed and fruit production, plant breeding, and plant research. Pollen quality parameters, viability and germination capacity, are analyzed by various staining methods or by in vitro germination assays, respectively. These methods are time-consuming, species-dependent, and require a lab environment. Furthermore, the obtained viability data are often poorly related to in vivo pollen germination and seed set. Here, we describe a quick, label-free method to analyze pollen using microfluidic chips inserted into an impedance flow cytometer (IFC). Using this approach, pollen quality parameters are determined by a single measurement in a species-independent manner. The advantage of this protocol is that pollen viability and germination can be analyzed quickly by a reliable and standardized method.
Technical advances in flow cytometry-based diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria

PubMed Central

Correia, Rodolfo Patussi; Bento, Laiz Cameirão; Bortolucci, Ana Carolina Apelle; Alexandre, Anderson Marega; Vaz, Andressa da Costa; Schimidell, Daniela; Pedro, Eduardo de Carvalho; Perin, Fabricio Simões; Nozawa, Sonia Tsukasa; Mendes, Cláudio Ernesto Albers; Barroso, Rodrigo de Souza; Bacal, Nydia Strachman

2016-01-01

ABSTRACT Objective: To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols. Methods: A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria. Results: Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection. Conclusion: High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones. PMID:27759825
Dissecting the Role of IGFBP-2 in Development of Acute Myeloid Leukemia

DTIC Science & Technology

2011-06-01

surface proteins on freshly isolated and cultured cells, as determined by flow cytometry ... Surface Immune Molecules on Phenotypic HSCs during Culture (A and B) A summary of the result of flow cytometry analysis of surface expression of indicated...from the distant implanted tumor were counted by flow cytometry analysis. The flow cytometry result was confirmed by counting GFP+ surface foci of
Flow cytometry: basic principles and applications.

PubMed

Adan, Aysun; Alizada, Günel; Kiraz, Yağmur; Baran, Yusuf; Nalbant, Ayten

2017-03-01

Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a single cell such as size and granularity simultaneously as the cell flows in suspension through a measuring device. Its working depends on the light scattering features of the cells under investigation, which may be derived from dyes or monoclonal antibodies targeting either extracellular molecules located on the surface or intracellular molecules inside the cell. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This review covers the general principles and selected applications of flow cytometry such as immunophenotyping of peripheral blood cells, analysis of apoptosis and detection of cytokines. Additionally, this report provides a basic understanding of flow cytometry technology essential for all users as well as the methods used to analyze and interpret the data. Moreover, recent progresses in flow cytometry have been discussed in order to give an opinion about the future importance of this technology.
Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus

PubMed Central

Gustafsson, Erika; Haas, Pieter-Jan; Walse, Björn; Hijnen, Marcel; Furebring, Christina; Ohlin, Mats; van Strijp, Jos AG; van Kessel, Kok PM

2009-01-01

Background The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity. Results In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31–121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding. Conclusion Conformational epitopes recognized by human antibodies have been mapped on the CHIPS surface and amino acid residues involved in both antibody and C5aR interaction could be defined. This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG. PMID:19284584
A smartphone controlled handheld microfluidic liquid handling system.

PubMed

Li, Baichen; Li, Lin; Guan, Allan; Dong, Quan; Ruan, Kangcheng; Hu, Ronggui; Li, Zhenyu

2014-10-21

Microfluidics and lab-on-a-chip technologies have made it possible to manipulate small volume liquids with unprecedented resolution, automation and integration. However, most current microfluidic systems still rely on bulky off-chip infrastructures such as compressed pressure sources, syringe pumps and computers to achieve complex liquid manipulation functions. Here, we present a handheld automated microfluidic liquid handling system controlled by a smartphone, which is enabled by combining elastomeric on-chip valves and a compact pneumatic system. As a demonstration, we show that the system can automatically perform all the liquid handling steps of a bead-based HIV1 p24 sandwich immunoassay on a multi-layer PDMS chip without any human intervention. The footprint of the system is 6 × 10.5 × 16.5 cm, and the total weight is 829 g including battery. Powered by a 12.8 V 1500 mAh Li battery, the system consumed 2.2 W on average during the immunoassay and lasted for 8.7 h. This handheld microfluidic liquid handling platform is generally applicable to many biochemical and cell-based assays requiring complex liquid manipulation and sample preparation steps such as FISH, PCR, flow cytometry and nucleic acid sequencing. In particular, the integration of this technology with read-out biosensors may help enable the realization of the long-sought Tricorder-like handheld in vitro diagnostic (IVD) systems.
Development of sugar chain-binding single-chain variable fragment antibody to adult T-cell leukemia cells using glyco-nanotechnology and phage display method.

PubMed

Muchima, Kaname; Todaka, Taro; Shinchi, Hiroyuki; Sato, Ayaka; Tazoe, Arisa; Aramaki, Rikiya; Kakitsubata, Yuhei; Yokoyama, Risa; Arima, Naomichi; Baba, Masanori; Wakao, Masahiro; Ito, Yuji; Suda, Yasuo

2018-04-01

Adult T-cell leukemia (ATL) is an intractable blood cancer caused by the infection of human T-cell leukemia virus type-1, and effective medical treatment is required. It is known that the structure and expression levels of cell surface sugar chains vary depending on cell states such as inflammation and cancer. Thus, it is expected that the antibody specific for ATL cell surface sugar chain would be an effective diagnostic tool and a strong candidate for the development of an anti-ATL drug. Here, we developed a stable sugar chain-binding single-chain variable fragment antibody (scFv) that can bind to ATL cells using a fibre-type Sugar Chip and phage display method. The fiber-type Sugar Chips were prepared using O-glycans released from ATL cell lines. The scFv-displaying phages derived from human B cells (diversity: 1.04 × 108) were then screened using the fiber-type Sugar Chips, and an O-glycan-binding scFv was obtained. The flow cytometry analysis revealed that the scFv predominantly bound to ATL cell lines. The sugar chain-binding properties of the scFv was evaluated by array-type Sugar Chip immobilized with a library of synthetic glycosaminoglycan disaccharide structures. Highly sulphated disaccharide structures were found to have high affinity to scFv.
An active, collaborative approach to learning skills in flow cytometry.

PubMed

Fuller, Kathryn; Linden, Matthew D; Lee-Pullen, Tracey; Fragall, Clayton; Erber, Wendy N; Röhrig, Kimberley J

2016-06-01

Advances in science education research have the potential to improve the way students learn to perform scientific interpretations and understand science concepts. We developed active, collaborative activities to teach skills in manipulating flow cytometry data using FlowJo software. Undergraduate students were given compensated clinical flow cytometry listmode output (FCS) files and asked to design a gating strategy to diagnose patients with different hematological malignancies on the basis of their immunophenotype. A separate cohort of research trainees was given uncompensated data files on which they performed their own compensation, calculated the antibody staining index, designed a sequential gating strategy, and quantified rare immune cell subsets. Student engagement, confidence, and perceptions of flow cytometry were assessed using a survey. Competency against the learning outcomes was assessed by asking students to undertake tasks that required understanding of flow cytometry dot plot data and gating sequences. The active, collaborative approach allowed students to achieve learning outcomes not previously possible with traditional teaching formats, for example, having students design their own gating strategy, without forgoing essential outcomes such as the interpretation of dot plots. In undergraduate students, favorable perceptions of flow cytometry as a field and as a potential career choice were correlated with student confidence but not the ability to perform flow cytometry data analysis. We demonstrate that this new pedagogical approach to teaching flow cytometry is beneficial for student understanding and interpretation of complex concepts. It should be considered as a useful new method for incorporating complex data analysis tasks such as flow cytometry into curricula. Copyright © 2016 The American Physiological Society.
Flow cytometric characterization of cerebrospinal fluid cells.

PubMed

de Graaf, Marieke T; de Jongste, Arjen H C; Kraan, Jaco; Boonstra, Joke G; Sillevis Smitt, Peter A E; Gratama, Jan W

2011-09-01

Flow cytometry facilitates the detection of a large spectrum of cellular characteristics on a per cell basis, determination of absolute cell numbers and detection of rare events with high sensitivity and specificity. White blood cell (WBC) counts in cerebrospinal fluid (CSF) are important for the diagnosis of many neurological disorders. WBC counting and differential can be performed by microscopy, hematology analyzers, or flow cytometry. Flow cytometry of CSF is increasingly being considered as the method of choice in patients suspected of leptomeningeal localization of hematological malignancies. Additionally, in several neuroinflammatory diseases such as multiple sclerosis and paraneoplastic neurological syndromes, flow cytometry is commonly performed to obtain insight into the immunopathogenesis of these diseases. Technically, the low cellularity of CSF samples, combined with the rapidly declining WBC viability, makes CSF flow cytometry challenging. Comparison of flow cytometry with microscopic and molecular techniques shows that each technique has its own advantages and is ideally combined. We expect that increasing the number of flow cytometric parameters that can be simultaneously studied within one sample, will further refine the information on CSF cell subsets in low-cellular CSF samples and enable to define cell populations more accurately. Copyright © 2011 International Clinical Cytometry Society.
Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods.

PubMed

Øbro, Nina F; Ryder, Lars P; Madsen, Hans O; Andersen, Mette K; Lausen, Birgitte; Hasle, Henrik; Schmiegelow, Kjeld; Marquart, Hanne V

2012-01-01

Reduction in minimal residual disease, measured by real-time quantitative PCR or flow cytometry, predicts prognosis in childhood B-cell precursor acute lymphoblastic leukemia. We explored whether cells reported as minimal residual disease by flow cytometry represent the malignant clone harboring clone-specific genomic markers (53 follow-up bone marrow samples from 28 children with B-cell precursor acute lymphoblastic leukemia). Cell populations (presumed leukemic and non-leukemic) were flow-sorted during standard flow cytometry-based minimal residual disease monitoring and explored by PCR and/or fluorescence in situ hybridization. We found good concordance between flow cytometry and genomic analyses in the individual flow-sorted leukemic (93% true positive) and normal (93% true negative) cell populations. Four cases with discrepant results had plausible explanations (e.g. partly informative immunophenotype and antigen modulation) that highlight important methodological pitfalls. These findings demonstrate that with sufficient experience, flow cytometry is reliable for minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia, although rare cases require supplementary PCR-based monitoring.
Topically Delivered Adipose Derived Stem Cells Show an Activated-Fibroblast Phenotype and Enhance Granulation Tissue Formation in Skin Wounds

DTIC Science & Technology

2013-01-31

have similar surface markers . We found that topically delivered ASCs are engrafted and proliferate in the wounds. We showed that transplanted ASCs...Material Command (W81XWH-10-2-0054). Flow cytometry was supported by the Northwestern University Flow Cytometry Facility and a Cancer Center Support...blasticidin. GFP expressing cells were further selected by flow cytometry using the Northwestern University Flow Cytometry Facility. Treatment of MSCs
Flexible planar microfluidic chip employing a light emitting diode and a PIN-photodiode for portable flow cytometers.

PubMed

Kettlitz, Siegfried W; Valouch, Sebastian; Sittel, Wiebke; Lemmer, Uli

2012-01-07

Detection of fluorescence particles is a key method of flow cytometry. We evaluate the performance of a design for a microfluidic fluorescence particle detection device. Due to the planar design with low layer thicknesses, we avoid optical components such as lenses or dichroic mirrors and substitute them with a shadow mask and colored film filters. A commercially available LED is used as the light source and a PIN-photodiode as detector. This design approach reduces component cost and power consumption and enables supplying the device with power from a standard USB port. From evaluation of this design, we obtain a maximum particle detection frequency of up to 600 particles per second at a sensitivity of better than 4.7 × 10(5) MESF (molecules of equivalent soluble fluorochrome) measured with particles for FITC sensitivity calibration. Lowering the flow rate increases the instrument sensitivity by an order of magnitude enabling the detection of particles with 4.5 × 10(4) MESF.

Small volume low mechanical stress cytometry using computer-controlled Braille display microfluidics.

PubMed

Tung, Yi-Chung; Torisawa, Yu-suke; Futai, Nobuyuki; Takayama, Shuichi

2007-11-01

This paper describes a micro flow cytometer system designed for efficient and non-damaging analysis of samples with small numbers of precious cells. The system utilizes actuation of Braille-display pins for micro-scale fluid manipulation and a fluorescence microscope with a CCD camera for optical detection. The microfluidic chip is fully disposable and is composed of a polydimethylsiloxane (PDMS) slab with microchannel features sealed against a thin deformable PDMS membrane. The channels are designed with diffusers to alleviate pulsatile flow behaviors inherent in pin actuator-based peristaltic pumping schemes to maximize hydrodynamic focusing of samples with minimal disturbances in the laminar streams within the channel. A funnel connected to the microfluidic channel is designed for efficient loading of samples with small number of cells and is also positioned on the chip to prevent physical damages of the samples by the squeezing actions of Braille pins during actuation. The sample loading scheme was characterized by both computational fluidic dynamics (CFD) simulation and experimental observation. A fluorescein solution was first used for flow field investigation, followed by use of fluorescence beads with known relative intensities for optical detection performance calibration. Murine myoblast cells (C2C12) were exploited to investigate cell viability for the sample loading scheme of the device. Furthermore, human promyelocytic leukemia (HL60) cells stained by hypotonic DNA staining buffer were also tested in the system for cell cycle analysis. The ability to efficiently analyze cellular samples where the number of cells is small was demonstrated by analyzing cells from a single embryoid body derived from mouse embryonic stem cells. Consequently, the designed microfluidic device reported in this paper is promising for easy-to-use, small sample size flow cytometric analysis, and has potential to be further integrated with other Braille display-based microfluidic devices to facilitate a multi-functional lab-on-a-chip for mammalian cell manipulations.
Biomimetic postcapillary expansions for enhancing rare blood cell separation on a microfluidic chip†

PubMed Central

Jain, Abhishek

2013-01-01

Blood cells naturally auto-segregate in postcapillary venules, with the erythrocytes (red blood cells, RBCs) aggregating near the axis of flow and the nucleated cells (NCs)—which include leukocytes, progenitor cells and, in cancer patients, circulating tumor cells—marginating toward the vessel wall. We have used this principle to design a microfluidic device that extracts nucleated cells (NCs) from whole blood. Fabricated using polydimethylsiloxane (PDMS) soft lithography, the biomimetic cell extraction device consists of rectangular microchannels that are 20–400 μm wide, 11 μm deep and up to 2 cm long. The key design feature is the use of repeated expansions/contractions of triangular geometry mimicking postcapillary venules, which enhance margination and optimize the extraction. The device operates on unprocessed whole blood and is able to extract 94 ± 4.5% of NCs with 45.75 ± 2.5-fold enrichment in concentration at a rate of 5 nl s−1. The device eliminates the need to preprocess blood via centrifugation or RBC lysis, and is ready to be implemented as the initial stage of lab-on-a-chip devices that require enriched nucleated cells. The potential downstream applications are numerous, encompassing all preclinical and clinical assays that operate on enriched NC populations and include on-chip flow cytometry PMID:21773633
Spectroscopic imaging system for high-throughput viability assessment of ovarian spheroids or microdissected tumor tissues (MDTs) in a microfluidic chip

NASA Astrophysics Data System (ADS)

St-Georges-Robillard, A.; Masse, M.; Kendall-Dupont, J.; Strupler, M.; Patra, B.; Jermyn, M.; Mes-Masson, A.-M.; Leblond, F.; Gervais, T.

2016-02-01

There is a growing effort in the biomicrosystems community to develop a personalized treatment response assay for cancer patients using primary cells, patient-derived spheroids, or live tissues on-chip. Recently, our group has developed a technique to cut tumors in 350 μm diameter microtissues and keep them alive on-chip, enabling multiplexed in vitro drug assays on primary tumor tissue. Two-photon microscopy, confocal microscopy and flow cytometry are the current standard to assay tissue chemosensitivity on-chip. While these techniques provide microscopic and molecular information, they are not adapted for high-throughput analysis of microtissues. We present a spectroscopic imaging system that allows rapid quantitative measurements of multiple fluorescent viability markers simultaneously by using a liquid crystal tunable filter to record fluorescence and transmittance spectra. As a proof of concept, 24 spheroids composed of ovarian cancer cell line OV90 were formed in a microfluidic chip, stained with two live cell markers (CellTrackerTM Green and Orange), and imaged. Fluorescence images acquired were normalized to the acquisition time and gain of the camera, dark noise was removed, spectral calibration was applied, and spatial uniformity was corrected. Spectral un-mixing was applied to separate each fluorophore's contribution. We have demonstrated that rapid and simultaneous viability measurements on multiple spheroids can be achieved, which will have a significant impact on the prediction of a tumor's response to multiple treatment options. This technique may be applied as well in drug discovery to assess the potential of a drug candidate directly on human primary tissue.
Comparative exploration of multidimensional flow cytometry software: a model approach evaluating T cell polyfunctional behavior.

PubMed

Spear, Timothy T; Nishimura, Michael I; Simms, Patricia E

2017-08-01

Advancement in flow cytometry reagents and instrumentation has allowed for simultaneous analysis of large numbers of lineage/functional immune cell markers. Highly complex datasets generated by polychromatic flow cytometry require proper analytical software to answer investigators' questions. A problem among many investigators and flow cytometry Shared Resource Laboratories (SRLs), including our own, is a lack of access to a flow cytometry-knowledgeable bioinformatics team, making it difficult to learn and choose appropriate analysis tool(s). Here, we comparatively assess various multidimensional flow cytometry software packages for their ability to answer a specific biologic question and provide graphical representation output suitable for publication, as well as their ease of use and cost. We assessed polyfunctional potential of TCR-transduced T cells, serving as a model evaluation, using multidimensional flow cytometry to analyze 6 intracellular cytokines and degranulation on a per-cell basis. Analysis of 7 parameters resulted in 128 possible combinations of positivity/negativity, far too complex for basic flow cytometry software to analyze fully. Various software packages were used, analysis methods used in each described, and representative output displayed. Of the tools investigated, automated classification of cellular expression by nonlinear stochastic embedding (ACCENSE) and coupled analysis in Pestle/simplified presentation of incredibly complex evaluations (SPICE) provided the most user-friendly manipulations and readable output, evaluating effects of altered antigen-specific stimulation on T cell polyfunctionality. This detailed approach may serve as a model for other investigators/SRLs in selecting the most appropriate software to analyze complex flow cytometry datasets. Further development and awareness of available tools will help guide proper data analysis to answer difficult biologic questions arising from incredibly complex datasets. © Society for Leukocyte Biology.
In Vivo Flow Cytometry: A Horizon of Opportunities

PubMed Central

Tuchin, Valery V.; Tárnok, Attila; Zharov, Vladimir P.

2012-01-01

Flow cytometry has been a fundamental tool of biological discovery for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents studying cells in their native environment. These problems can be overcome by use of in vivo flow cytometry which provides detection and imaging of circulating normal and abnormal cells directlyin blood or lymph flow. The goal of this mini-review is to provide a brief history, features and challenges of this new generation of flow cytometry methods and instruments. Spectrum of possibilities of in vivo flow cytometry in biological science (e.g., cell metabolism, immune function, or apoptosis) and medical fields (e.g., cancer, infection, cardiovascular disorder) including integrated photoacoustic-photothermal theranostics of circulating abnormal cells are discussed with focus on recent advances of this new platform. PMID:21915991
Separating the signal from the noise: Expanding flow cytometry into the sub-micron range.

EPA Science Inventory

Cytometry Part A Special Section: Separating the signal from the noise: Expanding flow cytometry into the sub-micron range. The current Cytometry Part A Special Section presents three studies that utilize cytometers to study sub-micron particles. The three studies involve the 1...
Fishing on chips: up-and-coming technological advances in analysis of zebrafish and Xenopus embryos.

PubMed

Zhu, Feng; Skommer, Joanna; Huang, Yushi; Akagi, Jin; Adams, Dany; Levin, Michael; Hall, Chris J; Crosier, Philip S; Wlodkowic, Donald

2014-11-01

Biotests performed on small vertebrate model organisms provide significant investigative advantages as compared with bioassays that employ cell lines, isolated primary cells, or tissue samples. The main advantage offered by whole-organism approaches is that the effects under study occur in the context of intact physiological milieu, with all its intercellular and multisystem interactions. The gap between the high-throughput cell-based in vitro assays and low-throughput, disproportionally expensive and ethically controversial mammal in vivo tests can be closed by small model organisms such as zebrafish or Xenopus. The optical transparency of their tissues, the ease of genetic manipulation and straightforward husbandry, explain the growing popularity of these model organisms. Nevertheless, despite the potential for miniaturization, automation and subsequent increase in throughput of experimental setups, the manipulation, dispensing and analysis of living fish and frog embryos remain labor-intensive. Recently, a new generation of miniaturized chip-based devices have been developed for zebrafish and Xenopus embryo on-chip culture and experimentation. In this work, we review the critical developments in the field of Lab-on-a-Chip devices designed to alleviate the limits of traditional platforms for studies on zebrafish and clawed frog embryo and larvae. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.
FuGEFlow: data model and markup language for flow cytometry.

PubMed

Qian, Yu; Tchuvatkina, Olga; Spidlen, Josef; Wilkinson, Peter; Gasparetto, Maura; Jones, Andrew R; Manion, Frank J; Scheuermann, Richard H; Sekaly, Rafick-Pierre; Brinkman, Ryan R

2009-06-16

Flow cytometry technology is widely used in both health care and research. The rapid expansion of flow cytometry applications has outpaced the development of data storage and analysis tools. Collaborative efforts being taken to eliminate this gap include building common vocabularies and ontologies, designing generic data models, and defining data exchange formats. The Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard was recently adopted by the International Society for Advancement of Cytometry. This standard guides researchers on the information that should be included in peer reviewed publications, but it is insufficient for data exchange and integration between computational systems. The Functional Genomics Experiment (FuGE) formalizes common aspects of comprehensive and high throughput experiments across different biological technologies. We have extended FuGE object model to accommodate flow cytometry data and metadata. We used the MagicDraw modelling tool to design a UML model (Flow-OM) according to the FuGE extension guidelines and the AndroMDA toolkit to transform the model to a markup language (Flow-ML). We mapped each MIFlowCyt term to either an existing FuGE class or to a new FuGEFlow class. The development environment was validated by comparing the official FuGE XSD to the schema we generated from the FuGE object model using our configuration. After the Flow-OM model was completed, the final version of the Flow-ML was generated and validated against an example MIFlowCyt compliant experiment description. The extension of FuGE for flow cytometry has resulted in a generic FuGE-compliant data model (FuGEFlow), which accommodates and links together all information required by MIFlowCyt. The FuGEFlow model can be used to build software and databases using FuGE software toolkits to facilitate automated exchange and manipulation of potentially large flow cytometry experimental data sets. Additional project documentation, including reusable design patterns and a guide for setting up a development environment, was contributed back to the FuGE project. We have shown that an extension of FuGE can be used to transform minimum information requirements in natural language to markup language in XML. Extending FuGE required significant effort, but in our experiences the benefits outweighed the costs. The FuGEFlow is expected to play a central role in describing flow cytometry experiments and ultimately facilitating data exchange including public flow cytometry repositories currently under development.
The role of RhD agglutination for the detection of weak D red cells by anti-D flow cytometry.

PubMed

Grey, D E; Davies, J I; Connolly, M; Fong, E A; Erber, W N

2005-04-01

Anti-D flow cytometry is an accurate method for quantifying feto-maternal haemorrhage (FMH). However, weak D red cells with <1000 RhD sites are not detectable using this methodology but are immunogenic. As quantitation of RhD sites is not practical, an alternative approach is required to identify those weak D fetal red cells where anti-D flow cytometry is inappropriate. We describe a simple algorithm based on RhD agglutination and flow cytometry peak separation. All weak D (n = 34) gave weak agglutination with RUM-1 on immediate spin (grading
Flow Cytometry and Solid Organ Transplantation: A Perfect Match

PubMed Central

Maguire, Orla; Tario, Joseph D.; Shanahan, Thomas C.; Wallace, Paul K.; Minderman, Hans

2015-01-01

In the field of transplantation, flow cytometry serves a well-established role in pre-transplant crossmatching and monitoring immune reconstitution following hematopoietic stem cell transplantation. The capabilities of flow cytometers have continuously expanded and this combined with more detailed knowledge of the constituents of the immune system, their function and interaction and newly developed reagents to study these parameters have led to additional utility of flow cytometry-based analyses, particularly in the post-transplant setting. This review discusses the impact of flow cytometry on managing alloantigen reactions, monitoring opportunistic infections and graft rejection and gauging immunosuppression in the context of solid organ transplantation. PMID:25296232
Scalable clustering algorithms for continuous environmental flow cytometry.

PubMed

Hyrkas, Jeremy; Clayton, Sophie; Ribalet, Francois; Halperin, Daniel; Armbrust, E Virginia; Howe, Bill

2016-02-01

Recent technological innovations in flow cytometry now allow oceanographers to collect high-frequency flow cytometry data from particles in aquatic environments on a scale far surpassing conventional flow cytometers. The SeaFlow cytometer continuously profiles microbial phytoplankton populations across thousands of kilometers of the surface ocean. The data streams produced by instruments such as SeaFlow challenge the traditional sample-by-sample approach in cytometric analysis and highlight the need for scalable clustering algorithms to extract population information from these large-scale, high-frequency flow cytometers. We explore how available algorithms commonly used for medical applications perform at classification of such a large-scale, environmental flow cytometry data. We apply large-scale Gaussian mixture models to massive datasets using Hadoop. This approach outperforms current state-of-the-art cytometry classification algorithms in accuracy and can be coupled with manual or automatic partitioning of data into homogeneous sections for further classification gains. We propose the Gaussian mixture model with partitioning approach for classification of large-scale, high-frequency flow cytometry data. Source code available for download at https://github.com/jhyrkas/seaflow_cluster, implemented in Java for use with Hadoop. hyrkas@cs.washington.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
Measurement of Lipid Accumulation in Chlorella vulgaris via Flow Cytometry and Liquid-State ¹H NMR Spectroscopy for Development of an NMR-Traceable Flow Cytometry Protocol

PubMed Central

Bono Jr., Michael S.; Garcia, Ravi D.; Sri-Jayantha, Dylan V.; Ahner, Beth A.; Kirby, Brian J.

2015-01-01

In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r 2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols. PMID:26267664
Measurement of lipid accumulation in Chlorella vulgaris via flow cytometry and liquid-state ¹H NMR spectroscopy for development of an NMR-traceable flow cytometry protocol.

PubMed

Bono, Michael S; Garcia, Ravi D; Sri-Jayantha, Dylan V; Ahner, Beth A; Kirby, Brian J

2015-01-01

In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols.
Application of a Short Intracellular pH Method to Flow Cytometry for Determining Saccharomyces cerevisiae Vitality ▿

PubMed Central

Weigert, Claudia; Steffler, Fabian; Kurz, Tomas; Shellhammer, Thomas H.; Methner, Frank-Jürgen

2009-01-01

The measurement of yeast's intracellular pH (ICP) is a proven method for determining yeast vitality. Vitality describes the condition or health of viable cells as opposed to viability, which defines living versus dead cells. In contrast to fluorescence photometric measurements, which show only average ICP values of a population, flow cytometry allows the presentation of an ICP distribution. By examining six repeated propagations with three separate growth phases (lag, exponential, and stationary), the ICP method previously established for photometry was transferred successfully to flow cytometry by using the pH-dependent fluorescent probe 5,6-carboxyfluorescein. The correlation between the two methods was good (r2 = 0.898, n = 18). With both methods it is possible to track the course of growth phases. Although photometry did not yield significant differences between exponentially and stationary phases (P = 0.433), ICP via flow cytometry did (P = 0.012). Yeast in an exponential phase has a unimodal ICP distribution, reflective of a homogeneous population; however, yeast in a stationary phase displays a broader ICP distribution, and subpopulations could be defined by using the flow cytometry method. In conclusion, flow cytometry yielded specific evidence of the heterogeneity in vitality of a yeast population as measured via ICP. In contrast to photometry, flow cytometry increases information about the yeast population's vitality via a short measurement, which is suitable for routine analysis. PMID:19581482
A benchmark for evaluation of algorithms for identification of cellular correlates of clinical outcomes.

PubMed

Aghaeepour, Nima; Chattopadhyay, Pratip; Chikina, Maria; Dhaene, Tom; Van Gassen, Sofie; Kursa, Miron; Lambrecht, Bart N; Malek, Mehrnoush; McLachlan, G J; Qian, Yu; Qiu, Peng; Saeys, Yvan; Stanton, Rick; Tong, Dong; Vens, Celine; Walkowiak, Sławomir; Wang, Kui; Finak, Greg; Gottardo, Raphael; Mosmann, Tim; Nolan, Garry P; Scheuermann, Richard H; Brinkman, Ryan R

2016-01-01

The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of computational methods for identifying cell populations in multidimensional flow cytometry data. Here we report the results of FlowCAP-IV where algorithms from seven different research groups predicted the time to progression to AIDS among a cohort of 384 HIV+ subjects, using antigen-stimulated peripheral blood mononuclear cell (PBMC) samples analyzed with a 14-color staining panel. Two approaches (FlowReMi.1 and flowDensity-flowType-RchyOptimyx) provided statistically significant predictive value in the blinded test set. Manual validation of submitted results indicated that unbiased analysis of single cell phenotypes could reveal unexpected cell types that correlated with outcomes of interest in high dimensional flow cytometry datasets. © 2015 International Society for Advancement of Cytometry.
The development and validation of different decision-making tools to predict urine culture growth out of urine flow cytometry parameter.

PubMed

Müller, Martin; Seidenberg, Ruth; Schuh, Sabine K; Exadaktylos, Aristomenis K; Schechter, Clyde B; Leichtle, Alexander B; Hautz, Wolf E

2018-01-01

Patients presenting with suspected urinary tract infection are common in every day emergency practice. Urine flow cytometry has replaced microscopic urine evaluation in many emergency departments, but interpretation of the results remains challenging. The aim of this study was to develop and validate tools that predict urine culture growth out of urine flow cytometry parameter. This retrospective study included all adult patients that presented in a large emergency department between January and July 2017 with a suspected urinary tract infection and had a urine flow cytometry as well as a urine culture obtained. The objective was to identify urine flow cytometry parameters that reliably predict urine culture growth and mixed flora growth. The data set was split into a training (70%) and a validation set (30%) and different decision-making approaches were developed and validated. Relevant urine culture growth (respectively mixed flora growth) was found in 40.2% (7.2% respectively) of the 613 patients included. The number of leukocytes and bacteria in flow cytometry were highly associated with urine culture growth, but mixed flora growth could not be sufficiently predicted from the urine flow cytometry parameters. A decision tree, predictive value figures, a nomogram, and a cut-off table to predict urine culture growth from bacteria and leukocyte count were developed, validated and compared. Urine flow cytometry parameters are insufficient to predict mixed flora growth. However, the prediction of urine culture growth based on bacteria and leukocyte count is highly accurate and the developed tools should be used as part of the decision-making process of ordering a urine culture or starting an antibiotic therapy if a urogenital infection is suspected.
The development and validation of different decision-making tools to predict urine culture growth out of urine flow cytometry parameter

PubMed Central

Seidenberg, Ruth; Schuh, Sabine K.; Exadaktylos, Aristomenis K.; Schechter, Clyde B.; Leichtle, Alexander B.; Hautz, Wolf E.

2018-01-01

Objective Patients presenting with suspected urinary tract infection are common in every day emergency practice. Urine flow cytometry has replaced microscopic urine evaluation in many emergency departments, but interpretation of the results remains challenging. The aim of this study was to develop and validate tools that predict urine culture growth out of urine flow cytometry parameter. Methods This retrospective study included all adult patients that presented in a large emergency department between January and July 2017 with a suspected urinary tract infection and had a urine flow cytometry as well as a urine culture obtained. The objective was to identify urine flow cytometry parameters that reliably predict urine culture growth and mixed flora growth. The data set was split into a training (70%) and a validation set (30%) and different decision-making approaches were developed and validated. Results Relevant urine culture growth (respectively mixed flora growth) was found in 40.2% (7.2% respectively) of the 613 patients included. The number of leukocytes and bacteria in flow cytometry were highly associated with urine culture growth, but mixed flora growth could not be sufficiently predicted from the urine flow cytometry parameters. A decision tree, predictive value figures, a nomogram, and a cut-off table to predict urine culture growth from bacteria and leukocyte count were developed, validated and compared. Conclusions Urine flow cytometry parameters are insufficient to predict mixed flora growth. However, the prediction of urine culture growth based on bacteria and leukocyte count is highly accurate and the developed tools should be used as part of the decision-making process of ordering a urine culture or starting an antibiotic therapy if a urogenital infection is suspected. PMID:29474463
Imaging flow cytometry for phytoplankton analysis.

PubMed

Dashkova, Veronika; Malashenkov, Dmitry; Poulton, Nicole; Vorobjev, Ivan; Barteneva, Natasha S

2017-01-01

This review highlights the concepts and instrumentation of imaging flow cytometry technology and in particular its use for phytoplankton analysis. Imaging flow cytometry, a hybrid technology combining speed and statistical capabilities of flow cytometry with imaging features of microscopy, is rapidly advancing as a cell imaging platform that overcomes many of the limitations of current techniques and contributed significantly to the advancement of phytoplankton analysis in recent years. This review presents the various instrumentation relevant to the field and currently used for assessment of complex phytoplankton communities' composition and abundance, size structure determination, biovolume estimation, detection of harmful algal bloom species, evaluation of viability and metabolic activity and other applications. Also we present our data on viability and metabolic assessment of Aphanizomenon sp. cyanobacteria using Imagestream X Mark II imaging cytometer. Herein, we highlight the immense potential of imaging flow cytometry for microalgal research, but also discuss limitations and future developments. Copyright © 2016 Elsevier Inc. All rights reserved.
[Which technique should be used in the phenotyping of lymphocytic alveolitis: Immunocytochemistry or flow cytometry].

PubMed

Mlika, Mona; Kasmi, Rihem; Safra, Ines; Braham, Emna; Chebbi, Chokri; Mezni, Faouzi El

2017-10-01

Diffuse interstitial pneumonias are considered as a group of multiple affections characterized by challenging diagnoses because of the lack of specific clinical signs. Radiologic investigations highlight the diagnoses in most of the cases but bronchoalveolar lavage plays a key role in the diagnostic diagram. We aim to compare the immunocytochemical technique and the flow cytometry in the phenotyping of lymphocytic alveolitis. We described a series of 32 lymphocytic alveolitis, which were analyzed using immunocytochemistry and flow cytometry. We found a good reproducibility between the immunocytochemistry performed on smears and cytoblocks (kappa=0.7) and a poor reproducibility between immunocytochemistry and flow cytometry (kappa=0.35). Our study emphasized on the poor reproducibility between immunocytochemistry and flow cytometry. Further studies about the reliability of both techniques are needed especially in discordant cases. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
FuGEFlow: data model and markup language for flow cytometry

PubMed Central

Qian, Yu; Tchuvatkina, Olga; Spidlen, Josef; Wilkinson, Peter; Gasparetto, Maura; Jones, Andrew R; Manion, Frank J; Scheuermann, Richard H; Sekaly, Rafick-Pierre; Brinkman, Ryan R

2009-01-01

Background Flow cytometry technology is widely used in both health care and research. The rapid expansion of flow cytometry applications has outpaced the development of data storage and analysis tools. Collaborative efforts being taken to eliminate this gap include building common vocabularies and ontologies, designing generic data models, and defining data exchange formats. The Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard was recently adopted by the International Society for Advancement of Cytometry. This standard guides researchers on the information that should be included in peer reviewed publications, but it is insufficient for data exchange and integration between computational systems. The Functional Genomics Experiment (FuGE) formalizes common aspects of comprehensive and high throughput experiments across different biological technologies. We have extended FuGE object model to accommodate flow cytometry data and metadata. Methods We used the MagicDraw modelling tool to design a UML model (Flow-OM) according to the FuGE extension guidelines and the AndroMDA toolkit to transform the model to a markup language (Flow-ML). We mapped each MIFlowCyt term to either an existing FuGE class or to a new FuGEFlow class. The development environment was validated by comparing the official FuGE XSD to the schema we generated from the FuGE object model using our configuration. After the Flow-OM model was completed, the final version of the Flow-ML was generated and validated against an example MIFlowCyt compliant experiment description. Results The extension of FuGE for flow cytometry has resulted in a generic FuGE-compliant data model (FuGEFlow), which accommodates and links together all information required by MIFlowCyt. The FuGEFlow model can be used to build software and databases using FuGE software toolkits to facilitate automated exchange and manipulation of potentially large flow cytometry experimental data sets. Additional project documentation, including reusable design patterns and a guide for setting up a development environment, was contributed back to the FuGE project. Conclusion We have shown that an extension of FuGE can be used to transform minimum information requirements in natural language to markup language in XML. Extending FuGE required significant effort, but in our experiences the benefits outweighed the costs. The FuGEFlow is expected to play a central role in describing flow cytometry experiments and ultimately facilitating data exchange including public flow cytometry repositories currently under development. PMID:19531228

Visualization of Pulmonary Clearance Mechanisms via Noninvasive Optical Imaging Validated by Near-Infrared Flow Cytometry

PubMed Central

Zhou, Haiying; Gunsten, Sean P.; Zhegalova, Natalia G.; Bloch, Sharon; Achilefu, Samuel; Holley, J. Christopher; Schweppe, Daniel; Akers, Walter; Brody, Steven L.; Eades, William; Berezin, Mikhail Y.

2016-01-01

In vivo optical imaging with near-infrared (NIR) probes is an established method of diagnostics in preclinical and clinical studies. However, the specificities of these probes are difficult to validate ex vivo due to the lack of NIR flow cytometry. To address this limitation, we modified a flow cytometer to include an additional NIR channel using a 752 nm laser line. The flow cytometry system was tested using NIR microspheres and cell lines labeled with a combination of visible range and NIR fluorescent dyes. The approach was verified in vivo in mice evaluated for immune response in lungs after intratracheal delivery of the NIR contrast agent. Flow cytometry of cells obtained from the lung bronchoalveolar lavage demonstrated that the NIR dye was taken up by pulmonary macrophages as early as four-hours post-injection. This combination of optical imaging with NIR flow cytometry extends the capability of imaging and enables complementation of in vivo imaging with cell-specific studies. PMID:25808737
Multinode acoustic focusing for parallel flow cytometry

PubMed Central

Piyasena, Menake E.; Suthanthiraraj, Pearlson P. Austin; Applegate, Robert W.; Goumas, Andrew M.; Woods, Travis A.; López, Gabriel P.; Graves, Steven W.

2012-01-01

Flow cytometry can simultaneously measure and analyze multiple properties of single cells or particles with high sensitivity and precision. Yet, conventional flow cytometers have fundamental limitations with regards to analyzing particles larger than about 70 microns, analyzing at flow rates greater than a few hundred microliters per minute, and providing analysis rates greater than 50,000 per second. To overcome these limits, we have developed multi-node acoustic focusing flow cells that can position particles (as small as a red blood cell and as large as 107 microns in diameter) into as many as 37 parallel flow streams. We demonstrate the potential of such flow cells for the development of high throughput, parallel flow cytometers by precision focusing of flow cytometry alignment microspheres, red blood cells, and the analysis of CD4+ cellular immunophenotyping assay. This approach will have significant impact towards the creation of high throughput flow cytometers for rare cell detection applications (e.g. circulating tumor cells), applications requiring large particle analysis, and high volume flow cytometry. PMID:22239072
Imaging cytometry in a plastic ultra-mobile system

NASA Astrophysics Data System (ADS)

Martínez Vázquez, R.; Trotta, G.; Paturzo, M.; Volpe, A.; Bernava, G.; Basile, V.; Ancona, A.; Ferraro, P.; Fassi, I.; Osellame, R.

2017-03-01

We present a cost-effective and highly-portable plastic prototype that can be interfaced with a cell phone to implement an optofluidic imaging cytometry platform. It is based on a PMMA microfluidic chip that fits inside an opto-mechanical platform fabricated by a 3D printer. The fluorescence excitation and imaging is performed using the LED and the CMOS from the cell phone increasing the compactness of the system. A custom developed application is used to analyze the images and provide a value of particle concentration.
Biomarkers of Selenium Chemoprevention of Prostate Cancer

DTIC Science & Technology

2005-01-01

than Se-Met in inhibiting Flow Kit from BD Pharmigen (San Diego, CA). Stained cells were then quantified by flow cytometry , and the data were analyzed...decrease in Quantitation of Apoptosis by Flow Cytometry . PC-3 cells were plated at cell number accumulation by MSA was related to cell cycle arrest, we... flow exposed to either 5 or 10Mm MSA for 48 or 72 h. Adherent cells harvested by mild cytometry of ethanol-permeabilized cells stained with Pl. Synchro
A Study of the Effects of High Power Pulsed 2450 MHz Microwaves, ELF modulated Microwaves, and ELF Fields on Human Lymphocytes and Selected Cell Lines

DTIC Science & Technology

1993-01-27

Considerable effect was expended in investigating shifts in intercellular calcium of one particular cell line, Jurket, using flow cytometry methods. No...culture. The following analysis were used to characterize the immortalized cell lines: flow cytometry , electron microscopy, two-dimensional protein gel...further characterized by flow cytometry , electron microscopy, two dimensional protein electrophoresis and nuclear run-off assay. Flow cytometric analysis of
Small lasers in flow cytometry.

PubMed

Telford, William G

2004-01-01

Laser technology has made tremendous advances in recent years, particularly in the area of diode and diode-pumped solid state sources. Flow cytometry has been a direct beneficiary of these advances, as these small, low-maintenance, inexpensive lasers with reasonable power outputs are integrated into flow cytometers. In this chapter we review the contribution and potential of solid-state lasers to flow cytometry, and show several examples of these novel sources integrated into production flow cytometers. Technical details and critical parameters for successful application of these lasers for biomedical analysis are reviewed.
Cytometry standards continuum

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Spidlen, Josef; Brinkman, Ryan R.

2008-02-01

Introduction: The International Society for Analytical Cytology, ISAC, is developing a new combined flow and image Analytical Cytometry Standard (ACS). This standard needs to serve both the research and clinical communities. The clinical medicine and clinical research communities have a need to exchange information with hospital and other clinical information systems. Methods: 1) Prototype the standard by creating CytometryML and a RAW format for binary data. 2) Join the ISAC Data Standards Task Force. 3) Create essential project documentation. 4) Cooperate with other groups by assisting in the preparation of the DICOM Supplement 122: Specimen Module and Pathology Service-Object Pair Classes. Results: CytometryML has been created and serves as a prototype and source of experience for the following: the Analytical Cytometry Standard (ACS) 1.0, the ACS container, Minimum Information about a Flow Cytometry Experiment (MIFlowCyt), and Requirements for a Data File Standard Format to Describe Flow Cytometry and Related Analytical Cytology Data. These requirements provide a means to judge the appropriateness of design elements and to develop tests for the final ACS. The requirements include providing the information required for understanding and reproducing a cytometry experiment or clinical measurement, and for a single standard for both flow and digital microscopic cytometry. Schemas proposed by other members of the ISAC Data Standards Task Force (e.g, Gating-ML) have been independently validated and have been integrated with CytometryML. The use of netCDF as an element of the ACS container has been proposed by others and a suggested method of its use is proposed.
Fundamentals of flow cytometry.

PubMed

Jaroszeski, M J; Radcliff, G

1999-02-01

Flow cytometers are instruments that are used primarily to measure the physical and biochemical characteristics of biological particles. This technology is used to perform measurements on whole cells as well as prepared cellular constituents, such as nuclei and organelles. Flow cytometers are investigative tools for a broad range of scientific disciplines because they make measurements on thousands of individual cells/particles in a matter of seconds. This is a unique advantage relative to other detection instruments that provide bulk particle measurements. Flow cytometry is a complex and highly technical field; therefore, a basic understanding of the technology is essential for all users. The purpose of this article is to provide fundamental information about the instrumentation used for flow cytometry as well as the methods used to analyze and interpret data. This information will provide a foundation to use flow cytometry effectively as a research tool.
Flow Cytometry with Gold Nanoparticles and their Clusters as scattering Contrast Agents: FDTD Simulation of Light-Cell Interaction

PubMed Central

Tanev, Stoyan; Sun, Wenbo; Pond, James; Tuchin, Valery V.; Zharov, Vladimir P.

2010-01-01

The formulation of the Finite-Difference Time-Domain (FDTD) approach is presented in the framework of its potential applications to in vivo flow cytometry based on light scattering. The consideration is focused on comparison of light scattering by a single biological cell alone in controlled refractive index matching conditions and by cells labeled by gold nanoparticles. The optical schematics including phase contrast (OPCM) microscopy as a prospective modality for in vivo flow cytometry is also analyzed. The validation of the FDTD approach for the simulation of flow cytometry may open a new avenue in the development of advanced cytometric techniques based on scattering effects from nanoscale targets. PMID:19670359
Diagnosis of Plasma Cell Dyscrasias and Monitoring of Minimal Residual Disease by Multiparametric Flow Cytometry

PubMed Central

Soh, Kah Teong; Tario, Joseph D.; Wallace, Paul K.

2018-01-01

Synopsis Plasma cell dyscrasia (PCD) is a heterogeneous disease which has seen a tremendous change in outcomes due to improved therapies. Over the last few decades, multiparametric flow cytometry has played an important role in the detection and monitoring of PCDs. Flow cytometry is a high sensitivity assay for early detection of minimal residual disease (MRD) that correlates well with progression-free survival and overall survival. Before flow cytometry can be effectively implemented in the clinical setting sample preparation, panel configuration, analysis, and gating strategies must be optimized to ensure accurate results. Current consensus methods and reporting guidelines for MRD testing are discussed. PMID:29128071
Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

DOEpatents

McCutchen-Maloney, Sandra L.

2002-01-01

DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.
Monitoring survival and function of transfused platelets in Bernard-Soulier syndrome by flow cytometry and a cone and plate(let) analyzer (Impact-R).

PubMed

Panzer, Simon; Eichelberger, Beate; Koren, Daniela; Kaufmann, Karin; Male, Christoph

2007-01-01

Bernard-Soulier syndrome (BSS) patients may repeatedly require transfusion of platelets (PLTs). The hemostatic competence of transfused PLTs requires monitoring. Flow cytometry and a cone and plate(let) analyzer (Impact-R, DiaMed) were used to monitor survival and function of transfused PLTs in a 7-year-old girl with BSS undergoing surgery. Flow cytometry was applied to differentiate autologous PLTs from transfused PLTs by staining for CD42b. The Impact, which measures PLT adhesion and aggregation in response to high shear stress, was used to evaluate PLT function. Transfused PLTs were detectable by flow cytometry for 1 week after transfusion. While the patient's PLTs did not respond to high shear stress before transfusion, a normal response was documented by the Impact on the day after transfusion and 1 week thereafter. Transfused PLTs were detectable by flow cytometry, and their functional activity was demonstrated by the Impact.
Evaluation of Leishmania species reactivity in human serologic diagnosis of leishmaniasis.

PubMed

Silvestre, Ricardo; Santarém, Nuno; Teixeira, Lúcia; Cunha, Joana; Schallig, Henk; Cordeiro-da-Silva, Anabela

2009-08-01

The sensitivities and specificities of IgG-ELISA and IgG flow cytometry based techniques using different Leishmania species were determined using sera collected from 40 cutaneous or visceral leishmaniasis patients. The flow cytometry technique, using promastigote parasite forms, performed better than total soluble extract IgG-ELISA. At the species level, the use of Leishmania amazonensis and Leishmania major as antigens in enzyme linked immunosorbent assay (ELISA) decreased the overall sensitivity. To assess the specificity of these tests, sera from malaria, toxoplasmosis, amoebiasis, schistosomiasis, and leprosy patients were used. We also included sera from Leishmania non-infected endemic individuals. The cutaneous species displayed a decreased specificity in both assays. Although more sensitive, flow cytometry using promastigote parasite forms generally presented lower levels of specificity when compared with total extract of IgG-ELISA. Overall, the results of the study show the potential of IgG flow cytometry for the diagnosis of leishmaniasis. Although highly sensitive, a refinement of the flow cytometry method should be performed to improve the overall specificity.
A Method for the Interpretation of Flow Cytometry Data Using Genetic Algorithms.

PubMed

Angeletti, Cesar

2018-01-01

Flow cytometry analysis is the method of choice for the differential diagnosis of hematologic disorders. It is typically performed by a trained hematopathologist through visual examination of bidimensional plots, making the analysis time-consuming and sometimes too subjective. Here, a pilot study applying genetic algorithms to flow cytometry data from normal and acute myeloid leukemia subjects is described. Initially, Flow Cytometry Standard files from 316 normal and 43 acute myeloid leukemia subjects were transformed into multidimensional FITS image metafiles. Training was performed through introduction of FITS metafiles from 4 normal and 4 acute myeloid leukemia in the artificial intelligence system. Two mathematical algorithms termed 018330 and 025886 were generated. When tested against a cohort of 312 normal and 39 acute myeloid leukemia subjects, both algorithms combined showed high discriminatory power with a receiver operating characteristic (ROC) curve of 0.912. The present results suggest that machine learning systems hold a great promise in the interpretation of hematological flow cytometry data.
Characterization of neutrophils and macrophages from ex vivo cultured murine bone marrow for morphologic maturation and functional responses by imaging flow cytometry

PubMed Central

Pelletier, Margery G. H.; Szymczak, Klaudia; Barbeau, Anna M.; Prata, Gianna N.; O’Fallon, Kevin S.; Gaines, Peter

2016-01-01

Neutrophils and macrophages differentiate from common myeloid progenitors in the bone marrow, where they undergo nuclear morphologic changes during maturation. During this process, both cell types acquire critical innate immune functions that include phagocytosis of pathogens, and for neutrophils the release of nuclear material called nuclear extracellular traps (NETs). Primary cells used to study these functions are typically purified from mature mouse tissues, but bone marrow-derived ex vivo cultures provide more abundant numbers of progenitors and functionally mature cells. Routine analyses of these cells use conventional microscopy and flow cytometry, which present limitations; microscopy is laborious and subjective, whereas flow cytometry lacks spatial resolution. Here we describe methods to generate enriched populations of neutrophils or macrophages from cryopreserved mouse bone marrow cultured ex vivo, and to use imaging flow cytometry that combines the resolution of microscopy with flow cytometry to analyze cells for morphologic features, phagocytosis, and NETosis. PMID:27663441
Mesenchymal Stem Cells for Vascular Target Discovery in Breast Cancer-Associated Angiogenesis

DTIC Science & Technology

2004-09-01

Matrigel plug and sorted by flow cytometry . Sorting of these retrieved cells based on co-expression of the GFP marker and cell- surface endothelial...express the green fluorescent protein (GFP) and clonal MSC populations can be isolated and phenotypically and genotypically analyzed by flow cytometry ...monoclonal populations of these GFP+ murine MSCs and conducted flow cytometry analysis to determine their phenotype. Specifically, we determined if
DNA polymorphism identity determination using flow cytometry

DOEpatents

Nolan, John P.; White, P. Scott; Cai, Hong

2001-01-01

DNA polymorphism identity determination using flow cytometry. Primers designed to be immobilized on microspheres are allowed to anneal to the DNA strand under investigation, and are extended by either DNA polymerase using fluorescent dideoxynucleotides or ligated by DNA ligase to fluorescent reporter oligonucleotides. The fluorescence of either the dideoxynucleotide or the reporter oligonucleotide attached to the immobilized primer is measured by flow cytometry, thereby identifying the nucleotide polymorphism on the DNA strand.
Early Detection of NSCLC Using Stromal Markers in Peripheral Blood

DTIC Science & Technology

2016-09-01

circulating myeloid cells, flow cytometry, RNA -sequencing, expression profiling. 3. ACCOMPLISHMENTS:  What were the major goals of the project...Subtask 2: Flow cytometry sorting of circulating myeloid cells. Subtask 3: RNA -Sequencing Subtask 4: RNA -seq data analysis Subtask 5: Feasible RT-PCR...accomplished the patient recruitment, flow cytometry sorting of circulating myeloid cells, RNA -sequencing of the samples. During the RNA - seq data analysis, we
Aptamer-fluorescent silica nanoparticles bioconjugates based dual-color flow cytometry for specific detection of Staphylococcus aureus.

PubMed

He, Xiaoxiao; Li, Yuhong; He, Dinggen; Wang, Kemin; Shangguan, Jingfang; Shi, Hui

2014-07-01

This paper describes a sensitive and specific determination strategy for Staphylococcus aureus (S. aureus) detection using aptamer recognition and fluorescent silica nanoparticles (FSiNPs) label based dual-color flow cytometry assay (Aptamer/FSiNPs-DCFCM). In the protocol, an aptamer, having high affinity to S. aureus, was first covalently immobilized onto chloropropyl functionalized FSiNPs through a click chemistry approach to generate aptamer-nanoparticles bioconjugates (Aptamer/FSiNPs). Next, S. aureus was incubated with Aptamer/FSiNPs, and then stained with SYBR Green I (a special staining material for the duplex DNA). Upon target binding and nucleic acid staining with SYBR Green I, the S. aureus was determined using two-color flow cytometry. The method took advantage of the specificity of aptamer, signal amplification of FSiNPs label and decreased false positives of two-color flow cytometry assay. It was demonstrated that these Aptamer/FSiNPs could efficiently recognize and fluorescently label target S. aureus. Through multiparameter determination with flow cytometry, this assay allowed for detection of as low as 1.5 x 10(2) and 7.6 x 10(2) cells mL(-1) S. aureus in buffer and spiked milk, respectively, with higher sensitivity than the Aptamer/FITC based flow cytometry.
CD200 is a useful diagnostic marker for identifying atypical chronic lymphocytic leukemia by flow cytometry.

PubMed

Ting, Y S; Smith, S A B C; Brown, D A; Dodds, A J; Fay, K C; Ma, D D F; Milliken, S; Moore, J J; Sewell, W A

2018-05-27

Immunophenotyping by flow cytometry is routinely employed in distinguishing between chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Inclusion of CD200 has been reported to contribute to more reliable differentiation between CLL and MCL. We investigated the value of CD200 in assessment of atypical CLL cases. CD200 expression on mature B cell neoplasms was studied by eight-color flow cytometry in combination with a conventional panel of flow cytometry markers. The study included 70 control samples, 63 samples with CLL or atypical CLL phenotype, 6 MCL samples, and 40 samples of other mature B cell neoplasms. All CLL samples were positive for CD200, whereas MCL samples were dim or negative for CD200. Of the CLL samples, 7 were atypical by conventional flow cytometry, with Matutes scores ≤3. These cases were tested for evidence of a t(11;14) translocation, characteristic of MCL, and all were negative, consistent with their classification as atypical CLL. All these atypical CLL samples were strongly positive for CD200. CD200 proved to be a useful marker for differentiation between CLL and MCL by flow cytometry. In particular, CD200 was useful in distinguishing CLL samples with atypical immunophenotypes from MCL. © 2018 John Wiley & Sons Ltd.

DNA flow cytometry of human spermatozoa: consistent stoichiometric staining of sperm DNA using a novel decondensation protocol.

PubMed

Kovács, Tamás; Békési, Gyöngyi; Fábián, Akos; Rákosy, Zsuzsa; Horváth, Gábor; Mátyus, László; Balázs, Margit; Jenei, Attila

2008-10-01

Rapid flow cytometric measurement of the frequency of aneuploid human sperms is in increasing demand but development of an exploitable method is hindered by difficulties of stoichiometric staining of sperm DNA. An aggressive decondensation protocol is needed after which cell integrity still remains intact. We used flow cytometry to examine the effect of lithium diiodosalicylate (LIS, chaotropic agent) on fluorescence intensity of propidium iodide-treated human spermatozoa from 10 normozoospermic men. When flow cytometric identification of diploid spermatozoa was achieved, validation was performed after sorting by three-color FISH. In contrast with the extremely variable histograms of nondecondensed sperms, consistent identification of haploid and diploid spermatozoa was possible if samples were decondensed with LIS prior to flow cytometry. A 76-fold enrichment of diploid sperms was observed in the sorted fractions by FISH. A significant correlation was found between the proportion of sorted cells and of diploid sperms by FISH. Application of LIS during the preparation of sperm for flow cytometry appears to ensure the stoichiometric staining of sperm DNA, making quantification of aneuploid sperm percentage possible. To our knowledge this is the first report in terms of separating spermatozoa with confirmedly abnormal chromosomal content. High correlation between the proportion of cells identified as having double DNA content by flow cytometry and diploid sperm by FISH allows rapid calculation of diploidy rate. Copyright 2008 International Society for Advancement of Cytometry.
High-throughput autofluorescence flow cytometry of breast cancer metabolism (Conference Presentation)

NASA Astrophysics Data System (ADS)

Shah, Amy T.; Cannon, Taylor M.; Higginbotham, Jim N.; Skala, Melissa C.

2016-02-01

Tumor heterogeneity poses challenges for devising optimal treatment regimens for cancer patients. In particular, subpopulations of cells can escape treatment and cause relapse. There is a need for methods to characterize tumor heterogeneity of treatment response. Cell metabolism is altered in cancer (Warburg effect), and cells use the autofluorescent cofactor NADH in numerous metabolic reactions. Previous studies have shown that microscopy measurements of NADH autofluorescence are sensitive to treatment response in breast cancer, and these techniques typically assess hundreds of cells per group. An alternative approach is flow cytometry, which measures fluorescence on a single-cell level and is attractive for characterizing tumor heterogeneity because it achieves high-throughput analysis and cell sorting in millions of cells per group. Current applications for flow cytometry rely on staining with fluorophores. This study characterizes flow cytometry measurements of NADH autofluorescence in breast cancer cells. Preliminary results indicate flow cytometry of NADH is sensitive to cyanide perturbation, which inhibits oxidative phosphorylation, in nonmalignant MCF10A cells. Additionally, flow cytometry is sensitive to higher NADH intensity for HER2-positive SKBr3 cells compared with triple-negative MDA-MB-231 cells. These results agree with previous microscopy studies. Finally, a mixture of SKBr3 and MDA-MB-231 cells were sorted into each cell type using NADH intensity. Sorted cells were cultured, and microscopy validation showed the expected morphology for each cell type. Ultimately, flow cytometry could be applied to characterize tumor heterogeneity based on treatment response and sort cell subpopulations based on metabolic profile. These achievements could enable individualized treatment strategies and improved patient outcomes.
Ultrasensitive automated RNA in situ hybridization for kappa and lambda light chain mRNA detects B-cell clonality in tissue biopsies with performance comparable or superior to flow cytometry.

PubMed

Guo, Ling; Wang, Zhen; Anderson, Courtney M; Doolittle, Emerald; Kernag, Siobhan; Cotta, Claudiu V; Ondrejka, Sarah L; Ma, Xiao-Jun; Cook, James R

2018-03-01

The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin-fixed, paraffin-embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells but are often insufficiently sensitive to detect the much lower abundance of light chains present in B-cells. We describe an ultrasensitive RNA in situ hybridization assay that has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain-restricted B-cells in 85 (42%) vs 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified restricted B-cells in 74 (89%) vs 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases owing to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphological features in formalin-fixed, paraffin-embedded tissues with a clinical sensitivity similar or superior to flow cytometry.
Ultrasensitive Automated RNA in situ Hybridization for Kappa and Lambda Light Chain mRNA Detects B-cell Clonality in Tissue Biopsies with Performance Comparable or Superior to Flow Cytometry

PubMed Central

Guo, Ling; Wang, Zhen; Anderson, Courtney M.; Doolittle, Emerald; Kernag, Siobhan; Cotta, Claudiu V.; Ondrejka, Sarah L.; Ma, Xiao-Jun; Cook, James R.

2017-01-01

The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin fixed paraffin embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells, but are often insufficiently sensitive to detect the much lower abundance of light chains present in B cells. We describe an ultrasensitive RNA in situ hybridization assay which has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain restricted B-cells in 85 (42%) vs. 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified a restricted B-cells in 74 (89%) vs. 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases due to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry, and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphologic features in formalin fixed, paraffin embedded tissues with a clinical sensitivity similar or superior to flow cytometry. PMID:29052600
Integration of semiconductor quantum dots into nano-bio-chip systems for enumeration of CD4+ T cell counts at the point-of-need†‡

PubMed Central

Jokerst, Jesse V.; Floriano, Pierre N.; Christodoulides, Nicolaos; Simmons, Glennon W.; McDevitt, John T.

2010-01-01

Recent humanitarian efforts have led to the widespread release of antiretroviral drugs for the treatment of the more than 33 million HIV afflicted people living in resource-scarce settings. Here, the enumeration of CD4+ T lymphocytes is required to establish the level at which the immune system has been compromised. The gold standard method used in developed countries, based on flow cytometry, though widely accepted and accurate, is precluded from widespread use in resource-scarce settings due to its high expense, high technical requirements, difficulty in operation-maintenance and the lack of portability for these sophisticated laboratory-confined systems. As part of continuing efforts to develop practical diagnostic instrumentation, the integration of semiconductor nanocrystals (quantum dots, QDs) into a portable microfluidic-based lymphocyte capture and detection device is completed. This integrated system is capable of isolating and counting selected lymphocyte sub-populations (CD3+CD4+) from whole blood samples. By combining the unique optical properties of the QDs with the sample handling capabilities and cost effectiveness of novel microfluidic systems, a practical, portable lymphocyte measurement modality that correlates nicely with flow cytometry (R2 = 0.97) has been developed. This QD-based system reduces the optical requirements significantly relative to molecular fluorophores and the mini-CD4 counting device is projected to be suitable for use in both point-of-need and resource-scarce settings. PMID:19023471
Two-Photon Flow Cytometry

NASA Technical Reports Server (NTRS)

Zhog, Cheng Frank; Ye, Jing Yong; Norris, Theodore B.; Myc, Andrzej; Cao, Zhengyl; Bielinska, Anna; Thomas, Thommey; Baker, James R., Jr.

2004-01-01

Flow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantitation is achieved by assuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow such as is obtained via hydrodynamic focusing. In this work, we demonstrate a two-beam, two- channel detection and two-photon excitation flow cytometry (T(sup 3)FC) system that enables multi-dye analysis to be performed very simply, with greatly relaxed requirements on the fluid flow. Two-photon excitation using a femtosecond near-infrared (NIR) laser has the advantages that it enables simultaneous excitation of multiple dyes and achieves very high signal-to-noise ratio through simplified filtering and fluorescence background reduction. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling of cells by targeted nanoparticles with multiple fluorophores enables normalization of the fluorescence signal and thus ratiometric measurements under nonuniform excitation. Quantitative size measurements can also be done even under conditions of nonuniform flow via a two-beam layout. This innovative detection scheme not only considerably simplifies the fluid flow system and the excitation and collection optics, it opens the way to quantitative cytometry in simple and compact microfluidics systems, or in vivo. Real-time detection of fluorescent microbeads in the vasculature of mouse ear demonstrates the ability to do flow cytometry in vivo. The conditions required to perform quantitative in vivo cytometry on labeled cells will be presented.
One-dimensional acoustic standing waves in rectangular channels for flow cytometry.

PubMed

Austin Suthanthiraraj, Pearlson P; Piyasena, Menake E; Woods, Travis A; Naivar, Mark A; Lόpez, Gabriel P; Graves, Steven W

2012-07-01

Flow cytometry has become a powerful analytical tool for applications ranging from blood diagnostics to high throughput screening of molecular assemblies on microsphere arrays. However, instrument size, expense, throughput, and consumable use limit its use in resource poor areas of the world, as a component in environmental monitoring, and for detection of very rare cell populations. For these reasons, new technologies to improve the size and cost-to-performance ratio of flow cytometry are required. One such technology is the use of acoustic standing waves that efficiently concentrate cells and particles to the center of flow channels for analysis. The simplest form of this method uses one-dimensional acoustic standing waves to focus particles in rectangular channels. We have developed one-dimensional acoustic focusing flow channels that can be fabricated in simple capillary devices or easily microfabricated using photolithography and deep reactive ion etching. Image and video analysis demonstrates that these channels precisely focus single flowing streams of particles and cells for traditional flow cytometry analysis. Additionally, use of standing waves with increasing harmonics and in parallel microfabricated channels is shown to effectively create many parallel focused streams. Furthermore, we present the fabrication of an inexpensive optical platform for flow cytometry in rectangular channels and use of the system to provide precise analysis. The simplicity and low-cost of the acoustic focusing devices developed here promise to be effective for flow cytometers that have reduced size, cost, and consumable use. Finally, the straightforward path to parallel flow streams using one-dimensional multinode acoustic focusing, indicates that simple acoustic focusing in rectangular channels may also have a prominent role in high-throughput flow cytometry. Copyright © 2012 Elsevier Inc. All rights reserved.
Genomic Instability at Premalignant and Early Stages of Breast Cancer Development

DTIC Science & Technology

1999-08-01

by routine DNA flow cytometry vation. ERBB2 expression was detected with a to determine DNA index (DI). commercially available antibody (Oncogene Sci...supplements the information gained from ic microsatellite primers. We observed that the ploidy analysis by DNA flow cytometry alone. In DNA so obtained...preserved the proportionality of many cases where flow cytometry could not be per- the different alleles as found in the original sample. formed because the
Inhibition of Breast Cancer-Induced Angiogenesis by a Diverged Homeobox Gene

DTIC Science & Technology

2006-05-01

and then harvested for flow cytometry using appropriate antibodies. Ad.Gax blocked the expression of VCAM-1, E-selectin, and ICAM-1. DOD Idea Award...solution. Bands were visualized by chemiluminescence using the ECL-Plus reagent (Amersham, Piscataway, NJ). Flow Cytometry Cells were harvested after...33), all of whose down-regulation we have confirmed using real time quantitative RT-PCR, Western blot, and flow cytometry (Fig. 5). Moreover, Gax
Probing Tumor Microenvironment With In Vivo Phage Display

DTIC Science & Technology

2014-10-01

C). (C) Dot plots showing mCherry expression on the X axis and fibroblast activation protein ( FAP ) or rabbit isotype control staining in the Y...by flow cytometry-based cell sorting using an antibody against fibroblast activation protein ( FAP ). During the optimization steps, flow cytometry...expression of αvβ3 and αvβ5 integrins, neuropilin-1 (NRP-1), and fibroblast activation protein ( FAP ) in hb6011 CAFs was analyzed by flow cytometry
Flow Cytometry Technician | Center for Cancer Research

Cancer.gov

PROGRAM DESCRIPTION The Basic Science Program (BSP) pursues independent, multidisciplinary research in basic and applied molecular biology, immunology, retrovirology, cancer biology, and human genetics. Research efforts and support are an integral part of the Center for Cancer Research (CCR) at the Frederick National Laboratory for Cancer Research (FNLCR). KEY ROLES/RESPONSIBILITIES The Flow Cytometry Core (Flow Core) of the Cancer and Inflammation Program (CIP) is a service core which supports the research efforts of the CCR by providing expertise in the field of flow cytometry (using analyzers and sorters) with the goal of gaining a more thorough understanding of the biology of cancer and cancer cells. The Flow Core provides service to 12-15 CIP laboratories and more than 22 non-CIP laboratories. Flow core staff provide technical advice on the experimental design of applications, which include immunological phenotyping, cell function assays, and cell cycle analysis. Work is performed per customer requirements, and no independent research is involved. The Flow Cytometry Technician will be responsible for: Monitor performance of and maintain high dimensional flow cytometer analyzers and cell sorters Operate high dimensional flow cytometer analyzers and cell sorters Monitoring lab supply levels and order lab supplies, perform various record keeping responsibilities Assist in the training of scientific end users on the use of flow cytometry in their research, as well as how to operate and troubleshoot the bench-top analyzer instruments Experience with sterile technique and tissue culture
Interferon regulatory factor 1 and histone H4 acetylation in systemic lupus erythematosus

PubMed Central

Leung, Yiu Tak; Shi, Lihua; Maurer, Kelly; Song, Li; Zhang, Zhe; Petri, Michelle; Sullivan, Kathleen E

2015-01-01

Histone acetylation modulates gene expression and has been described as increased in systemic lupus erythematosus (SLE). We investigated interferon regulatory factor 1 (IRF1) interactions that influence H4 acetylation (H4ac) in SLE. Intracellular flow cytometry for H4 acetylated lysine (K) 5, K8, K12, and K16 was performed. Histone acetylation was defined in monocytes and T cells from controls and SLE patients. RNA-Seq studies were performed on monocytes to look for an imbalance in histone acetyltransferases and histone deacetylase enzyme expression. Expression levels were validated using real-time quantitative RT-PCR. IRF1 induction of H4ac was evaluated using D54MG cells overexpressing IRF1. IRF1 protein interactions were studied using co-immunoprecipitation assays. IRF1-dependent recruitment of histone acetyltransferases to target genes was examined by ChIP assays using p300 antibody. Flow cytometry data showed significantly increased H4K5, H4K8, H4K12, and H4K16 acetylation in SLE monocytes. HDAC3 and HDAC11 gene expression were decreased in SLE monocytes. PCAF showed significantly higher gene expression in SLE than controls. IRF1-overexpressing D54MG cells were associated with significantly increased H4K5, H4K8, and H4K12 acetylation compared to vector-control D54MG cells both globally and at specific target genes. Co-immunoprecipitation studies using D54MG cells revealed IRF1 protein-protein interactions with PCAF, P300, CBP, GCN5, ATF2, and HDAC3. ChIP experiments demonstrated increased p300 recruitment to known IRF1 targets in D54MG cells overexpressing IRF1. In contrast, p300 binding to IRF1 targets decreased in D54MG cells with IRF1 knockdown. SLE appears to be associated with an imbalance in histone acetyltransferases and histone deacetylase enzymes favoring pathologic H4 acetylation. Furthermore, IRF1 directly interacts with chromatin modifying enzymes, supporting a model where recruitment to specific target genes is mediated in part by IRF1. PMID:25611806
Analysis of single mammalian cells on-chip.

PubMed

Sims, Christopher E; Allbritton, Nancy L

2007-04-01

A goal of modern biology is to understand the molecular mechanisms underlying cellular function. The ability to manipulate and analyze single cells is crucial for this task. The advent of microengineering is providing biologists with unprecedented opportunities for cell handling and investigation on a cell-by-cell basis. For this reason, lab-on-a-chip (LOC) technologies are emerging as the next revolution in tools for biological discovery. In the current discussion, we seek to summarize the state of the art for conventional technologies in use by biologists for the analysis of single, mammalian cells, and then compare LOC devices engineered for these same single-cell studies. While a review of the technical progress is included, a major goal is to present the view point of the practicing biologist and the advances that might increase adoption by these individuals. The LOC field is expanding rapidly, and we have focused on areas of broad interest to the biology community where the technology is sufficiently far advanced to contemplate near-term application in biological experimentation. Focus areas to be covered include flow cytometry, electrophoretic analysis of cell contents, fluorescent-indicator-based analyses, cells as small volume reactors, control of the cellular microenvironment, and single-cell PCR.
Near infrared lasers in flow cytometry.

PubMed

Telford, William G

2015-07-01

Technology development in flow cytometry has closely tracked laser technology, the light source that flow cytometers almost exclusively use to excite fluorescent probes. The original flow cytometers from the 1970s and 1980s used large water-cooled lasers to produce only one or two laser lines at a time. Modern cytometers can take advantage of the revolution in solid state laser technology to use almost any laser wavelength ranging from the ultraviolet to the near infrared. Commercial cytometers can now be equipped with many small solid state lasers, providing almost any wavelength needed for cellular analysis. Flow cytometers are now equipped to analyze 20 or more fluorescent probes simultaneously, requiring multiple laser wavelengths. Instrument developers are now trying to increase this number by designing fluorescent probes that can be excited by laser wavelength at the "edges" of the visible light range, in the near ultraviolet and near-infrared region. A variety of fluorescent probes have been developed that excite with violet and long wavelength ultraviolet light; however, the near-infrared range (660-800 nm) has yet seen only exploitation in flow cytometry. Fortunately, near-infrared laser diodes and other solid state laser technologies appropriate for flow cytometry have been in existence for some time, and can be readily incorporated into flow cytometers to accelerate fluorescent probe development. The near infrared region represents one of the last "frontiers" to maximize the number of fluorescent probes that can be analyzed by flow cytometry. In addition, near infrared fluorescent probes used in biomedical tracking and imaging could also be employed for flow cytometry with the correct laser wavelengths. This review describes the available technology, including lasers, fluorescent probes and detector technology optimal for near infrared signal detection. Published by Elsevier Inc.
Blockade of Tumor Cell TGF-Betas: A Strategy to Reverse Antiestrogen Resistance in Human Breast Cancer

DTIC Science & Technology

2002-01-01

the TM- FKHRL1 construct exhibited exclusive nuclear localization Cell Cycle Analysis by Flow Cytometry of the HA-tagged mutant under any experimental...distribution as measured by flow cytometry (Figure 8A). ALS AND METHODS. Consistent with its antiapoptotic effect, these results, addi- tion of TGFI3... flow cytometry . Under these conditions more than 95% of selected cells expressed GFP at the time of experiments. Immunoblot Analysis. Cells were
Slow-Adhering Stem Cells Derived from Injured Skeletal Muscle Have Improved Regenerative Capacity

DTIC Science & Technology

2011-08-01

images. Flow Cytometry Assay of Stem Cell Markers SASCs (1 105) isolated from noninjured or injured muscle were collected and washed twice with...muscle. Results of flow cytometry further verified Sca-1 and CD34 expression in isolated SASCs, and a greater percentage of cells were positive for Sca-1...from both injured and control noninjured muscle were analyzed using flow cytometry for the immunofluorescent signal of Sca-1 and CD34. Results
Augmenting Trastuzumab Therapy against Breast Cancer through Selective Activation of NK Cells

DTIC Science & Technology

2014-12-01

purity as defined by CD3-CD56+ flow cytometry ) and activation (>50% expression of CD137). Breast cancer cell lines including MCF7 (A and E...purity as defined by CD3-CD56+ flow cytometry ) and activation (>50% expression of CD137). Chromium-labeled breast cancer cell lines including MCF7 (A...and Whiteside, T.L. 2007. A novel multiparametric flow cytometry -based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons
Hypoxia and Prx1 in Malignant Progression of Prostate Cancer

DTIC Science & Technology

2006-09-01

Species (ROS) Formation The rate of ROS formation was determined by flow cytometry analysis using the probe 20,70-dichlorofluorescin diacetate (DCFH-DA...DA were subjected to 4-h hypoxia treatment. After the indicated time, fluorescent cells were analyzed by flow cytometry . Western Blot Analysis Equal...species (ROS) generation was measured by flow cytometry at 0.5, 1, 2, 3, 6, 12, or 24 h after hypoxia treatment. The rate of ROS generation increased
Significances of red cell bound immunoglobulin G as detected by flow cytometry in patients with Coombs-negative immune hemolysis.

PubMed

Thedsawad, A; Taka, O; Wanachiwanawin, W

2016-04-01

This study was to investigate the use of flow cytometry for detection and quantitation of red blood cells (RBC) bound IgG in immune hemolysis of patients with autoimmune hemolytic anaemia (AIHA) and systematic lupus erythematosus (SLE). Two to ten percent of patients with warm-autoimmune hemolytic anaemia (WAIHA) exhibit a negative direct Coombs test. Flow cytometry has been applied to detect RBC bound IgG with high accuracy, reproducibility and sensitivity. In this study 45 and 75 patients with AIHA and SLE, respectively were evaluated for RBC bound IgG by direct Coombs test and flow cytometry. Seventy-one percent (32/45) and 31% (23/75) of patients with AIHA and SLE respectively, had laboratory evidence of hemolysis. A positive flow cytometry, as defined by mean fluorescent intensity (MFI) values >0·21 and IgG molecules >28, was found in 4 of 32 (12·5%) and 4 of 23 (17·4%) patients with AIHA and SLE who had hemolysis with a negative direct Coombs test. There were very strong and strong correlations between the strength of direct Coombs test with MFI values and IgG molecules in patients with AIHA and SLE, respectively. Flow cytometry can be applied in the diagnosis of Coombs-negative hemolytic anaemia in patients with AIHA and SLE. © 2016 British Blood Transfusion Society.
Immunophenotyping of acute leukaemias by flow cytometry: a review.

PubMed

Pamnani, R

2009-12-01

To provide an overview of the utility of flow cytometry for phenotyping of acute leukaemias and selection-of monoclonal antibodies. The literature review was obtained through internet, journals and chapters in the relevant books. Relevant articles and chapters on immunophenotyping of acute leukaemias were selected from respected international journals and books in the field of haematology and were reviewed. Complete articles relevant to the topic were selected and reviewed and the necessary information extracted for this review. Flow cytometry has been used extensively in recent years to characterise haemopoeitic malignancies and done routinely in the developed world. This technique has greatly improved the diagnosis and classification of haemopoeitic malignancies and has been recommended by World Health Organisation classification (WHO) of tumours of haemopoeitic and lymphoid tissue. Application of flow cytometry for the diagnosis of leukaemias has been recently introduced in Kenya and is currently being undertaken in research using limited but appropriate panels of monoclonal antibodies. It is hoped that findings of this research will inform the use of flow cytometry as an ancillary diagnostic technique in our resource-constrained set up.

A classification tree approach for improving the utilization of flow cytometry testing of blood specimens for B-cell non-Hodgkin lymphoproliferative disorders.

PubMed

Healey, Ryan; Naugler, Christopher; de Koning, Lawrence; Patel, Jay L

2015-01-01

We sought to improve the diagnostic efficiency of flow cytometry investigation on blood by developing data-driven ordering guidelines. Our goal was to improve flow cytometry utilization by decreasing negative testing, therefore reducing healthcare costs. We investigated several laboratory tests performed alongside flow cytometry to identify biomarkers useful in excluding non-leukemic bloods. Test results and patient demographic features were subjected to receiver-operator characteristic (ROC) curve, logistic regression and classification tree analyses to find significant predictors and develop decision rules. Our data show that, in the absence of a compelling clinical indication, flow cytometry testing is largely non-informative on bloods from patients less than 50 years of age having an absolute lymphocyte count (ALC) below 5.0 × 10(9)/L. For patients over age 50 having an ALC below this value, a ferritin value above 450 μg/L is counter-indicative of B-cell clonality. Using these guidelines, 26% of cases were correctly predicted as negative with greater than 97% accuracy.
DNA Detection by Flow Cytometry using PNA-Modified Metal-Organic Framework Particles.

PubMed

Mejia-Ariza, Raquel; Rosselli, Jessica; Breukers, Christian; Manicardi, Alex; Terstappen, Leon W M M; Corradini, Roberto; Huskens, Jurriaan

2017-03-23

A DNA-sensing platform is developed by exploiting the easy surface functionalization of metal-organic framework (MOF) particles and their highly parallelized fluorescence detection by flow cytometry. Two strategies were employed to functionalize the surface of MIL-88A, using either covalent or non-covalent interactions, resulting in alkyne-modified and biotin-modified MIL-88A, respectively. Covalent surface coupling of an azide-dye and the alkyne-MIL-88A was achieved by means of a click reaction. Non-covalent streptavidin-biotin interactions were employed to link biotin-PNA to biotin-MIL-88A particles mediated by streptavidin. Characterization by confocal imaging and flow cytometry demonstrated that DNA can be bound selectively to the MOF surface. Flow cytometry provided quantitative data of the interaction with DNA. Making use of the large numbers of particles that can be simultaneously processed by flow cytometry, this MOF platform was able to discriminate between fully complementary, single-base mismatched, and randomized DNA targets. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
qFlow Cytometry-Based Receptoromic Screening: A High-Throughput Quantification Approach Informing Biomarker Selection and Nanosensor Development.

PubMed

Chen, Si; Weddell, Jared; Gupta, Pavan; Conard, Grace; Parkin, James; Imoukhuede, Princess I

2017-01-01

Nanosensor-based detection of biomarkers can improve medical diagnosis; however, a critical factor in nanosensor development is deciding which biomarker to target, as most diseases present several biomarkers. Biomarker-targeting decisions can be informed via an understanding of biomarker expression. Currently, immunohistochemistry (IHC) is the accepted standard for profiling biomarker expression. While IHC provides a relative mapping of biomarker expression, it does not provide cell-by-cell readouts of biomarker expression or absolute biomarker quantification. Flow cytometry overcomes both these IHC challenges by offering biomarker expression on a cell-by-cell basis, and when combined with calibration standards, providing quantitation of biomarker concentrations: this is known as qFlow cytometry. Here, we outline the key components for applying qFlow cytometry to detect biomarkers within the angiogenic vascular endothelial growth factor receptor family. The key aspects of the qFlow cytometry methodology include: antibody specificity testing, immunofluorescent cell labeling, saturation analysis, fluorescent microsphere calibration, and quantitative analysis of both ensemble and cell-by-cell data. Together, these methods enable high-throughput quantification of biomarker expression.
Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

PubMed Central

Hossain, Md. Sharoare; Johannisson, Anders; Wallgren, Margareta; Nagy, Szabolcs; Siqueira, Amanda Pimenta; Rodriguez-Martinez, Heriberto

2011-01-01

Flow cytometry is now a recognized methodology within animal spermatology, and has moved from being a research tool to become routine in the assessment of animal semen destined to breeding. The availability of ‘bench-top' flow cytometers and of newer and versatile markers for cell structure and function had allowed the instrumentation to measure more sperm parameters, from viability to reactiveness when exposed to exogenous stimuli, and to increase our capabilities to sort spermatozoa for potential fertilizing capacity, or chromosomal sex. The present review summarizes the state of the art regarding flow cytometry applied to animal andrology, albeit keeping an open comparative intent. It critically evaluates the present and future capabilities of flow cytometry for the diagnostics of potential fertility and for the development of current reproductive technologies such as sperm freezing, sperm selection and sperm sorting. The flow cytometry methods will probably further revolutionize our understanding of the sperm physiology and their functionality, and will undoubtedly extend its application in isolating many uncharacterized features of spermatozoa. However, continuous follow-up of the methods is a necessity owing to technical developments and the complexity of mapping spermatozoa. PMID:21478895
CytometryML: a markup language for analytical cytology

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Leif, Stephanie H.; Leif, Suzanne B.

2003-06-01

Cytometry Markup Language, CytometryML, is a proposed new analytical cytology data standard. CytometryML is a set of XML schemas for encoding both flow cytometry and digital microscopy text based data types. CytometryML schemas reference both DICOM (Digital Imaging and Communications in Medicine) codes and FCS keywords. These schemas provide representations for the keywords in FCS 3.0 and will soon include DICOM microscopic image data. Flow Cytometry Standard (FCS) list-mode has been mapped to the DICOM Waveform Information Object. A preliminary version of a list mode binary data type, which does not presently exist in DICOM, has been designed. This binary type is required to enhance the storage and transmission of flow cytometry and digital microscopy data. Index files based on Waveform indices will be used to rapidly locate the cells present in individual subsets. DICOM has the advantage of employing standard file types, TIF and JPEG, for Digital Microscopy. Using an XML schema based representation means that standard commercial software packages such as Excel and MathCad can be used to analyze, display, and store analytical cytometry data. Furthermore, by providing one standard for both DICOM data and analytical cytology data, it eliminates the need to create and maintain special purpose interfaces for analytical cytology data thereby integrating the data into the larger DICOM and other clinical communities. A draft version of CytometryML is available at www.newportinstruments.com.
Flow cytometry with gold nanoparticles and their clusters as scattering contrast agents: FDTD simulation of light-cell interaction.

PubMed

Tanev, Stoyan; Sun, Wenbo; Pond, James; Tuchin, Valery V; Zharov, Vladimir P

2009-09-01

The formulation of the finite-difference time-domain (FDTD) approach is presented in the framework of its potential applications to in-vivo flow cytometry based on light scattering. The consideration is focused on comparison of light scattering by a single biological cell alone in controlled refractive-index matching conditions and by cells labeled by gold nanoparticles. The optical schematics including phase contrast (OPCM) microscopy as a prospective modality for in-vivo flow cytometry is also analyzed. The validation of the FDTD approach for the simulation of flow cytometry may open up a new avenue in the development of advanced cytometric techniques based on scattering effects from nanoscale targets. 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The role of flow cytometry in companion animal diagnostic medicine.

PubMed

Tarrant, Jacqueline M

2005-11-01

Flow cytometry is a powerful tool for characterising the composition of complex cell populations. The accuracy and precision of this technology for describing and enumerating cells exceeds traditional methods. The number of diagnostic veterinary laboratories with access to a dedicated machine is increasing, and there is the potential to offer a clinical flow cytometry service. The improved availability of monoclonal antibodies (mAb) to cell markers expressed by the leukocytes of companion animals, permits the implementation of comprehensive mAb panels suitable for diagnosis of lympho- and myeloproliferative disease. Reticulated erythrocyte and platelet quantification, antiglobulin assays for immune-mediated cytopenias, lymphocyte subset analysis, and immunophenotyping of lymphoma and leukemia, have been validated for companion animal samples on the flow cytometer. It is now timely to consider the role of flow cytometry in diagnostic practice, and the requirement for quality assurance and standardization of testing procedures.
FlowSOM: Using self-organizing maps for visualization and interpretation of cytometry data.

PubMed

Van Gassen, Sofie; Callebaut, Britt; Van Helden, Mary J; Lambrecht, Bart N; Demeester, Piet; Dhaene, Tom; Saeys, Yvan

2015-07-01

The number of markers measured in both flow and mass cytometry keeps increasing steadily. Although this provides a wealth of information, it becomes infeasible to analyze these datasets manually. When using 2D scatter plots, the number of possible plots increases exponentially with the number of markers and therefore, relevant information that is present in the data might be missed. In this article, we introduce a new visualization technique, called FlowSOM, which analyzes Flow or mass cytometry data using a Self-Organizing Map. Using a two-level clustering and star charts, our algorithm helps to obtain a clear overview of how all markers are behaving on all cells, and to detect subsets that might be missed otherwise. R code is available at https://github.com/SofieVG/FlowSOM and will be made available at Bioconductor. © 2015 International Society for Advancement of Cytometry.
Web-based analysis and publication of flow cytometry experiments.

PubMed

Kotecha, Nikesh; Krutzik, Peter O; Irish, Jonathan M

2010-07-01

Cytobank is a Web-based application for storage, analysis, and sharing of flow cytometry experiments. Researchers use a Web browser to log in and use a wide range of tools developed for basic and advanced flow cytometry. In addition to providing access to standard cytometry tools from any computer, Cytobank creates a platform and community for developing new analysis and publication tools. Figure layouts created on Cytobank are designed to allow transparent access to the underlying experiment annotation and data processing steps. Since all flow cytometry files and analysis data are stored on a central server, experiments and figures can be viewed or edited by anyone with the proper permission, from any computer with Internet access. Once a primary researcher has performed the initial analysis of the data, collaborators can engage in experiment analysis and make their own figure layouts using the gated, compensated experiment files. Cytobank is available to the scientific community at http://www.cytobank.org. (c) 2010 by John Wiley & Sons, Inc.
Web-Based Analysis and Publication of Flow Cytometry Experiments

PubMed Central

Kotecha, Nikesh; Krutzik, Peter O.; Irish, Jonathan M.

2014-01-01

Cytobank is a web-based application for storage, analysis, and sharing of flow cytometry experiments. Researchers use a web browser to log in and use a wide range of tools developed for basic and advanced flow cytometry. In addition to providing access to standard cytometry tools from any computer, Cytobank creates a platform and community for developing new analysis and publication tools. Figure layouts created on Cytobank are designed to allow transparent access to the underlying experiment annotation and data processing steps. Since all flow cytometry files and analysis data are stored on a central server, experiments and figures can be viewed or edited by anyone with the proper permissions from any computer with Internet access. Once a primary researcher has performed the initial analysis of the data, collaborators can engage in experiment analysis and make their own figure layouts using the gated, compensated experiment files. Cytobank is available to the scientific community at www.cytobank.org PMID:20578106
Confocal Microscopy and Flow Cytometry System Performance: Assessment of QA Parameters that affect data Quanitification

EPA Science Inventory

Flow and image cytometers can provide useful quantitative fluorescence data. We have devised QA tests to be used on both a flow cytometer and a confocal microscope to assure that the data is accurate, reproducible and precise. Flow Cytometry: We have provided two simple perform...
Therapy of Prostate Cancer Using a Human Antibody Targeting the Type 1 Insulin-Like Growth Factor Receptor (IGF-IR)

DTIC Science & Technology

2009-09-01

euthanized, tumors harvested and portions processed for IHC, Western blot, flow cytometry , culture, and RNA analysis. If not enough tissue is available...temperature for 60 minutes. Samples were analyzed by flow cytometry using a BD FACScan. Data were analyzed with CellQuestPRO software. Evaluation of BrdUrd...were approved by the University of Washington Institutional Animal Care and Use Committee (IACUC). Flow cytometry . To measure tumor IGF-IR expression
Micro and Nano-mediated 3D Cardiac Tissue Engineering

DTIC Science & Technology

2009-10-01

in both flow cytometry and Western Blot applications. The CD34 antigen is important in stem cell research due to its widespread use in identifying...for further characterization. We generated a pCD34 expressing CHO cell line (CHO-CD34) and analyzed pCD34 expression by flow cytometry (Figure 1A... flow cytometry using the 3G7 antibody and co-stained with an anti-CD31 antibody (AbD Serotec; FITC conjugated). CD31 (PECAM) is a pan endothelial
Dose-Dependent Thresholds of 10-ns Electric Pulse Induced Plasma Membrane Disruption and Cytotoxicity in Multiple Cell Lines

DTIC Science & Technology

2011-01-01

normalized to parallel controls. Flow Cytometry and Confocal Microscopy Upon exposure to 10-ns EP, aliquots of the cellular suspension were added to a tube...Survival data was processed and plotted using GrapherH software (Golden Software, Golden, Colorado). Flow cytometry results were processed in C6 software...Accuri Cytometers, Inc., Ann Arbor, MI) and FCSExpress software (DeNovo Software, Los Angeles, CA). Final analysis and presentation of flow cytometry
Sensitivity of Breast Cancer Stem Cells to TRA-8 Anti-DR5 Monoclonal Antibody

DTIC Science & Technology

2012-02-01

cytotoxicity and reduction in BrCSC marker expression. A. 2LMP cells were sorted using flow cytometry for CD44+/CD24-/ALDHhigh. Cells were pre...cells were sorted using flow cytometry for ALDH? cells and allowed to form primary tumorspheres for 3 days. After tumorspheres were mechanically...n =5 ) Day Fig. 5 Effect of ex vivo treatment of BrCSC enriched cells on tumorgenicity in NOD/SCID mice. 2LMP cells were sorted using flow cytometry
Canonical Wnt Signaling as a Specific Marker of Normal and Tumorigenic Mammary Stem Cells

DTIC Science & Technology

2013-02-01

get enough sorted mammary cells for the transplantation experiments. We are currently working with our Flow Cytometry Core to sort Lin-/CD24+/CD49...activity our flow cytometry data suggests t here is a 2-fold increase in the number of FOG+ MEC’s in BATgal animals compared to contro ls which...this populat ion of cells is enriched for stem cell activity. Flow cytometry will determine the percentage of FOG+ cells within pre-neoplastic BATgai
Articular Cartilage Repair Through Muscle Cell-Based Tissue Engineering

DTIC Science & Technology

2010-03-01

results suggest that sFlt-1 has more of an enhancing effect in vivo. With cell markers and flow cytometry , investiga- tors at our laboratory have...were analyzed by flow cytometry . They were immunostained by desmin, vimentin and MyoD and their chondrogenic potential was evaluated under the...M1, M2, and M3) and 3 F-MDSC populations (F1, F2, and F3) were characterized by flow cytometry for CD34 and Sca1 expression. MDSCs were labeled with
Dynamic analysis of apoptosis using cyanine SYTO probes: From classical to microfluidic cytometry

PubMed Central

Wlodkowic, Donald; Skommer, Joanna; Faley, Shannon; Darzynkiewicz, Zbigniew; Cooper, Jonathan M.

2013-01-01

Cell death is a stochastic process, often initiated and/or executed in a multi-pathway/multi-organelle fashion. Therefore, high-throughput single-cell analysis platforms are required to provide detailed characterization of kinetics and mechanisms of cell death in heterogeneous cell populations. However, there is still a largely unmet need for inert fluorescent probes, suitable for prolonged kinetic studies. Here, we compare the use of innovative adaptation of unsymmetrical SYTO dyes for dynamic real-time analysis of apoptosis in conventional as well as microfluidic chip-based systems. We show that cyanine SYTO probes allow non-invasive tracking of intracellular events over extended time. Easy handling and “stain–no wash” protocols open up new opportunities for high-throughput analysis and live-cell sorting. Furthermore, SYTO probes are easily adaptable for detection of cell death using automated microfluidic chip-based cytometry. Overall, the combined use of SYTO probes and state-of-the-art Lab-on-a-Chip platform emerges as a cost effective solution for automated drug screening compared to conventional Annexin V or TUNEL assays. In particular, it should allow for dynamic analysis of samples where low cell number has so far been an obstacle, e.g. primary cancer stems cells or circulating minimal residual tumors. PMID:19298813
Flow Cytometry Scientist | Center for Cancer Research

Cancer.gov

PROGRAM DESCRIPTION The Basic Science Program (BSP) pursues independent, multidisciplinary research in basic and applied molecular biology, immunology, retrovirology, cancer biology, and human genetics. Research efforts and support are an integral part of the Center for Cancer Research (CCR) at the Frederick National Laboratory for Cancer Research (FNLCR). KEY ROLES/RESPONSIBILITIES The Flow Cytometry Core (Flow Core) in the Cancer and Inflammation Program (CIP) is a service core which supports the research efforts of the CCR by providing expertise in the field of flow cytometry (using analyzers and sorters) with the goal of gaining a more thorough understanding of the biology of the immune system, cancer, and inflammation processes. The Flow Core provides service to 12-15 CIP laboratories and more than 22 non-CIP laboratories. Flow core staff provide technical advice on the experimental design of applications, which include immunological phenotyping, cell function assays, and cell cycle analysis. Work is performed per customer requirements, and no independent research is involved. The Flow Cytometry Scientist will be responsible for: Daily management of the Flow Cytometry Core, to include the supervision and guidance of technical staff members Monitor performance of and maintain high dimensional flow cytometer analyzers and cell sorters Operate high dimensional flow cytometer analyzers and cell sorters Provide scientific expertise to the user community and facilitate the development of cutting edge technologies Interact with Flow Core users and customers, and provide technical and scientific advice, and guidance regarding their experiments, including possible collaborations Train staff and scientific end users on the use of flow cytometry in their research, as well as teach them how to operate and troubleshoot the bench-top analyzer instruments Prepare and deliver lectures, as well as one-on-one training sessions, with customers/users Ensure that protocols are up-to-date, and appropriately adhered to Experience with sterile technique and tissue culture
Spaceflight Flow Cytometry: Design Challenges and Applications

NASA Technical Reports Server (NTRS)

Pappas, Dimitri; Kao, Shih-Hsin; Jeevarajan, Antony S.

2004-01-01

Future space exploration missions will require analytical technology capable of providing both autonomous medical care to the crew and investigative capabilities to researchers. While several promising candidate technologies exist for further development, flow cytometry is an attractive technology as it offers both crew health and a wide array of biochemistry and immunology assays. While flow cytometry has been widely used for cellular analysis in both clinical and research settings, the requirements for proper operation in spaceflight impose constraints on any instrument designs. The challenges of designing a spaceflight-ready flow cytometer are discussed, as well as some preliminary results using a prototype system.

High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles

NASA Astrophysics Data System (ADS)

Regmi, Raju; Mohan, Kavya; Mondal, Partha Pratim

2014-09-01

Visualization of intracellular organelles is achieved using a newly developed high throughput imaging cytometry system. This system interrogates the microfluidic channel using a sheet of light rather than the existing point-based scanning techniques. The advantages of the developed system are many, including, single-shot scanning of specimens flowing through the microfluidic channel at flow rate ranging from micro- to nano- lit./min. Moreover, this opens-up in-vivo imaging of sub-cellular structures and simultaneous cell counting in an imaging cytometry system. We recorded a maximum count of 2400 cells/min at a flow-rate of 700 nl/min, and simultaneous visualization of fluorescently-labeled mitochondrial network in HeLa cells during flow. The developed imaging cytometry system may find immediate application in biotechnology, fluorescence microscopy and nano-medicine.
Role of CD81 and CD58 in minimal residual disease detection in pediatric B lymphoblastic leukemia.

PubMed

Tsitsikov, E; Harris, M H; Silverman, L B; Sallan, S E; Weinberg, O K

2018-06-01

Minimal residual disease (MRD) in B lymphoblastic leukemia has been demonstrated to be a powerful predictor of clinical outcome in numerous studies in both children and adults. In this study, we evaluated 86 pediatric patients with both diagnostic and remission flow cytometry studies and compared expression of CD81, CD58, CD19, CD34, CD20, and CD38 in the detection of MRD. We evaluated 86 patients with B lymphoblastic leukemia who had both diagnostic studies and remission studies for the presence of MRD using multicolor flow cytometry. We established our detection limit for identifying abnormal lymphoblasts using serial dilutions. We also compared flow cytometry findings with molecular MRD detection in a subset of patients. We found that we can resolve differences between hematogones and lymphoblasts in 85 of 86 cases using a combination of CD45, CD19, CD34, CD10, CD20, CD38, CD58, and CD81. Our detection limit using flow cytometry is 0.002% for detecting a population of abnormal B lymphoblasts. Comparison with MRD assessment by molecular methods showed a high concordance rate with flow cytometry findings. Our study highlights importance of using multiple markers to detect MRD in B lymphoblastic leukemia. Our findings indicate that including both CD58 and CD81 markers in addition to CD19, CD34, CD20, CD38, and CD10 are helpful in MRD detection by flow cytometry. © 2018 John Wiley & Sons Ltd.
An Active, Collaborative Approach to Learning Skills in Flow Cytometry

ERIC Educational Resources Information Center

Fuller, Kathryn; Linden, Matthew D.; Lee-Pullen, Tracey; Fragall, Clayton; Erber, Wendy N.; Röhrig, Kimberley J.

2016-01-01

Advances in science education research have the potential to improve the way students learn to perform scientific interpretations and understand science concepts. We developed active, collaborative activities to teach skills in manipulating flow cytometry data using FlowJo software. Undergraduate students were given compensated clinical flow…
Multiplex polymerase chain reaction on FTA cards vs. flow cytometry for B-lymphocyte clonality.

PubMed

Dictor, Michael; Skogvall, Ingela; Warenholt, Janina; Rambech, Eva

2007-01-01

Two-colour flow cytometry was compared with multiplex PCR with capillary electrophoresis for clonality determination in specific categories of B-cell lymphoma. FTA cards were evaluated for preserving DNA from node imprints and expediting molecular analysis. A single-tube multiplex PCR targeted IGH and lymphoma-specific translocations in DNA extracted from 180 frozen lymphoid tissues and DNA bound to FTA cards from 192 fresh tissues and 137 aspirates. PCR results were compared with flow cytometry in the extracted and aspirated samples. Overall, single-tube multiplex PCR sensitivity was equivalent in the sample groups (intergroup range 79%-91%). False negatives were associated with tumour origin in the follicle centre. Multiplex PCR and flow cytometry were equally sensitive and together detected 98% of B-cell lymphomas. Additional two-tube targeting of IGK suggested an overall molecular sensitivity >90%. False positive (pseudoclonal) single-tube multiplex PCR was associated with necrosis and sparse lymphocytes. Multiplex PCR using template DNA bound to an FTA card effectively detects B-lymphocyte clonality, obviates DNA extraction and refrigeration, and can be used without diminished sensitivity in fine needle aspirates or node imprints as a replacement for or complement to flow cytometry at any point in the diagnostic work-up.
CytometryML, an XML format based on DICOM and FCS for analytical cytology data.

PubMed

Leif, Robert C; Leif, Suzanne B; Leif, Stephanie H

2003-07-01

Flow Cytometry Standard (FCS) was initially created to standardize the software researchers use to analyze, transmit, and store data produced by flow cytometers and sorters. Because of the clinical utility of flow cytometry, it is necessary to have a standard consistent with the requirements of medical regulatory agencies. We extended the existing mapping of FCS to the Digital Imaging and Communications in Medicine (DICOM) standard to include list-mode data produced by flow cytometry, laser scanning cytometry, and microscopic image cytometry. FCS list-mode was mapped to the DICOM Waveform Information Object. We created a collection of Extensible Markup Language (XML) schemas to express the DICOM analytical cytologic text-based data types except for large binary objects. We also developed a cytometry markup language, CytometryML, in an open environment subject to continuous peer review. The feasibility of expressing the data contained in FCS, including list-mode in DICOM, was demonstrated; and a preliminary mapping for list-mode data in the form of XML schemas and documents was completed. DICOM permitted the creation of indices that can be used to rapidly locate in a list-mode file the cells that are members of a subset. DICOM and its coding schemes for other medical standards can be represented by XML schemas, which can be combined with other relevant XML applications, such as Mathematical Markup Language (MathML). The use of XML format based on DICOM for analytical cytology met most of the previously specified requirements and appears capable of meeting the others; therefore, the present FCS should be retired and replaced by an open, XML-based, standard CytometryML. Copyright 2003 Wiley-Liss, Inc.
Flow cytometry in the post fluorescence era.

PubMed

Nolan, Garry P

2011-12-01

While flow cytometry once enabled researchers to examine 10--15 cell surface parameters, new mass flow cytometry technology enables interrogation of up to 45 parameters on a single cell. This new technology has increased understanding of cell expression and how cells differentiate during hematopoiesis. Using this information, knowledge of leukemia cell biology has also increased. Other new technologies, such as SPADE analysis and single cell network profiling (SCNP), are enabling researchers to put different cancers into more biologically similar categories and have the potential to enable more personalized medicine. Copyright © 2011. Published by Elsevier Ltd.
Novel Therapeutic and Prophylactic Modalities to Protect U.S. Armed Forces Against Major Biological Threat Agents

DTIC Science & Technology

2004-10-01

using flow cytometry after staining with CBA kit produced by BD-Pharmingen. The CBA kit can simultaneously test 5 inflammatory cytokines that include...or TLR4 transfected cells using flow cytometry . CHOK1 and CHOR1.1 cells were plated out in a 24-well dish and transfected 24 h later with either TLR2...with PE-labeled anti-hTLR2 antibody or PE-isotype control antibody (eBiosciences, CA), and cells were analyzed by flow cytometry . 39 The expression
Rapid susceptibility testing of fungi by flow cytometry using vital staining.

PubMed Central

Wenisch, C; Linnau, K F; Parschalk, B; Zedtwitz-Liebenstein, K; Georgopoulos, A

1997-01-01

A 1-h assay for antifungal susceptibility testing measuring the impairment of fungal metabolic activity was developed. Yeast viability was analyzed by flow cytometry with a novel fluorescent probe, FUN-1, which emits a red fluorescence when the yeast is metabolically active. For nine Candida albicans strains tested, this method yielded results comparable to those obtained by the standard M27 procedure for amphotericin B, flucytosine, fluconazole, and ketoconazole. Whether the flow cytometry antifungal susceptibility test results correlate with the in vivo activities of the drugs remains to determined. PMID:8968873
Examination pf Potential Anti-Tumor Activity of N-Thiolated B-Lactam Antibiotics in Nude Mice Bearing Human Breast Tumors

DTIC Science & Technology

2005-08-01

temperature in the dark, and then analyzed by flow cytometry within 3 hr of staining. 2.7. Caspase-3/-7 activity assay To measure cell-free caspase-3/-7...were treated with 50 mM of lactam 12 for the indicated hours. (A) Measurement of sub-G1 DNA content by flow cytometry analysis. The percentage of sub...Daniel for critical reading of the manuscript. We also appreciate the assistance of the Flow Cytometry Core at H. Lee Moffitt Cancer Center
The use of flow cytometry to monitor chitin synthesis in regenerating protoplasts of Candida albicans.

PubMed

Hector, R F; Braun, P C; Hart, J T; Kamarck, M E

1990-01-01

Flow cytometry was used to monitor chitin synthesis in regenerating protoplasts of the yeast Candida albicans. Comparisons of cells stained with Calcofluor White, a fluorochrome with known affinity for chitin, and cells incubated in the presence of N-[3H]-acetylglucosamine, the precursor substrate for chitin, showed a linear relationship between fluorescence and incorporation of label over time. Changes in both the fluorescence and light scatter of regenerating protoplasts treated with inhibitors of fungal chitin synthase were also quantitated by flow cytometry.
Suppression of Antimicrobial Peptide Expression by Ureaplasma Species

PubMed Central

Crabb, Donna M.; Dai, Yuling; Chen, Yuying; Waites, Ken B.; Atkinson, T. Prescott

2014-01-01

Ureaplasma species commonly colonize the adult urogenital tract and are implicated in invasive diseases of adults and neonates. Factors that permit the organisms to cause chronic colonization or infection are poorly understood. We sought to investigate whether host innate immune responses, specifically, antimicrobial peptides (AMPs), are involved in determining the outcome of Ureaplasma infections. THP-1 cells, a human monocytoid tumor line, were cocultured with Ureaplasma parvum and U. urealyticum. Gene expression levels of a variety of host defense genes were quantified by real-time PCR. In vitro antimicrobial activities of synthetic AMPs against Ureaplasma spp. were determined using a flow cytometry-based assay. Chromosomal histone modifications in host defense gene promoters were tested by chromatin immunoprecipitation (ChIP). DNA methylation status in the AMP promoter regions was also investigated. After stimulation with U. parvum and U. urealyticum, the expression of cell defense genes, including the AMP genes (DEFB1, DEFA5, DEFA6, and CAMP), was significantly downregulated compared to that of TNFA and IL-8, which were upregulated. In vitro flow cytometry-based antimicrobial assay revealed that synthetic peptides LL-37, hBD-3, and hBD-1 had activity against Ureaplasma spp. Downregulation of the AMP genes was associated with chromatin modification alterations, including the significantly decreased histone H3K9 acetylation with U. parvum infection. No DNA methylation status changes were detected upon Ureaplasma infection. In conclusion, AMPs have in vitro activity against Ureaplasma spp., and suppression of AMP expression might be important for the organisms to avoid this aspect of the host innate immune response and to establish chronic infection and colonization. PMID:24491573
Suppression of antimicrobial peptide expression by ureaplasma species.

PubMed

Xiao, Li; Crabb, Donna M; Dai, Yuling; Chen, Yuying; Waites, Ken B; Atkinson, T Prescott

2014-04-01

Ureaplasma species commonly colonize the adult urogenital tract and are implicated in invasive diseases of adults and neonates. Factors that permit the organisms to cause chronic colonization or infection are poorly understood. We sought to investigate whether host innate immune responses, specifically, antimicrobial peptides (AMPs), are involved in determining the outcome of Ureaplasma infections. THP-1 cells, a human monocytoid tumor line, were cocultured with Ureaplasma parvum and U. urealyticum. Gene expression levels of a variety of host defense genes were quantified by real-time PCR. In vitro antimicrobial activities of synthetic AMPs against Ureaplasma spp. were determined using a flow cytometry-based assay. Chromosomal histone modifications in host defense gene promoters were tested by chromatin immunoprecipitation (ChIP). DNA methylation status in the AMP promoter regions was also investigated. After stimulation with U. parvum and U. urealyticum, the expression of cell defense genes, including the AMP genes (DEFB1, DEFA5, DEFA6, and CAMP), was significantly downregulated compared to that of TNFA and IL-8, which were upregulated. In vitro flow cytometry-based antimicrobial assay revealed that synthetic peptides LL-37, hBD-3, and hBD-1 had activity against Ureaplasma spp. Downregulation of the AMP genes was associated with chromatin modification alterations, including the significantly decreased histone H3K9 acetylation with U. parvum infection. No DNA methylation status changes were detected upon Ureaplasma infection. In conclusion, AMPs have in vitro activity against Ureaplasma spp., and suppression of AMP expression might be important for the organisms to avoid this aspect of the host innate immune response and to establish chronic infection and colonization.
In vivo plant flow cytometry: A first proof-of-concept

PubMed Central

Nedosekin, Dmitry A.; Khodakovskaya, Mariya V.; Biris, Alexandru S.; Wang, Daoyuan; Xu, Yang; Villagarcia, Hector; Galanzha, Ekaterina I.; Zharov, Vladimir P.

2011-01-01

In vivo flow cytometry has facilitated advances in the ultrasensitive detection of tumor cells, bacteria, nanoparticles, dyes, and other normal and abnormal objects directly in blood and lymph circulatory systems. Here, we propose in vivo plant flow cytometry for the real-time noninvasive study of nanomaterial transport in xylem and phloem plant vascular systems. As a proof of this concept, we demonstrate in vivo real-time photoacoustic monitoring of quantum dot-carbon nanotube conjugate uptake and uptake by roots and spreading through stem to leaves in a tomato plant. In addition, in vivo scanning cytometry using multimodal photoacoustic, photothermal, and fluorescent detection schematics provided multiplex detection and identification of nanoparticles accumulated in plant leaves in the presence of intensive absorption, scattering, and autofluorescent backgrounds. The use of a portable fiber-based photoacoustic flow cytometer for studies of plant vasculature was demonstrated. These integrated cytometry modalities using both endogenous and exogenous contrast agents have a potential to open new avenues of in vivo study of the nutrients, products of photosynthesis and metabolism, nanoparticles, infectious agents, and other objects transported through plant vasculature. PMID:21905208
Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

NASA Astrophysics Data System (ADS)

Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

2016-09-01

Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis.
Isolation of purified oocyst walls and sporocysts from Toxoplasma gondii.

PubMed

Everson, William V; Ware, Michael W; Dubey, J P; Lindquist, H D Alan

2002-01-01

Toxoplasma gondii oocysts are environmentally resistant and can infect virtually all warm-blooded hosts, including humans and livestock. Little is known about the biochemical basis for this resistance of oocysts, and mechanism for excystation of T. gondii sporozoites. The objective of the present study was to evaluate different methods (mechanical fragmentation, gradients, flow cytometry) to separate and purify T. gondii oocyst walls and sporocysts. Oocyst walls were successfully separated and purified using iodixanol gradients. Sporocysts were successfully separated and purified using iodixanol and Percoll gradients. Purification was also achieved by flow cytometry. Flow cytometry with fluorescence-activated cell sorting (FACS) yielded analytical quantities of oocyst walls and intact sporocysts. Flow cytometry with FACS also proved useful for quantitation of purity obtained following iodixanol gradient fractionation. Methods reported in this paper will be useful for analytical purposes, such as proteomic analysis of components unique to this life cycle stage, development of detection methods, or excystation studies.
Methodology and application of flow cytometry for investigation of human malaria parasites.

PubMed

Grimberg, Brian T

2011-03-31

Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using drugs or antibodies, have relied on the use of microscopy or radioactive hypoxanthine uptake. These are considered gold standards for measuring the effectiveness of antimalarial treatments, however, these methods have well known shortcomings. With the advent of flow cytometry coupled with the use of fluorescent DNA stains allowed for increased speed, reproducibility, and qualitative estimates of the effectiveness of antibodies and drugs to limit malaria parasite growth which addresses the challenges of traditional techniques. Because materials and machines available to research facilities are so varied, different methods have been developed to investigate malaria parasites by flow cytometry. This review is intended to serve as a reference guide for advanced users and importantly, as a primer for new users, to support expanded use and improvements to malaria flow cytometry, particularly in endemic countries. Copyright © 2011 Elsevier B.V. All rights reserved.
Alternatives to current flow cytometry data analysis for clinical and research studies.

PubMed

Gondhalekar, Carmen; Rajwa, Bartek; Patsekin, Valery; Ragheb, Kathy; Sturgis, Jennifer; Robinson, J Paul

2018-02-01

Flow cytometry has well-established methods for data analysis based on traditional data collection techniques. These techniques typically involved manual insertion of tube samples into an instrument that, historically, could only measure 1-3 colors. The field has since evolved to incorporate new technologies for faster and highly automated sample preparation and data collection. For example, the use of microwell plates on benchtop instruments is now a standard on virtually every new instrument, and so users can easily accumulate multiple data sets quickly. Further, because the user must carefully define the layout of the plate, this information is already defined when considering the analytical process, expanding the opportunities for automated analysis. Advances in multi-parametric data collection, as demonstrated by the development of hyperspectral flow-cytometry, 20-40 color polychromatic flow cytometry, and mass cytometry (CyTOF), are game-changing. As data and assay complexity increase, so too does the complexity of data analysis. Complex data analysis is already a challenge to traditional flow cytometry software. New methods for reviewing large and complex data sets can provide rapid insight into processes difficult to define without more advanced analytical tools. In settings such as clinical labs where rapid and accurate data analysis is a priority, rapid, efficient and intuitive software is needed. This paper outlines opportunities for analysis of complex data sets using examples of multiplexed bead-based assays, drug screens and cell cycle analysis - any of which could become integrated into the clinical environment. Copyright © 2017. Published by Elsevier Inc.
Flow Cytometry Sorting to Separate Viable Giant Viruses from Amoeba Co-culture Supernatants

PubMed Central

Khalil, Jacques Y. B.; Langlois, Thierry; Andreani, Julien; Sorraing, Jean-Marc; Raoult, Didier; Camoin, Laurence; La Scola, Bernard

2017-01-01

Flow cytometry has contributed to virology but has faced many drawbacks concerning detection limits, due to the small size of viral particles. Nonetheless, giant viruses changed many concepts in the world of viruses, as a result of their size and hence opened up the possibility of using flow cytometry to study them. Recently, we developed a high throughput isolation of viruses using flow cytometry and protozoa co-culture. Consequently, isolating a viral mixture in the same sample became more common. Nevertheless, when one virus multiplies faster than others in the mixture, it is impossible to obtain a pure culture of the minority population. Here, we describe a robust sorting system, which can separate viable giant virus mixtures from supernatants. We tested three flow cytometry sorters by sorting artificial mixtures. Purity control was assessed by electron microscopy and molecular biology. As proof of concept, we applied the sorting system to a co-culture supernatant taken from a sample containing a viral mixture that we couldn't separate using end point dilution. In addition to isolating the quick-growing Mimivirus, we sorted and re-cultured a new, slow-growing virus, which we named “Cedratvirus.” The sorting assay presented in this paper is a powerful and versatile tool for separating viral populations from amoeba co-cultures and adding value to the new field of flow virometry. PMID:28111619
Flow Cytometry Sorting to Separate Viable Giant Viruses from Amoeba Co-culture Supernatants.

PubMed

Khalil, Jacques Y B; Langlois, Thierry; Andreani, Julien; Sorraing, Jean-Marc; Raoult, Didier; Camoin, Laurence; La Scola, Bernard

2016-01-01

Flow cytometry has contributed to virology but has faced many drawbacks concerning detection limits, due to the small size of viral particles. Nonetheless, giant viruses changed many concepts in the world of viruses, as a result of their size and hence opened up the possibility of using flow cytometry to study them. Recently, we developed a high throughput isolation of viruses using flow cytometry and protozoa co-culture. Consequently, isolating a viral mixture in the same sample became more common. Nevertheless, when one virus multiplies faster than others in the mixture, it is impossible to obtain a pure culture of the minority population. Here, we describe a robust sorting system, which can separate viable giant virus mixtures from supernatants. We tested three flow cytometry sorters by sorting artificial mixtures. Purity control was assessed by electron microscopy and molecular biology. As proof of concept, we applied the sorting system to a co-culture supernatant taken from a sample containing a viral mixture that we couldn't separate using end point dilution. In addition to isolating the quick-growing Mimivirus , we sorted and re-cultured a new, slow-growing virus, which we named "Cedratvirus." The sorting assay presented in this paper is a powerful and versatile tool for separating viral populations from amoeba co-cultures and adding value to the new field of flow virometry.
Flow cytometric HyPer-based assay for hydrogen peroxide.

PubMed

Lyublinskaya, O G; Antonov, S A; Gorokhovtsev, S G; Pugovkina, N A; Kornienko, Ju S; Ivanova, Ju S; Shatrova, A N; Aksenov, N D; Zenin, V V; Nikolsky, N N

2018-05-30

HyPer is a genetically encoded fluorogenic sensor for hydrogen peroxide which is generally used for the ratiometric imaging of H 2 O 2 fluxes in living cells. Here, we demonstrate the advantages of HyPer-based ratiometric flow cytometry assay for H 2 O 2 , by using K562 and human mesenchymal stem cell lines expressing HyPer. We show that flow cytometry analysis is suitable to detect HyPer response to submicromolar concentrations of extracellularly added H 2 O 2 that is much lower than concentrations addressed previously in the other HyPer-based assays (such as cell imaging or fluorimetry). Suggested technique is also much more sensitive to hydrogen peroxide than the widespread flow cytometry assay exploiting H 2 O 2 -reactive dye H 2 DCFDA and, contrary to the H 2 DCFDA-based assay, can be employed for the kinetic studies of H 2 O 2 utilization by cells, including measurements of the rate constants of H 2 O 2 removal. In addition, flow cytometry multi-parameter ratiometric measurements enable rapid and high-throughput detection of endogenously generated H 2 O 2 in different subpopulations of HyPer-expressing cells. To sum up, HyPer can be used in multi-parameter flow cytometry studies as a highly sensitive indicator of intracellular H 2 O 2 . Copyright © 2018. Published by Elsevier Inc.

Label-free counting of circulating cells by in vivo photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Zhou, Quanyu; Yang, Ping; Wang, Qiyan; Pang, Kai; Zhou, Hui; He, Hao; Wei, Xunbin

2018-02-01

Melanoma, developing from melanocytes, is the most serious type of skin cancer. Circulating melanoma cells, the prognosis marker for metastasis, are present in the circulation at the early stage. Thus, quantitative detection of rare circulating melanoma cells is essential for monitoring tumor metastasis and prognosis evaluation. Compared with in vitro assays, in vivo flow cytometry is able to identify circulating tumor cells without drawing blood. Here, we built in vivo photoacoustic flow cytometry based on the high absorption coefficient of melanoma cells, which is applied to labelfree counting of circulating melanoma cells in tumor-bearing mice.
In Vivo Myeloperoxidase Imaging and Flow Cytometry Analysis of Intestinal Myeloid Cells.

PubMed

Hülsdünker, Jan; Zeiser, Robert

2016-01-01

Myeloperoxidase (MPO) imaging is a non-invasive method to detect cells that produce the enzyme MPO that is most abundant in neutrophils, macrophages, and inflammatory monocytes. While lacking specificity for any of these three cell types, MPO imaging can provide guidance for further flow cytometry-based analysis of tissues where these cell types reside. Isolation of leukocytes from the intestinal tract is an error-prone procedure. Here, we describe a protocol for intestinal leukocyte isolation that works reliable in our hands and allows for flow cytometry-based analysis, in particular of neutrophils.
Critical Role of CD8 T Cells in Mediating Sex-Based Differences in a Murine Model of Lupus

DTIC Science & Technology

2009-08-21

into female transfers (fF) mice that was reduced in CD8 depleted fF mice. Flow cytometry analysis showed increased numbers of splenic...splenocytes were first analyzed by flow cytometry for CD4 and CD8 T cells and F1 mice received either: a) unfractionated splenocytes (CD8 intactF1...using magnetic beads purchased from Invitrogen (Carlsbad, CA) according to the manufacturer’s instructions. Flow cytometry analysis before cell
Acoustofluidic bacteria separation

NASA Astrophysics Data System (ADS)

Li, Sixing; Ma, Fen; Bachman, Hunter; Cameron, Craig E.; Zeng, Xiangqun; Huang, Tony Jun

2017-01-01

Bacterial separation from human blood samples can help with the identification of pathogenic bacteria for sepsis diagnosis. In this work, we report an acoustofluidic device for label-free bacterial separation from human blood samples. In particular, we exploit the acoustic radiation force generated from a tilted-angle standing surface acoustic wave (taSSAW) field to separate Escherichia coli from human blood cells based on their size difference. Flow cytometry analysis of the E. coli separated from red blood cells shows a purity of more than 96%. Moreover, the label-free electrochemical detection of the separated E. coli displays reduced non-specific signals due to the removal of blood cells. Our acoustofluidic bacterial separation platform has advantages such as label-free separation, high biocompatibility, flexibility, low cost, miniaturization, automation, and ease of in-line integration. The platform can be incorporated with an on-chip sensor to realize a point-of-care sepsis diagnostic device.
Value of HIV patients with regular follow-up as in-house internal controls of flow cytometry measurement of lymphocyte subsets.

PubMed

de Carvalho Bittencourt, Marcelo; Kohler, Chantal; Henard, Sandrine; Rabaud, Christian; Béné, Marie C; Faure, Gilbert C

2013-01-01

Quality assessment in flow cytometry cannot obey the same rules as those applicable to the measurement of chemical analytes. However, regular follow-up of known patients may provide a robust in-house control of cell subsets evaluation. Sequential blood samples assessed for 32 HIV patients over several years and showing good stability were retrospectively assessed to establish coefficient of variations of the percentages of CD3+, CD4+, CD8+ cells, and CD4+ absolute counts (ACs). Mean relative standard variations for the whole cohort were of 0.04, 0.14, 0.08, and 0.18 for CD3%, CD4%, CD8%, and CD4 ACs, respectively. In-house follow-up of regularly checked compliant patients is a good alternative to traditional and costly repeatability and reproducibility studies for the validation of routine flow cytometry. © 2013 International Clinical Cytometry Society. Copyright © 2013 International Clinical Cytometry Society.
Value of HIV patients with regular follow-up as in-house internal controls of flow cytometry measurement of lymphocyte subsets.

PubMed

de Carvalho Bittencourt, Marcelo; Kohler, Chantal; Henard, Sandrine; Rabaud, Christian; Béné, Marie C; Faure, Gilbert C

2013-07-08

Background. Quality assessment in flow cytometry cannot obey the same rules as those applicable to the measurement of chemical analytes. However, regular follow-up of known patients may provide a robust in-house control of cell subsets evaluation. Methods. Sequential blood samples assessed for 32 HIV patients over several years and showing good stability were retrospectively assessed to establish coefficient of variations of the percentages of CD3+, CD4+, CD8+ cells and CD4+ absolute counts. Results. Mean relative standard variations for the whole cohort were of 0.04, 0.14, 0.08 and 0.18 for CD3%, CD4% CD8% and CD4 absolute counts respectively. Discussion. In-house follow up of regularly checked compliant patients is a good alternative to traditional and costly repeatability and reproducibility studies for the validation of routine flow cytometry. © 2013 Clinical Cytometry Society. Copyright © 2013 Clinical Cytometry Society.
Cooperative suction by vertical capillary array pump for controlling flow profiles of microfluidic sensor chips.

PubMed

Horiuchi, Tsutomu; Hayashi, Katsuyoshi; Seyama, Michiko; Inoue, Suzuyo; Tamechika, Emi

2012-10-18

A passive pump consisting of integrated vertical capillaries has been developed for a microfluidic chip as an useful component with an excellent flow volume and flow rate. A fluidic chip built into a passive pump was used by connecting the bottoms of all the capillaries to a top surface consisting of a thin layer channel in the microfluidic chip where the thin layer channel depth was smaller than the capillary radius. As a result the vertical capillaries drew fluid cooperatively rather than independently, thus exerting the maximum suction efficiency at every instance. This meant that a flow rate was realized that exhibited little variation and without any external power or operation. A microfluidic chip built into this passive pump had the ability to achieve a quasi-steady rather than a rapidly decreasing flow rate, which is a universal flow characteristic in an ordinary capillary.
Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation.

PubMed

Binsker, Ulrike; Palankar, Raghavendra; Wesche, Jan; Kohler, Thomas P; Prucha, Josephine; Burchhardt, Gerhard; Rohde, Manfred; Schmidt, Frank; Bröker, Barbara M; Mamat, Uwe; Pané-Farré, Jan; Graf, Anica; Ebner, Patrick; Greinacher, Andreas; Hammerschmidt, Sven

2018-04-01

Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection. Schattauer GmbH Stuttgart.
OpenCyto: An Open Source Infrastructure for Scalable, Robust, Reproducible, and Automated, End-to-End Flow Cytometry Data Analysis

PubMed Central

Finak, Greg; Frelinger, Jacob; Jiang, Wenxin; Newell, Evan W.; Ramey, John; Davis, Mark M.; Kalams, Spyros A.; De Rosa, Stephen C.; Gottardo, Raphael

2014-01-01

Flow cytometry is used increasingly in clinical research for cancer, immunology and vaccines. Technological advances in cytometry instrumentation are increasing the size and dimensionality of data sets, posing a challenge for traditional data management and analysis. Automated analysis methods, despite a general consensus of their importance to the future of the field, have been slow to gain widespread adoption. Here we present OpenCyto, a new BioConductor infrastructure and data analysis framework designed to lower the barrier of entry to automated flow data analysis algorithms by addressing key areas that we believe have held back wider adoption of automated approaches. OpenCyto supports end-to-end data analysis that is robust and reproducible while generating results that are easy to interpret. We have improved the existing, widely used core BioConductor flow cytometry infrastructure by allowing analysis to scale in a memory efficient manner to the large flow data sets that arise in clinical trials, and integrating domain-specific knowledge as part of the pipeline through the hierarchical relationships among cell populations. Pipelines are defined through a text-based csv file, limiting the need to write data-specific code, and are data agnostic to simplify repetitive analysis for core facilities. We demonstrate how to analyze two large cytometry data sets: an intracellular cytokine staining (ICS) data set from a published HIV vaccine trial focused on detecting rare, antigen-specific T-cell populations, where we identify a new subset of CD8 T-cells with a vaccine-regimen specific response that could not be identified through manual analysis, and a CyTOF T-cell phenotyping data set where a large staining panel and many cell populations are a challenge for traditional analysis. The substantial improvements to the core BioConductor flow cytometry packages give OpenCyto the potential for wide adoption. It can rapidly leverage new developments in computational cytometry and facilitate reproducible analysis in a unified environment. PMID:25167361
OpenCyto: an open source infrastructure for scalable, robust, reproducible, and automated, end-to-end flow cytometry data analysis.

PubMed

Finak, Greg; Frelinger, Jacob; Jiang, Wenxin; Newell, Evan W; Ramey, John; Davis, Mark M; Kalams, Spyros A; De Rosa, Stephen C; Gottardo, Raphael

2014-08-01

Flow cytometry is used increasingly in clinical research for cancer, immunology and vaccines. Technological advances in cytometry instrumentation are increasing the size and dimensionality of data sets, posing a challenge for traditional data management and analysis. Automated analysis methods, despite a general consensus of their importance to the future of the field, have been slow to gain widespread adoption. Here we present OpenCyto, a new BioConductor infrastructure and data analysis framework designed to lower the barrier of entry to automated flow data analysis algorithms by addressing key areas that we believe have held back wider adoption of automated approaches. OpenCyto supports end-to-end data analysis that is robust and reproducible while generating results that are easy to interpret. We have improved the existing, widely used core BioConductor flow cytometry infrastructure by allowing analysis to scale in a memory efficient manner to the large flow data sets that arise in clinical trials, and integrating domain-specific knowledge as part of the pipeline through the hierarchical relationships among cell populations. Pipelines are defined through a text-based csv file, limiting the need to write data-specific code, and are data agnostic to simplify repetitive analysis for core facilities. We demonstrate how to analyze two large cytometry data sets: an intracellular cytokine staining (ICS) data set from a published HIV vaccine trial focused on detecting rare, antigen-specific T-cell populations, where we identify a new subset of CD8 T-cells with a vaccine-regimen specific response that could not be identified through manual analysis, and a CyTOF T-cell phenotyping data set where a large staining panel and many cell populations are a challenge for traditional analysis. The substantial improvements to the core BioConductor flow cytometry packages give OpenCyto the potential for wide adoption. It can rapidly leverage new developments in computational cytometry and facilitate reproducible analysis in a unified environment.
Organizing the Cellular and Molecular Heterogeneity in High-Grade Serous Ovarian Cancer by Mass Cytometry

DTIC Science & Technology

2014-10-01

Bendall SC, Sung P, Nolan GP, Arvin AM. Single-cell mass cytometry analysis of human tonsil T cell remodeling by varicella zoster virus. Cell Rep...Perspectives on Flow Cytometry 2013, September 20, 2013, Mass Cytometry and Cell Cycle, Mexico City, Mexico (by Web Conference) Nolan: Nuclear
Flow cytometry microscopy and hyperspectral imaging of microcystis, cyanobacteria and algae

EPA Science Inventory

The detection of algae and cyanobacteria is an important step in assessing water quality. Studies were initiated using microscopy, flow cytometry and hyperspectral imaging with two fresh water species that could be grown in the laboratory: Microcystis Aeruginosa (cyanobacteria),...
Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate

NASA Technical Reports Server (NTRS)

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

1999-01-01

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

PubMed

Atkuri, Kondala R; Stevens, Jeffrey C; Neubert, Hendrik

2015-02-01

Advanced single-cell analysis technologies (e.g., mass cytometry) that help in multiplexing cellular measurements in limited-volume primary samples are critical in bridging discovery efforts to successful drug approval. Mass cytometry is the state-of-the-art technology in multiparametric single-cell analysis. Mass cytometers (also known as cytometry by time-of-flight or CyTOF) combine the cellular analysis principles of traditional fluorescence-based flow cytometry with the selectivity and quantitative power of inductively coupled plasma-mass spectrometry. Standard flow cytometry is limited in the number of parameters that can be measured owing to the overlap in signal when detecting fluorescently labeled antibodies. Mass cytometry uses antibodies tagged to stable isotopes of rare earth metals, which requires minimal signal compensation between the different metal tags. This unique feature enables researchers to seamlessly multiplex up to 40 independent measurements on single cells. In this overview we first present an overview of mass cytometry and compare it with traditional flow cytometry. We then discuss the emerging and potential applications of CyTOF technology in the pharmaceutical industry, including quantitative and qualitative deep profiling of immune cells and their applications in assessing drug immunogenicity, extensive mapping of signaling networks in single cells, cell surface receptor quantification and multiplexed internalization kinetics, multiplexing sample analysis by barcoding, and establishing cell ontologies on the basis of phenotype and/or function. We end with a discussion of the anticipated impact of this technology on drug development lifecycle with special emphasis on the utility of mass cytometry in deciphering a drug's pharmacokinetics and pharmacodynamics relationship. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.
Cytometry metadata in XML

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Leif, Stephanie H.

2016-04-01

Introduction: The International Society for Advancement of Cytometry (ISAC) has created a standard for the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt 1.0). CytometryML will serve as a common metadata standard for flow and image cytometry (digital microscopy). Methods: The MIFlowCyt data-types were created, as is the rest of CytometryML, in the XML Schema Definition Language (XSD1.1). The datatypes are primarily based on the Flow Cytometry and the Digital Imaging and Communication (DICOM) standards. A small section of the code was formatted with standard HTML formatting elements (p, h1, h2, etc.). Results:1) The part of MIFlowCyt that describes the Experimental Overview including the specimen and substantial parts of several other major elements has been implemented as CytometryML XML schemas (www.cytometryml.org). 2) The feasibility of using MIFlowCyt to provide the combination of an overview, table of contents, and/or an index of a scientific paper or a report has been demonstrated. Previously, a sample electronic publication, EPUB, was created that could contain both MIFlowCyt metadata as well as the binary data. Conclusions: The use of CytometryML technology together with XHTML5 and CSS permits the metadata to be directly formatted and together with the binary data to be stored in an EPUB container. This will facilitate: formatting, data- mining, presentation, data verification, and inclusion in structured research, clinical, and regulatory documents, as well as demonstrate a publication's adherence to the MIFlowCyt standard, promote interoperability and should also result in the textual and numeric data being published using web technology without any change in composition.
ggCyto: Next Generation Open-Source Visualization Software for Cytometry.

PubMed

Van, Phu; Jiang, Wenxin; Gottardo, Raphael; Finak, Greg

2018-06-01

Open source software for computational cytometry has gained in popularity over the past few years. Efforts such as FlowCAP, the Lyoplate and Euroflow projects have highlighted the importance of efforts to standardize both experimental and computational aspects of cytometry data analysis. The R/BioConductor platform hosts the largest collection of open source cytometry software covering all aspects of data analysis and providing infrastructure to represent and analyze cytometry data with all relevant experimental, gating, and cell population annotations enabling fully reproducible data analysis. Data visualization frameworks to support this infrastructure have lagged behind. ggCyto is a new open-source BioConductor software package for cytometry data visualization built on ggplot2 that enables ggplot-like functionality with the core BioConductor flow cytometry data structures. Amongst its features are the ability to transform data and axes on-the-fly using cytometry-specific transformations, plot faceting by experimental meta-data variables, and partial matching of channel, marker and cell populations names to the contents of the BioConductor cytometry data structures. We demonstrate the salient features of the package using publicly available cytometry data with complete reproducible examples in a supplementary material vignette. https://bioconductor.org/packages/devel/bioc/html/ggcyto.html. gfinak@fredhutch.org. Supplementary data are available at Bioinformatics online and at http://rglab.org/ggcyto/.
Genome-wide analysis of replication timing by next-generation sequencing with E/L Repli-seq.

PubMed

Marchal, Claire; Sasaki, Takayo; Vera, Daniel; Wilson, Korey; Sima, Jiao; Rivera-Mulia, Juan Carlos; Trevilla-García, Claudia; Nogues, Coralin; Nafie, Ebtesam; Gilbert, David M

2018-05-01

This protocol is an extension to: Nat. Protoc. 6, 870-895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early- and late-replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and subnuclear position. Moreover, RT is regulated during development and is altered in diseases. Here, we describe E/L Repli-seq, an extension of our Repli-chip protocol. E/L Repli-seq is a rapid, robust and relatively inexpensive protocol for analyzing RT by next-generation sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse-labeled with BrdU, and early and late S-phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNA from early versus late S-phase fractions. The results are comparable to those of Repli-chip, with the additional benefits of genome-wide sequence information and an increased dynamic range. We also provide computational pipelines for downstream analyses, for parsing phased genomes using single-nucleotide polymorphisms (SNPs) to analyze RT allelic asynchrony, and for direct comparison to Repli-chip data. This protocol can be performed in up to 3 d before sequencing, and requires basic cellular and molecular biology skills, as well as a basic understanding of Unix and R.
Flow Cytometry, Microscopy and Hyperspectral Imaging of microcystis, Cyanobacteria, and Algae- SETAC

EPA Science Inventory

The detection of cyanobacteria algae, and picoplankton, in water is an important step in assessing water quality. Studies were initiated using fluorescence microscopy, flow cytometry and hyperspectral imaging with two fresh water species that were cultured in the laboratory:Micr...
Flow cytometry enables identification of sporophytic eliciting stress treatments in gametic cells.

PubMed

Ribalta, F M; Croser, J S; Ochatt, S J

2012-01-01

Flow cytometry was used to quantify the effect of individual and combined stress treatments on elicitation of androgenesis by analyzing the relative nuclear DNA content of in vitro cultured microspores of Pisum sativum L. Differences in relative nuclear DNA content of microspores within anthers after stress treatments were clearly evident from the flow cytometry profiles, and permitted us to predict whether a combination of stresses were elicitors or enhancers of androgenesis. This is the first report to assess the effect of various stress treatments in a plant species based on relative nuclear DNA content and to use this information to categorize them as 'elicitors' or 'enhancers'. Flow cytometry represents a simple, quick and reliable way to analyze and discriminate the effect of various stress treatments on elicitation of androgenesis. These results form a solid basis for further efforts designed to enhance responses and to extend double haploid technology to other legumes. Copyright © 2011 Elsevier GmbH. All rights reserved.
Integration of lyoplate based flow cytometry and computational analysis for standardized immunological biomarker discovery.

PubMed

Villanova, Federica; Di Meglio, Paola; Inokuma, Margaret; Aghaeepour, Nima; Perucha, Esperanza; Mollon, Jennifer; Nomura, Laurel; Hernandez-Fuentes, Maria; Cope, Andrew; Prevost, A Toby; Heck, Susanne; Maino, Vernon; Lord, Graham; Brinkman, Ryan R; Nestle, Frank O

2013-01-01

Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases.

Integration of Lyoplate Based Flow Cytometry and Computational Analysis for Standardized Immunological Biomarker Discovery

PubMed Central

Villanova, Federica; Di Meglio, Paola; Inokuma, Margaret; Aghaeepour, Nima; Perucha, Esperanza; Mollon, Jennifer; Nomura, Laurel; Hernandez-Fuentes, Maria; Cope, Andrew; Prevost, A. Toby; Heck, Susanne; Maino, Vernon; Lord, Graham; Brinkman, Ryan R.; Nestle, Frank O.

2013-01-01

Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases. PMID:23843942
Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation.

PubMed

Böttcher, S; Ritgen, M; Pott, C; Brüggemann, M; Raff, T; Stilgenbauer, S; Döhner, H; Dreger, P; Kneba, M

2004-10-01

The clinically most suitable method for minimal residual disease (MRD) detection in chronic lymphocytic leukemia is still controversial. We prospectively compared MRD assessment in 158 blood samples of 74 patients with CLL after stem cell transplantation (SCT) using four-color flow cytometry (MRD flow) in parallel with consensus IgH-PCR and ASO IgH real-time PCR (ASO IgH RQ-PCR). In 25 out of 106 samples (23.6%) with a polyclonal consensus IgH-PCR pattern, MRD flow still detected CLL cells, proving higher sensitivity of flow cytometry over PCR-genescanning with consensus IgH-primers. Of 92 samples, 14 (15.2%) analyzed in parallel by MRD flow and by ASO IgH RQ-PCR were negative by our flow cytometric assay but positive by PCR, thus demonstrating superior sensitivity of RQ-PCR with ASO primers. Quantitative MRD levels measured by both methods correlated well (r=0.93). MRD detection by flow and ASO IgH RQ-PCR were equally suitable to monitor MRD kinetics after allogeneic SCT, but the PCR method detected impending relapses after autologous SCT earlier. An analysis of factors that influence sensitivity and specificity of flow cytometry for MRD detection allowed to devise further improvements of this technique.
Differential gene expression profiling of Actinobacillus pleuropneumoniae during induction of primary alveolar macrophage apoptosis in piglets.

PubMed

Wang, Lei; Qin, Wanhai; Ruidong, Zhai; Liu, Shiting; Zhang, Hu; Sun, Changjiang; Feng, Xin; Gu, Jingmin; Du, Chongtao; Han, Wenyu; Langford, P R; Lei, Liancheng

2015-01-01

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the causative agent of porcine pleuropneumonia, a disease that causes serious problems for the swine industry. Successful infection by this bacterium requires breaking the first line of defence in the lungs, the primary alveolar macrophages (PAMs). Therefore, exploring A. pleuropneumoniae-PAM interactions will provide vital groundwork for the scientific control of this infectious disease, which has been little studied up to now. In this work, PAMs were isolated from piglets and co-incubated with A. pleuropneumoniae serovar 5b strain L20 in vitro, and their interaction, PAM cell death, and differential gene expression of A. pleuropneumoniae in response to PAM cell death were observed and analysed using confocal microscopy, electron microscopy, RT-PCR, Western blot, flow cytometry and the use of a gene expression profile chip. A. pleuropneumoniae quickly adhered to and invaded PAMs, inducing apoptosis, which was confirmed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The highest percentage of apoptosis in cells was confirmed using flow cytometry when the cells were infected at a multiplicity of infection (MOI) of 10 and incubated for 5 h, with higher expression of activated caspase-3 as measured by Western blot. Using microarray gene chips with 2868 probes containing nearly all of the genomic sequence of A. pleuropneumoniae serotype 5b strain L20, a total of 185 bacterial genes were found to be differentially expressed (including 92 up-regulated and 93 down-regulated genes) and involved in the process of apoptosis, as compared with the expression of control bacteria cultured without PAMs in BHI medium (mean expression ratios >1.5-fold, p < 0.05). The up-regulated genes are involved in energy metabolism, gene transcription and translation, virulence related gene such as LPS, Trimeric Autotransporter Adhesin, RTX and similar genes. The down-regulated genes are involved in amino acid, cofactor, and vitamin metabolism, and also include ABC transporters. These data demonstrate that A. pleuropneumoniae induces apoptosis of PAMs and undergoes complex changes in gene transcription, including expression changes in known and potential virulence factors. Some potentially novel virulence targets have been identified, suggesting new strategies for the development of vaccines and medicines for both preventive and clinical use. Copyright © 2014 Elsevier Ltd. All rights reserved.
Immune Response to Mycobacterial Infection: Lessons from Flow Cytometry

PubMed Central

Rovina, Nikoletta; Panagiotou, Marios; Koulouris, Nikolaos G.

2013-01-01

Detecting and treating active and latent tuberculosis are pivotal elements for effective infection control; yet, due to their significant inherent limitations, the diagnostic means for these two stages of tuberculosis (TB) to date remain suboptimal. This paper reviews the current diagnostic tools for mycobacterial infection and focuses on the application of flow cytometry as a promising method for rapid and reliable diagnosis of mycobacterial infection as well as discrimination between active and latent TB: it summarizes diagnostic biomarkers distinguishing the two states of infection and also features of the distinct immune response against Mycobacterium tuberculosis (Mtb) at certain stages of infection as revealed by flow cytometry to date. PMID:24376464
Immune response to mycobacterial infection: lessons from flow cytometry.

PubMed

Rovina, Nikoletta; Panagiotou, Marios; Pontikis, Konstantinos; Kyriakopoulou, Magdalini; Koulouris, Nikolaos G; Koutsoukou, Antonia

2013-01-01

Detecting and treating active and latent tuberculosis are pivotal elements for effective infection control; yet, due to their significant inherent limitations, the diagnostic means for these two stages of tuberculosis (TB) to date remain suboptimal. This paper reviews the current diagnostic tools for mycobacterial infection and focuses on the application of flow cytometry as a promising method for rapid and reliable diagnosis of mycobacterial infection as well as discrimination between active and latent TB: it summarizes diagnostic biomarkers distinguishing the two states of infection and also features of the distinct immune response against Mycobacterium tuberculosis (Mtb) at certain stages of infection as revealed by flow cytometry to date.
Flow cytometry as a rapid test for detection of penicillin resistance directly in bacterial cells in Enterococcus faecalis and Staphylococcus aureus.

PubMed

Jarzembowski, T; Wiśniewska, K; Józwik, A; Bryl, E; Witkowski, J

2008-08-01

We studied the usefulness of flow cytometry for detection of penicillin resistance in E. faecalis and S. aureus by direct binding of commercially available fluorescent penicillin, Bocillin FL, to cells obtained from culture. There were significantly lower percentages of fluorescent cells and median and mean fluorescence values per particle in penicillin-resistant than in penicillin-sensitive strains of both species observed. The method allows rapid detection of penicillin resistance in S. aureus and E. faecalis. The results encourage further investigations on the detection of antibiotic resistance in bacteria using flow cytometry.
Flow cytometry: a promising technique for the study of silicone oil-induced particulate formation in protein formulations.

PubMed

Ludwig, D Brett; Trotter, Joseph T; Gabrielson, John P; Carpenter, John F; Randolph, Theodore W

2011-03-15

Subvisible particles in formulations intended for parenteral administration are of concern in the biopharmaceutical industry. However, monitoring and control of subvisible particulates can be complicated by formulation components, such as the silicone oil used for the lubrication of prefilled syringes, and it is difficult to differentiate microdroplets of silicone oil from particles formed by aggregated protein. In this study, we demonstrate the ability of flow cytometry to resolve mixtures comprising subvisible bovine serum albumin (BSA) aggregate particles and silicone oil emulsion droplets with adsorbed BSA. Flow cytometry was also used to investigate the effects of silicone oil emulsions on the stability of BSA, lysozyme, abatacept, and trastuzumab formulations containing surfactant, sodium chloride, or sucrose. To aid in particle characterization, the fluorescence detection capabilities of flow cytometry were exploited by staining silicone oil with BODIPY 493/503 and model proteins with Alexa Fluor 647. Flow cytometric analyses revealed that silicone oil emulsions induced the loss of soluble protein via protein adsorption onto the silicone oil droplet surface. The addition of surfactant prevented protein from adsorbing onto the surface of silicone oil droplets. There was minimal formation of homogeneous protein aggregates due to exposure to silicone oil droplets, although oil droplets with surface-adsorbed trastuzumab exhibited flocculation. The results of this study demonstrate the utility of flow cytometry as an analytical tool for monitoring the effects of subvisible silicone oil droplets on the stability of protein formulations. Copyright © 2010 Elsevier Inc. All rights reserved.
Use of flow cytometry to monitor cell damage and predict fermentation activity of dried yeasts.

PubMed

Attfield, P V; Kletsas, S; Veal, D A; van Rooijen, R; Bell, P J

2000-08-01

Viable dried yeast is used as an inoculum for many fermentations in the baking and wine industries. The fermentative activity of yeast in bread dough or grape must is a critical parameter of process efficiency. Here, it is shown that fluorescent stains and flow cytometry can be used in concert to predict the abilities of populations of dried bakers' and wine yeasts to ferment after rehydration. Fluorescent dyes that stain cells only if they have damaged membrane potential (oxonol) or have increased membrane permeability (propidium iodide) were used to analyse, by flow cytometry, populations of rehydrated yeasts. A strong relationship (r2 = 0.99) was found between the percentages of populations staining with the oxonol and the degree of cell membrane damage as measured by the more traditional method of leakage of intracellular compounds. There were also were good negative relationships (r2 > or = 0.83) between fermentation by rehydrated bakers' or wine dry yeasts and percentage of populations staining with either oxonol or propidium iodide. Fluorescent staining with flow cytometry confirmed that factors such as vigour of dried yeast mixing in water, soaking before stirring, rehydration in water or fermentation medium and temperature of rehydration have profound effects on subsequent yeast vitality. These experiments indicate the potential of flow cytometry as a rapid means of predicting the fermentation performance of dried bakers' and wine yeasts.
Novel Methods of Determining Urinary Calculi Composition: Petrographic Thin Sectioning of Calculi and Nanoscale Flow Cytometry Urinalysis

PubMed Central

Gavin, Carson T; Ali, Sohrab N; Tailly, Thomas; Olvera-Posada, Daniel; Alenezi, Husain; Power, Nicholas E; Hou, Jinqiang; St. Amant, Andre H; Luyt, Leonard G; Wood, Stephen; Wu, Charles; Razvi, Hassan; Leong, Hon S

2016-01-01

Accurate determination of urinary stone composition has significant bearing on understanding pathophysiology, choosing treatment modalities and preventing recurrence. A need exists for improved methods to determine stone composition. Urine of 31 patients with known renal calculi was examined with nanoscale flow cytometry and the calculi collected during surgery subsequently underwent petrographic thin sectioning with polarized and fluorescent microscopy. Fluorescently labeled bisphosphonate probes (Alendronate-fluorescein/Alendronate-Cy5) were developed for nanoscale flow cytometry to enumerate nanocrystals that bound the fluorescent probes. Petrographic sections of stones were also imaged by fluorescent and polarized light microscopy with composition analysis correlated to alendronate +ve nanocrystal counts in corresponding urine samples. Urine samples from patients with Ca2+ and Mg2+ based calculi exhibited the highest alendronate +ve nanocrystal counts, ranging from 100–1000 nm in diameter. This novel urine based assay was in agreement with composition determined by petrographic thin sections with Alendronate probes. In some cases, high alendronate +ve nanocrystal counts indicated a Ca2+ or Mg2+ composition, as confirmed by petrographic analysis, overturning initial spectrophotometric diagnosis of stone composition. The combination of nanoscale flow cytometry and petrographic thin sections offer an alternative means for determining stone composition. Nanoscale flow cytometry of alendronate +ve nanocrystals alone may provide a high-throughput means of evaluating stone burden. PMID:26771074
Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM).

PubMed

Tang, Anson H L; Lai, Queenie T K; Chung, Bob M F; Lee, Kelvin C M; Mok, Aaron T Y; Yip, G K; Shum, Anderson H C; Wong, Kenneth K Y; Tsia, Kevin K

2017-06-28

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.
Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris

PubMed Central

Chioccioli, Maurizio; Hankamer, Ben; Ross, Ian L.

2014-01-01

Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD750) for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate) medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl) to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD750 measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary during growth. PMID:24832156
Carbocysteine counteracts the effects of cigarette smoke on cell growth and on the SIRT1/FoxO3 axis in bronchial epithelial cells.

PubMed

Pace, E; Di Vincenzo, S; Ferraro, M; Bruno, A; Dino, P; Bonsignore, M R; Battaglia, S; Saibene, F; Lanata, L; Gjomarkaj, M

2016-08-01

Cigarette smoke may accelerate cellular senescence by increasing oxidative stress. Altered proliferation and altered expression of anti-aging factors, including SIRT1 and FoxO3, characterise cellular senescence. The effects of carbocysteine on the SIRT1/FoxO3 axis and on downstream molecular mechanisms in human bronchial epithelial cells exposed to cigarette smoke are largely unknown. Aim of this study was to explore whether carbocysteine modulated SIRT1/FoxO3 axis, and downstream molecular mechanisms associated to cellular senescence, in a bronchial epithelial cell line (16-HBE) exposed to cigarette smoke. 16HBE cells were stimulated with/without cigarette smoke extracts (CSE) and carbocysteine. Flow cytometry and clonogenic assay were used to assess cell proliferation; western blot analysis was used for assessing nuclear expression of SIRT1 and FoxO3. The nuclear co-localization of SIRT1 and FoxO3 was assessed by fluorescence microscopy. Beta galactosidase (a senescence marker) and SIRT1 activity were assessed by specific staining and colorimetric assays, respectively. ChiP Assay and flow cytometry were used for assessing survivin gene regulation and protein expression, respectively. CSE decreased cell proliferation, the nuclear expression of SIRT1 and FoxO3 and increased beta galactosidase staining. CSE, reduced SIRT1 activity and FoxO3 localization on survivin promoter thus increasing survivin expression. In CSE stimulated bronchial epithelial cells carbocysteine reverted these phenomena by increasing cell proliferation, and SIRT1 and FoxO3 nuclear expression, and by reducing beta galactosidase staining and survivin expression. The study shows for the first time that carbocysteine may revert some senescence processes induced by oxidative stress due to cigarette smoke exposure. Copyright © 2016 Elsevier Inc. All rights reserved.
A high-throughput flow cytometry-on-a-CMOS platform for single-cell dielectric spectroscopy at microwave frequencies.

PubMed

Chien, Jun-Chau; Ameri, Ali; Yeh, Erh-Chia; Killilea, Alison N; Anwar, Mekhail; Niknejad, Ali M

2018-06-06

This work presents a microfluidics-integrated label-free flow cytometry-on-a-CMOS platform for the characterization of the cytoplasm dielectric properties at microwave frequencies. Compared with MHz impedance cytometers, operating at GHz frequencies offers direct intracellular permittivity probing due to electric fields penetrating through the cellular membrane. To overcome the detection challenges at high frequencies, the spectrometer employs on-chip oscillator-based sensors, which embeds simultaneous frequency generation, electrode excitation, and signal detection capabilities. By employing an injection-locking phase-detection technique, the spectrometer offers state-of-the-art sensitivity, achieving a less than 1 aFrms capacitance detection limit (or 5 ppm in frequency-shift) at a 100 kHz noise filtering bandwidth, enabling high throughput (>1k cells per s), with a measured cellular SNR of more than 28 dB. With CMOS/microfluidics co-design, we distribute four sensing channels at 6.5, 11, 17.5, and 30 GHz in an arrayed format whereas the frequencies are selected to center around the water relaxation frequency at 18 GHz. An issue in the integration of CMOS and microfluidics due to size mismatch is also addressed through introducing a cost-efficient epoxy-molding technique. With 3-D hydrodynamic focusing microfluidics, we perform characterization on four different cell lines including two breast cell lines (MCF-10A and MDA-MB-231) and two leukocyte cell lines (K-562 and THP-1). After normalizing the higher frequency signals to the 6.5 GHz ones, the size-independent dielectric opacity shows a differentiable distribution at 17.5 GHz between normal (0.905 ± 0.160, mean ± std.) and highly metastatic (1.033 ± 0.107) breast cells with p ≪ 0.001.
Emerging metrology for high-throughput nanomaterial genotoxicology.

PubMed

Nelson, Bryant C; Wright, Christa W; Ibuki, Yuko; Moreno-Villanueva, Maria; Karlsson, Hanna L; Hendriks, Giel; Sims, Christopher M; Singh, Neenu; Doak, Shareen H

2017-01-01

The rapid development of the engineered nanomaterial (ENM) manufacturing industry has accelerated the incorporation of ENMs into a wide variety of consumer products across the globe. Unintentionally or not, some of these ENMs may be introduced into the environment or come into contact with humans or other organisms resulting in unexpected biological effects. It is thus prudent to have rapid and robust analytical metrology in place that can be used to critically assess and/or predict the cytotoxicity, as well as the potential genotoxicity of these ENMs. Many of the traditional genotoxicity test methods [e.g. unscheduled DNA synthesis assay, bacterial reverse mutation (Ames) test, etc.,] for determining the DNA damaging potential of chemical and biological compounds are not suitable for the evaluation of ENMs, due to a variety of methodological issues ranging from potential assay interferences to problems centered on low sample throughput. Recently, a number of sensitive, high-throughput genotoxicity assays/platforms (CometChip assay, flow cytometry/micronucleus assay, flow cytometry/γ-H2AX assay, automated 'Fluorimetric Detection of Alkaline DNA Unwinding' (FADU) assay, ToxTracker reporter assay) have been developed, based on substantial modifications and enhancements of traditional genotoxicity assays. These new assays have been used for the rapid measurement of DNA damage (strand breaks), chromosomal damage (micronuclei) and for detecting upregulated DNA damage signalling pathways resulting from ENM exposures. In this critical review, we describe and discuss the fundamental measurement principles and measurement endpoints of these new assays, as well as the modes of operation, analytical metrics and potential interferences, as applicable to ENM exposures. An unbiased discussion of the major technical advantages and limitations of each assay for evaluating and predicting the genotoxic potential of ENMs is also provided. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society 2016.
Annual Progress Report FY-92. Volume 1

DTIC Science & Technology

1993-01-21

Billups, L Flow Cytom Resh Psychologist 12 0180 CS Hamm, C DCI 7 DESCRIPTION GRADE MOS BRANCH NAME ACTIVITY Kyle Metabolic Unit Nursing Service Supv...3349 Salata, Kalman PhD. Mitogen-Inducible T Suppressor Cell 13 Assay by Flow Cytometry (12/89) 3350 Salata, Kalman PhD. Flow Cytometric Analysis of...17 Immunotherapy (3/90) 3354 Salata, Kalman PhD. Two Way Mixed Lymphocyte Culture: 18 Analysis by Two Color Flow Cytometry (4/90) 3355 Salata, Kalman
Teaching the Microbial Growth Curve Concept Using Microalgal Cultures and Flow Cytometry

ERIC Educational Resources Information Center

Forget, Nathalie; Belzile, Claude; Rioux, Pierre; Nozais, Christian

2010-01-01

The microbial growth curve is widely studied within microbiology classes and bacteria are usually the microbial model used. Here, we describe a novel laboratory protocol involving flow cytometry to assess the growth dynamics of the unicellular microalgae "Isochrysis galbana." The algal model represents an appropriate alternative to…
Targeting the Nociceptin/Orphanin FQ Receptor for Scleroderma Therapy

DTIC Science & Technology

2015-12-01

bottom well; the number of migrating cells is quantified by flow cytometry. In the aortic ring assay, freshly isolated thoracic aorta rings will...quantified by flow cytometry. In the aortic ring assay, freshly isolated thoracic aorta rings will be harvested and mounted in a small-vessel myograph. KO
Flow cytometry of sputum: assessing inflammation and immune response elements in the bronchial airways**

EPA Science Inventory

Rationale: The evaluation of sputum leukocytes by flow cytometry is an opportunity to assess characteristics of cells residing in the central airways, yet it is hampered by certain inherent properties of sputum including mucus and large amounts of contaminating cells and debris. ...
Ferroptosis and Cell Death Analysis by Flow Cytometry.

PubMed

Chen, Daishi; Eyupoglu, Ilker Y; Savaskan, Nicolai

2017-01-01

Cell death and its recently discovered regulated form ferroptosis are characterized by distinct morphological, electrophysiological, and pharmacological features. In particular ferroptosis can be induced by experimental compounds and clinical drugs (i.e., erastin, sulfasalazine, sorafenib, and artesunate) in various cell types and cancer cells. Pharmacologically, this cell death process can be inhibited by iron chelators and lipid peroxidation inhibitors. Relevance of this specific cell death form has been found in different pathological conditions such as cancer, neurotoxicity, neurodegeneration, and ischemia. Distinguishing cell viability and cell death is essential for experimental and clinical applications and a key component in flow cytometry experiments. Dead cells can compromise the integrity of the data by nonspecific binding of antibodies and dyes. Therefore it is essential that dead cells are robustly and reproducibly identified and characterized by means of cytometry application. Here we describe a procedure to detect and quantify cell death and its specific form ferroptosis based on standard flow cytometry techniques.
Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds.

PubMed

Schuck, Desirée Cigaran; Ribeiro, Ramira Yuri; Nery, Arthur A; Ulrich, Henning; Garcia, Célia R S

2011-11-01

Melatonin and its derivatives modulate the Plasmodium falciparum and Plasmodium chabaudi cell cycle. Flow cytometry was employed together with the nucleic acid dye YOYO-1 allowing precise discrimination between mono- and multinucleated forms of P. falciparum-infected red blood cell. The use of YOYO-1 permitted excellent discrimination between uninfected and infected red blood cells as well as between early and late parasite stages. Fluorescence intensities of schizont-stage parasites were about 10-fold greater than those of ring-trophozoite form parasites. Melatonin and related indolic compounds including serotonin, N-acetyl-serotonin and tryptamine induced an increase in the percentage of multinucleated forms compared to non-treated control cultures. YOYO-1 staining of infected erythrocyte and subsequent flow cytometry analysis provides a powerful tool in malaria research for screening of bioactive compounds. Copyright © 2011 International Society for Advancement of Cytometry.

Semi-quantitative estimation of cellular SiO2 nanoparticles using flow cytometry combined with X-ray fluorescence measurements.

PubMed

Choi, Seo Yeon; Yang, Nuri; Jeon, Soo Kyung; Yoon, Tae Hyun

2014-09-01

In this study, we have demonstrated feasibility of a semi-quantitative approach for the estimation of cellular SiO2 nanoparticles (NPs), which is based on the flow cytometry measurements of their normalized side scattering intensity. In order to improve our understanding on the quantitative aspects of cell-nanoparticle interactions, flow cytometry, transmission electron microscopy, and X-ray fluorescence experiments were carefully performed for the HeLa cells exposed to SiO2 NPs with different core diameters, hydrodynamic sizes, and surface charges. Based on the observed relationships among the experimental data, a semi-quantitative cellular SiO2 NPs estimation method from their normalized side scattering and core diameters was proposed, which can be applied for the determination of cellular SiO2 NPs within their size-dependent linear ranges. © 2014 International Society for Advancement of Cytometry.
Flow-directed loading of block copolymer micelles with hydrophobic probes in a gas-liquid microreactor.

PubMed

Wang, Chih-Wei; Bains, Aman; Sinton, David; Moffitt, Matthew G

2013-07-02

We investigate the loading efficiencies of two chemically distinct hydrophobic fluorescent probes, pyrene and naphthalene, for self-assembly and loading of polystyrene-block-poly(acrylic acid) (PS-b-PAA) micelles in gas-liquid segmented microfluidic reactors under different chemical and flow conditions. On-chip loading efficiencies are compared to values obtained via off-chip dropwise water addition to a solution of copolymer and probe. On-chip, probe loading efficiencies depend strongly on the chemical probe, initial solvent, water content, and flow rate. For pyrene and naphthalene probes, maximum on-chip loading efficiencies of 73 ± 6% and 11 ± 3%, respectively, are obtained, in both cases using the more polar solvent (DMF), an intermediate water content (2 wt % above critical), and a low flow rate (∼5 μL/min); these values are compared to 81 ± 6% and 48 ± 2%, respectively, for off-chip loading. On-chip loading shows a significant improvement over the off-chip process where shear-induced formation of smaller micelles enables increased encapsulation of probe. As well, we show that on-chip loading allows off-chip release kinetics to be controlled via flow rate: compared to vehicles produced at ∼5 μL/min, pyrene release kinetics from vehicles produced at ∼50 μL/min showed a longer initial period of burst release, followed by slow release over a longer total period. These results demonstrate the necessity to match probes, solvents, and running conditions to achieve effective loading, which is essential information for further developing these on-chip platforms for manufacturing drug delivery formulations.
FlowCal: A user-friendly, open source software tool for automatically converting flow cytometry data from arbitrary to calibrated units

PubMed Central

Castillo-Hair, Sebastian M.; Sexton, John T.; Landry, Brian P.; Olson, Evan J.; Igoshin, Oleg A.; Tabor, Jeffrey J.

2017-01-01

Flow cytometry is widely used to measure gene expression and other molecular biological processes with single cell resolution via fluorescent probes. Flow cytometers output data in arbitrary units (a.u.) that vary with the probe, instrument, and settings. Arbitrary units can be converted to the calibrated unit molecules of equivalent fluorophore (MEF) using commercially available calibration particles. However, there is no convenient, non-proprietary tool available to perform this calibration. Consequently, most researchers report data in a.u., limiting interpretation. Here, we report a software tool named FlowCal to overcome current limitations. FlowCal can be run using an intuitive Microsoft Excel interface, or customizable Python scripts. The software accepts Flow Cytometry Standard (FCS) files as inputs and is compatible with different calibration particles, fluorescent probes, and cell types. Additionally, FlowCal automatically gates data, calculates common statistics, and produces publication quality plots. We validate FlowCal by calibrating a.u. measurements of E. coli expressing superfolder GFP (sfGFP) collected at 10 different detector sensitivity (gain) settings to a single MEF value. Additionally, we reduce day-to-day variability in replicate E. coli sfGFP expression measurements due to instrument drift by 33%, and calibrate S. cerevisiae mVenus expression data to MEF units. Finally, we demonstrate a simple method for using FlowCal to calibrate fluorescence units across different cytometers. FlowCal should ease the quantitative analysis of flow cytometry data within and across laboratories and facilitate the adoption of standard fluorescence units in synthetic biology and beyond. PMID:27110723
Role of receptor occupancy assays by flow cytometry in drug development.

PubMed

Stewart, Jennifer J; Green, Cherie L; Jones, Nicholas; Liang, Meina; Xu, Yuanxin; Wilkins, Danice E C; Moulard, Maxime; Czechowska, Kamila; Lanham, David; McCloskey, Thomas W; Ferbas, John; van der Strate, Barry W A; Högerkorp, Carl-Magnus; Wyant, Timothy; Lackey, Alan; Litwin, Virginia

2016-03-01

The measurement of the binding of a biotherapeutic to its cellular target, receptor occupancy (RO), is increasingly important in development of biologically-based therapeutic agents. Receptor occupancy (RO) assays by flow cytometry describe the qualitative and/or quantitative assessment of the binding of a therapeutic agent to its cell surface target. Such RO assays can be as simple as measuring the number of cell surface receptors bound by an antireceptor therapeutic agent or can be designed to address more complicated scenarios such as internalization or shedding events once a receptor engages the administered therapeutic agent. Data generated from RO assays can also be used to model whether given doses of an experimental therapeutic agent and their administration schedules lead to predicted levels of receptor occupancy and whether the receptor is modulated (up or down) on cells engaged by the therapeutic agent. There are a variety of approaches that can be used when undertaking RO assays and with the ability to measure distinct subsets in heterogeneous populations, flow cytometry is ideally suited to RO measurements. This article highlights the importance of RO assays on the flow cytometric platform in the development of biotherapeutic agents. © 2016 The Authors Cytometry Part B: Clinical Cytometry Published by Wiley Periodicals, Inc.
Microcapillary-Based Flow-Through Immunosensor and Displacement Immunoassay Using the Same.

DTIC Science & Technology

1997-04-28

an antibody. If desired, an electroosmotic 24 pump may be used to flow fluid through the microcapillary or 25 microcapillaries in the chip...8 for field use. 9 Fig. 1C shows a flow immunosensor chip 100. Buffer flow 10 through microcapillary passage 102 by virtue of an electroosmotic ...Power for an 23 electroosmotic pump or other fluid pump, as well as any other on- 24 chip components, may be provided by a battery incorporated into
Determination of allergenic load and pollen count of Cupressus arizonica pollen by flow cytometry using Cup a1 polyclonal antibody.

PubMed

Benítez, Francisco Moreno; Camacho, Antonio Letrán; Del Cuvillo Bernal, Alfonso; de Medina, Pedro Lobatón Sánchez; Cózar, Francisco J García; Romeu, Ma Luisa Espinazo

2013-07-10

Background: There is an increase in the incidence of pollen related allergy, thus information on pollen schedules would be a great asset for physicians to improve the clinical care of patients. Like cypress pollen sensitization shows a high prevalence among the causes of allergic rhinitis, and therefore it is of interest to use it like a model of study, distinguishing cypress pollen, pollen count and allergenic load level. In this work, we use a flow cytometry based technique to obtain both Cupressus arizonica pollen count and allergenic load, using specific rabbit polyclonal antibody Cup a1 and its comparison with optical microscopy technique measurement. Methods: Airborne samples were collected from Burkard Spore-Trap and Burkard Cyclone Cupressus arizonica pollen was studied using specific rabbit polyclonal antibody Cup a1, labelled with AlexaFluor ® 488 or 750 and analysed by Flow Cytometry in both an EPICS XL and Cyan ADP cytometers (Beckman Coulter ® ). Optical microscopy study was realized with a Leica optical microscope. Bland & Altman was used to determine agreement between both techniques measured. Results: We can identify three different populations based on rabbit polyclonal antibody Cup a1 staining. The main region (44.5%) had 97.3% recognition, a second region (25%) with 28% and a third region (30.5%) with 68% respectively. Immunofluorescence and confocal microscopy showed that main region corresponds to whole pollen grains, the second region are pollen without exine and the third region is constituted by smaller particles with allergenic properties. Pollen schedule shows a higher correlation measured by optical microscopy and flow cytometry in the pollen count with a p-value: 0.0008E -2 and 0.0002 with regard to smaller particles, so the Bland & Altman measurement showed a good correlation between them, p-value: 0,0003. Conclusion: Determination of pollen count and allergenic load by flow cytometry represents an important tool in the determination of airborne respiratory allergens. We showed that not only whole pollen but also smaller particles could induce allergic sensitization. This is the first study where flow cytometry is used for calculating pollen counts and allergenic load. © 2013 Clinical Cytometry Society. Copyright © 2013 Clinical Cytometry Society.
Guide to red fluorescent proteins and biosensors for flow cytometry.

PubMed

Piatkevich, Kiryl D; Verkhusha, Vladislav V

2011-01-01

Since the discovery of the first red fluorescent protein (RFP), named DsRed, 12 years ago, a wide pallet of red-shifted fluorescent proteins has been cloned and biotechnologically developed into monomeric fluorescent probes for optical microscopy. Several new types of monomeric RFPs that change the emission wavelength either with time, called fluorescent timers, or after a brief irradiation with violet light, known as photoactivatable proteins, have been also engineered. Moreover, RFPs with a large Stokes shift of fluorescence emission have been recently designed. Because of their distinctive excitation and fluorescence detection conditions developed specifically for microscopy, these fluorescent probes can be suboptimal for flow cytometry. Here, we have selected and summarized the advanced orange, red, and far-red fluorescent proteins with the properties specifically required for the flow cytometry applications. Their effective brightness was calculated for the laser sources available for the commercial flow cytometers and sorters. Compatibility of the fluorescent proteins of different colors in a multiparameter flow cytometry was determined. Novel FRET pairs, utilizing RFPs, RFP-based intracellular biosensors, and their application to a high-throughput screening, are also discussed. Copyright © 2011 Elsevier Inc. All rights reserved.
Structure of the Global Nanoscience and Nanotechnology Research Literature

DTIC Science & Technology

2006-01-01

Transistors, Nature, 424 (6949): 654-657, 2003. Joannopoulos, JD, Meade, RD, Winn, JN, Photonic Crystals: Molding the Flow of Light, Princeton...1.27 Force Microscopy 40 0.10 0.00 Electron Spectroscopy 40 0.10 0.00 Rutherford backscattering spectrometry 38 0.10 0.00 flow cytometry 36 0.09...Backscattering Spectroscopy/Spectrometry • Flow Cytometry • Spectrophotometry (UV-Visible) • Deep Level Transient Spectroscopy • Inductively
Mobile flow cytometer for mHealth.

PubMed

Balsam, Joshua; Bruck, Hugh Alan; Rasooly, Avraham

2015-01-01

Flow cytometry is used for cell counting and analysis in numerous clinical and environmental applications. However flow cytometry is not used in mHealth mainly because current flow cytometers are large, expensive, power-intensive devices designed to operate in a laboratory. Their design results in a lack of portability and makes them unsuitable for mHealth applications. Another limitation of current technology is the low volumetric throughput rates that are not suitable for rapid detection of rare cells.To address these limitations, we describe here a novel, low-cost, mobile flow cytometer based on wide-field imaging with a webcam for large volume and high throughput fluorescence detection of rare cells as a simulation for circulating tumor cells (CTCs) detection. The mobile flow cytometer uses a commercially available webcam capable of 187 frames per second video capture at a resolution of 320 × 240 pixels. For fluorescence detection, a 1 W 450 nm blue laser is used for excitation of Syto-9 fluorescently stained cells detected at 535 nm. A wide-field flow cell was developed for large volume analysis that allows for the linear velocity of target cells to be lower than in conventional hydrodynamic focusing flow cells typically used in cytometry. The mobile flow cytometer was found to be capable of detecting low concentrations at flow rates of 500 μL/min, suitable for rare cell detection in large volumes. The simplicity and low cost of this device suggests that it may have a potential clinical use for mHealth flow cytometry for resource-poor settings associated with global health.
Annual Progress Report FY-91. Volume 1 and 2.

DTIC Science & Technology

1992-03-12

Pulmonary Med Tech 11 0644 GS Berger, TA Allergy Microbiologist 12 0403 GS Billups, L Flow Cytom Chemist 12 1320 GS Vacant Pulmonary Kyle Metabolic Unit...Reactions 11 (11/89) 3349 Salata, Kalman PhD. Mitogen-Inducible T Suppressor Cell 12 Assay by Flow Cytometry (12/89) 3350 Salata, Kalman PhD. Flow ...3/90) 3354 Salata, Kalman PhD. Two Way Mixed Lymphocyte Culture: 17 Analysis by Two Color Flow Cytometry (4/90) 3355 Salata, Kalman PhD. Effect of
CytometryML and other data formats

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2006-02-01

Cytology automation and research will be enhanced by the creation of a common data format. This data format would provide the pathology and research communities with a uniform way for annotating and exchanging images, flow cytometry, and associated data. This specification and/or standard will include descriptions of the acquisition device, staining, the binary representations of the image and list-mode data, the measurements derived from the image and/or the list-mode data, and descriptors for clinical/pathology and research. An international, vendor-supported, non-proprietary specification will allow pathologists, researchers, and companies to develop and use image capture/analysis software, as well as list-mode analysis software, without worrying about incompatibilities between proprietary vendor formats. Presently, efforts to create specifications and/or descriptions of these formats include the Laboratory Digital Imaging Project (LDIP) Data Exchange Specification; extensions to the Digital Imaging and Communications in Medicine (DICOM); Open Microscopy Environment (OME); Flowcyt, an extension to the present Flow Cytometry Standard (FCS); and CytometryML. The feasibility of creating a common data specification for digital microscopy and flow cytometry in a manner consistent with its use for medical devices and interoperability with both hospital information and picture archiving systems has been demonstrated by the creation of the CytometryML schemas. The feasibility of creating a software system for digital microscopy has been demonstrated by the OME. CytometryML consists of schemas that describe instruments and their measurements. These instruments include digital microscopes and flow cytometers. Optical components including the instruments' excitation and emission parts are described. The description of the measurements made by these instruments includes the tagged molecule, data acquisition subsystem, and the format of the list-mode and/or image data. Many of the CytometryML data-types are based on the Digital Imaging and Communications in Medicine (DICOM). Binary files for images and list-mode data have been created and read.
Routine detection of Epstein-Barr virus specific T-cells in the peripheral blood by flow cytometry

NASA Technical Reports Server (NTRS)

Crucian, B. E.; Stowe, R. P.; Pierson, D. L.; Sams, C. F.

2001-01-01

The ability to detect cytomegalovirus-specific T-cells (CD4(+)) in the peripheral blood by flow cytometry has been recently described by Picker et al. In this method, cells are incubated with viral antigen and responding (cytokine producing) T-cells are then identified by flow cytometry. To date, this technique has not been reliably used to detect Epstein-Barr virus (EBV)-specific T-cells primarily due to the superantigen/mitogenic properties of the virus which non-specifically activate T-cells. By modifying culture conditions under which the antigens are presented, we have overcome this limitation and developed an assay to detect and quantitate EBV-specific T-cells. The detection of cytokine producing T-cells by flow cytometry requires an extremely strong signal (such as culture in the presence of PMA and ionomycin). Our data indicate that in modified culture conditions (early removal of viral antigen) the non-specific activation of T-cells by EBV is reduced, but antigen presentation will continue uninhibited. Using this method, EBV-specific T-cells may be legitimately detected using flow cytometry. No reduction in the numbers of antigen-specific T-cells was observed by the early removal of target antigen when verified using cytomegalovirus antigen (a virus with no non-specific T-cell activation properties). In EBV-seropositive individuals, the phenotype of the EBV-specific cytokine producing T-cells was evaluated using four-color flow cytometry and found to be CD45(+), CD3(+), CD4(+), CD45RA(-), CD69(+), CD25(-). This phenotype indicates the stimulation of circulating previously unactivated memory T-cells. No cytokine production was observed in CD4(+) T-cells from EBV-seronegative individuals, confirming the specificity of this assay. In addition, the use of four color cytometry (CD45, CD3, CD69, IFNgamma/IL-2) allows the total quantitative assessment of EBV-specific T-cells while monitoring the interference of EBV non-specific mitogenic activity. This method may have significant utility for the monitoring of the immune response to latent virus infection/reactivation.
Determination of allergenic load and pollen count of Cupressus arizonica pollen by flow cytometry using Cup a1 polyclonal antibody.

PubMed

Benítez, Francisco Moreno; Camacho, Antonio Letrán; del Cuvillo Bernal, Alfonso; de Medina, Pedro Lobatón Sánchez; García Cózar, Francisco J; Romeu, Marisa Espinazo

2014-01-01

There is an increase in the incidence of pollen related allergy, thus information on pollen schedules would be a great asset for physicians to improve the clinical care of patients. Like cypress pollen sensitization shows a high prevalence among the causes of allergic rhinitis, and therefore it is of interest to use it like a model of study, distinguishing cypress pollen, pollen count, and allergenic load level. In this work, we use a flow cytometry based technique to obtain both Cupressus arizonica pollen count and allergenic load, using specific rabbit polyclonal antibody Cup a1 and its comparison with optical microscopy technique measurement. Airborne samples were collected from Burkard Spore-Trap and Burkard Cyclone Cupressus arizonica pollen was studied using specific rabbit polyclonal antibody Cup a1, labeled with AlexaFluor(®) 488 or 750 and analysed by Flow Cytometry in both an EPICS XL and Cyan ADP cytometers (Beckman Coulter(®) ). Optical microscopy study was realized with a Leica optical microscope. Bland and Altman was used to determine agreement between both techniques measured. We can identify three different populations based on rabbit polyclonal antibody Cup a1 staining. The main region (44.5%) had 97.3% recognition, a second region (25%) with 28% and a third region (30.5%) with 68% respectively. Immunofluorescence and confocal microscopy showed that main region corresponds to whole pollen grains, the second region are pollen without exine and the third region is constituted by smaller particles with allergenic properties. Pollen schedule shows a higher correlation measured by optical microscopy and flow cytometry in the pollen count with a P-value: 0.0008 E(-2) and 0.0002 with regard to smaller particles, so the Bland and Altman measurement showed a good correlation between them, P-value: 0.0003. Determination of pollen count and allergenic load by flow cytometry represents an important tool in the determination of airborne respiratory allergens. We showed that not only whole pollen but also smaller particles could induce allergic sensitization. This is the first study where flow cytometry is used for calculating pollen counts and allergenic load. Copyright © 2013 Clinical Cytometry Society.
Micro flow-through PCR in a PMMA chip fabricated by KrF excimer laser.

PubMed

Yao, Liying; Liu, Baoan; Chen, Tao; Liu, Shibing; Zuo, Tiechuan

2005-09-01

As the third PCR technology, micro flow-through PCR chip can amplify DNA specifically in an exponential fashion in vitro. Nowadays many academies in the world have successfully amplified DNA using their own-made flow-through PCR chip. In this paper, the ablation principle of PMMA at 248 nm excimer laser was studied, then a PMMA based flow-through PCR chip with 20 cycles was fabricated by excimer laser at 19 kv and 18 mm/min. The chip was bonded together with another cover chip at 105( composite function)C, 160 N and 20 minutes. In the end, it was integrated with electrical thermal thin films and Pt 100 temperature sensors. The temperature controllers was built standard PID digital temperature controller, the temperature control precision was +/- 0.2( composite function)C. The temperature grads between the three temperature zones were 16.5 and 22.2( composite function)C respectively, the gaps between the temperature zones could realize heat insulation.
78 FR 5186 - Clinical Flow Cytometry in Hematologic Malignancies; Public Workshop; Request for Comments

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-24

... in the diagnosis of leukemia and lymphoma and more recently in the detection of minimal residual... chronic lymphocytic leukemia (CLL); (3) Third-party flow cytometry data analysis software; and (4... held February 27, 2013 (77 FR 76051, December 26, 2012). An FDA workshop for acute lymphocytic leukemia...
Applications of flow cytometry to toxicological mycotoxin effects in cultured mammalian cells: a review.

PubMed

Juan-García, Ana; Manyes, Lara; Ruiz, María-José; Font, Guillermina

2013-06-01

This review gives an overview of flow cytometry applications to toxicological studies of several physiological target sites of mycotoxins on different mammalian cell lines. Mycotoxins are secondary metabolites of fungi that may be present in food, feed, air and water. The increasing presence of mycotoxins in crops, their wide distribution in the food chain, and their potential for toxicity demonstrate the need for further knowledge. Flow cytometry has become a valuable tool in mycotoxin studies in recent years for the rapid analysis of single cells in a mixture. In toxicology, the power of these methods lies in the possibility of determining a wide range of cell parameters, providing valuable information to elucidate cell growth and viability, metabolic activity, mitochondrial membrane potential and membrane integrity mechanisms. There are studies using flow cytometry technique on Alternaria, Aspergillus, Fusarium and Penicillium mycotoxins including information about cell type, assay conditions and functional parameters. Most of the studies collected in the literature are on deoxynivalenol and zearalenone mycotoxins. Cell cycle analysis and apoptosis are the processes more widely investigated. Copyright © 2013 Elsevier Ltd. All rights reserved.
[Fiat Lux. May be no more true in cytometry! Go to mass and spectrum but still stay classic].

PubMed

Idziorek, Thierry; Cazareth, Julie; Blanc, Catherine; Jouy, Nathalie; Bourdely, Pierre; Corneau, Aurélien

2018-05-01

The last decade has been an era of accelerated technological progress for flow cytometry. New technologies have been developed such as mass cytometry in which standard fluorochromes have been replaced by lanthanide-based non-radioactive metals and by spectral cytometry that measures the complete fluorescence spectrum. In this review, we schematically describe conventional, mass and spectral cytometry and present the plus and minus of each technology. © 2018 médecine/sciences – Inserm.
[Clinical usefulness of urine-formed elements' information obtained from bacteria detection by flow cytometry method that uses nucleic acid staining].

PubMed

Nakagawa, Hiroko; Yuno, Tomoji; Itho, Kiichi

2009-03-01

Recently, specific detection method for Bacteria, by flow cytometry method using nucleic acid staining, was developed as a function of automated urine formed elements analyzer for routine urine testing. Here, we performed a basic study on this bacteria analysis method. In addition, we also have a comparison among urine sediment analysis, urine Gram staining and urine quantitative cultivation, the conventional methods performed up to now. As a result, the bacteria analysis with flow cytometry method that uses nucleic acid staining was excellent in reproducibility, and higher sensitivity compared with microscopic urinary sediment analysis. Based on the ROC curve analysis, which settled urine culture method as standard, cut-off level of 120/microL was defined and its sensitivity = 85.7%, specificity = 88.2%. In the analysis of scattergram, accompanied with urine culture method, among 90% of rod positive samples, 80% of dots were appeared in the area of 30 degrees from axis X. In addition, one case even indicated that analysis of bacteria by flow cytometry and scattergram of time series analysis might be helpful to trace the progress of causative bacteria therefore the information supposed to be clinically significant. Reporting bacteria information with nucleic acid staining flow cytometry method is expected to contribute to a rapid diagnostics and treatment of urinary tract infections. Besides, the contribution to screening examination of microbiology and clinical chemistry, will deliver a more efficient solution to urine analysis.
Flow Cytometry: Evolution of Microbiological Methods for Probiotics Enumeration.

PubMed

Pane, Marco; Allesina, Serena; Amoruso, Angela; Nicola, Stefania; Deidda, Francesca; Mogna, Luca

2018-05-14

The purpose of this trial was to verify that the analytical method ISO 19344:2015 (E)-IDF 232:2015 (E) is valid and reliable for quantifying the concentration of the probiotic Lactobacillus rhamnosus GG (ATCC 53103) in a finished product formulation. Flow cytometry assay is emerging as an alternative rapid method for microbial detection, enumeration, and population profiling. The use of flow cytometry not only permits the determination of viable cell counts but also allows for enumeration of damaged and dead cell subpopulations. Results are expressed as TFU (Total Fluorescent Units) and AFU (Active Fluorescent Units). In December 2015, the International Standard ISO 19344-IDF 232 "Milk and milk products-Starter cultures, probiotics and fermented products-Quantification of lactic acid bacteria by flow cytometry" was published. This particular ISO can be applied universally and regardless of the species of interest. Analytical method validation was conducted on 3 different industrial batches of L. rhamnosus GG according to USP39<1225>/ICH Q2R1 in term of: accuracy, precision (repeatability), intermediate precision (ruggedness), specificity, limit of quantification, linearity, range, robustness. The data obtained on the 3 batches of finished product have significantly demonstrated the validity and robustness of the cytofluorimetric analysis. On the basis of the results obtained, the ISO 19344:2015 (E)-IDF 232:2015 (E) "Quantification of lactic acid bacteria by flow cytometry" can be used for the enumeration of L. rhamnosus GG in a finished product formulation.
Data File Standard for Flow Cytometry, version FCS 3.1.

PubMed

Spidlen, Josef; Moore, Wayne; Parks, David; Goldberg, Michael; Bray, Chris; Bierre, Pierre; Gorombey, Peter; Hyun, Bill; Hubbard, Mark; Lange, Simon; Lefebvre, Ray; Leif, Robert; Novo, David; Ostruszka, Leo; Treister, Adam; Wood, James; Murphy, Robert F; Roederer, Mario; Sudar, Damir; Zigon, Robert; Brinkman, Ryan R

2010-01-01

The flow cytometry data file standard provides the specifications needed to completely describe flow cytometry data sets within the confines of the file containing the experimental data. In 1984, the first Flow Cytometry Standard format for data files was adopted as FCS 1.0. This standard was modified in 1990 as FCS 2.0 and again in 1997 as FCS 3.0. We report here on the next generation flow cytometry standard data file format. FCS 3.1 is a minor revision based on suggested improvements from the community. The unchanged goal of the standard is to provide a uniform file format that allows files created by one type of acquisition hardware and software to be analyzed by any other type.The FCS 3.1 standard retains the basic FCS file structure and most features of previous versions of the standard. Changes included in FCS 3.1 address potential ambiguities in the previous versions and provide a more robust standard. The major changes include simplified support for international characters and improved support for storing compensation. The major additions are support for preferred display scale, a standardized way of capturing the sample volume, information about originality of the data file, and support for plate and well identification in high throughput, plate based experiments. Please see the normative version of the FCS 3.1 specification in Supporting Information for this manuscript (or at http://www.isac-net.org/ in the Current standards section) for a complete list of changes.

Data File Standard for Flow Cytometry, Version FCS 3.1

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

Spidlen, Josef; Moore, Wayne; Parks, David

2009-11-10

The flow cytometry data file standard provides the specifications needed to completely describe flow cytometry data sets within the confines of the file containing the experimental data. In 1984, the first Flow Cytometry Standard format for data files was adopted as FCS 1.0. This standard was modified in 1990 as FCS 2.0 and again in 1997 as FCS 3.0. We report here on the next generation flow cytometry standard data file format. FCS 3.1 is a minor revision based on suggested improvements from the community. The unchanged goal of the standard is to provide a uniform file format that allowsmore » files created by one type of acquisition hardware and software to be analyzed by any other type. The FCS 3.1 standard retains the basic FCS file structure and most features of previous versions of the standard. Changes included in FCS 3.1 address potential ambiguities in the previous versions and provide a more robust standard. The major changes include simplified support for international characters and improved support for storing compensation. The major additions are support for preferred display scale, a standardized way of capturing the sample volume, information about originality of the data file, and support for plate and well identification in high throughput, plate based experiments. Please see the normative version of the FCS 3.1 specification in Supporting Information for this manuscript (or at http://www.isac-net.org/ in the Current standards section) for a complete list of changes.« less
Rapid assessment of viable but non-culturable Bacillus coagulans MTCC 5856 in commercial formulations using Flow cytometry.

PubMed

Majeed, Muhammed; Majeed, Shaheen; Nagabhushanam, Kalyanam; Punnapuzha, Ardra; Philip, Sheena; Mundkur, Lakshmi

2018-01-01

Accurate enumeration of bacterial count in probiotic formulation is imperative to ensure that the product adheres to regulatory standards and citation in consumer product label. Standard methods like plate count, can enumerate only replicating bacterial population under selected culture conditions. Viable but non culturable bacteria (VBNC) retain characteristics of living cells and can regain cultivability by a process known as resuscitation. This is a protective mechanism adapted by bacteria to evade stressful environmental conditions. B. coagulans MTCC 5856(LactoSpore®) is a probiotic endospore which can survive for decades in hostile environments without dividing. In the present study, we explored the use of flow cytometry to enumerate the viable count of B. coagulans MTCC 5856 under acidic and alkaline conditions, high temperature and in commercial formulations like compressed tablets and capsules. Flow cytometry (FCM) was comparable to plate count method when the spores were counted at physiological conditions. We show that VBNC state is induced in B. coagulans MTCC 5856by high temperature and acidic pH. The cells get resuscitated under physiological conditions and FCM was sensitive to detect the VBNC spores. Flow cytometry showed excellent ability to assess the viable spore count in commercial probiotic formulations of B. coagulans MTCC 5856. The results establish Flow cytometry as a reliable method to count viable bacteria in commercial probiotic preparations. Sporulation as well as existence as VBNC could contribute to the extreme stability of B. coagulans MTCC 5856.
Rapid assessment of viable but non-culturable Bacillus coagulans MTCC 5856 in commercial formulations using Flow cytometry

PubMed Central

Majeed, Muhammed; Majeed, Shaheen; Nagabhushanam, Kalyanam; Punnapuzha, Ardra; Philip, Sheena

2018-01-01

Accurate enumeration of bacterial count in probiotic formulation is imperative to ensure that the product adheres to regulatory standards and citation in consumer product label. Standard methods like plate count, can enumerate only replicating bacterial population under selected culture conditions. Viable but non culturable bacteria (VBNC) retain characteristics of living cells and can regain cultivability by a process known as resuscitation. This is a protective mechanism adapted by bacteria to evade stressful environmental conditions. B. coagulans MTCC 5856(LactoSpore®) is a probiotic endospore which can survive for decades in hostile environments without dividing. In the present study, we explored the use of flow cytometry to enumerate the viable count of B. coagulans MTCC 5856 under acidic and alkaline conditions, high temperature and in commercial formulations like compressed tablets and capsules. Flow cytometry (FCM) was comparable to plate count method when the spores were counted at physiological conditions. We show that VBNC state is induced in B. coagulans MTCC 5856by high temperature and acidic pH. The cells get resuscitated under physiological conditions and FCM was sensitive to detect the VBNC spores. Flow cytometry showed excellent ability to assess the viable spore count in commercial probiotic formulations of B. coagulans MTCC 5856. The results establish Flow cytometry as a reliable method to count viable bacteria in commercial probiotic preparations. Sporulation as well as existence as VBNC could contribute to the extreme stability of B. coagulans MTCC 5856. PMID:29474436
DOE Office of Scientific and Technical Information (OSTI.GOV)

Vasdekis, Andreas E.; Stephanopoulos, Gregory

The sampling and manipulation of cells down to the individual has been of substantial interest since the very beginning of Life Sciences. Herein, our objective is to highlight the most recent developments in single cell manipulation, as well as pioneering ones. First, flow-through methods will be discussed, namely methods in which the single cells flow continuously in an ordered manner during their analysis. This section will be followed by confinement techniques that enable cell isolation and confinement in one, two- or three-dimensions. Flow cytometry and droplet microfluidics are the two most common methods of flow-through analysis. While both are high-throughputmore » techniques, their difference lays in the fact that the droplet encapsulated cells experience a restricted and personal microenvironment, while in flow cytometry cells experience similar nutrient and stimuli initial concentrations. These methods are rather well established; however, they recently enabled immense strides in single cell phenotypic analysis, namely the identification and analysis of metabolically distinct individuals from an isogenic population using both droplet microfluidics and flow cytometry.« less
Impact of Two Measures of Micrometastatic Disease on Clinical Outcomes in Patients with Newly Diagnosed Ewing Sarcoma: A Report from the Children’s Oncology Group

PubMed Central

Vo, Kieuhoa T.; Edwards, Jeremy V.; Epling, C. Lorrie; Sinclair, Elizabeth; Hawkins, Douglas S.; Grier, Holcombe E.; Janeway, Katherine A.; Barnette, Phillip; McIlvaine, Elizabeth; Krailo, Mark D.; Barkauskas, Donald A.; Matthay, Katherine K.; Womer, Richard B.; Gorlick, Richard G.; Lessnick, Stephen L.; Mackall, Crystal L.; DuBois, Steven G.

2016-01-01

Purpose Flow cytometry and RT-PCR can detect occult Ewing sarcoma (ES) cells in the blood and bone marrow (BM). These techniques were used to evaluate the prognostic significance of micrometastatic disease in ES. Experimental Design Newly diagnosed patients with ES were enrolled on two prospective multi-center studies. In the flow cytometry cohort, patients were defined as “positive” for BM micrometastatic disease if their CD99+/CD45− values were above the upper limit in 22 control patients. In the PCR cohort, RT-PCR on blood or BM samples classified the patients as “positive” or “negative” for EWSR1/FLI1 translocations. The association between micrometastatic disease burden with clinical features and outcome was assessed. Co-expression of IGF-1R on detected tumor cells was performed in a subset of flow cytometry samples. Results The median total BM CD99+CD45− percent was 0.0012% (range 0–1.10%) in the flow cytometry cohort, with 14/109 (12.8%) of ES patients defined as “positive.” In the PCR cohort, 19.6% (44/225) patients were “positive” for any EWSR1/FLI1 translocation in blood or BM. There were no differences in baseline clinical features or event-free or overall survival between patients classified as “positive” vs. “negative” by either method. CD99+CD45− cells had significantly higher IGF-1R expression compared to CD45+ hematopoietic cells (mean geometric mean fluorescence intensity 982.7 vs. 190.9; p<0.001). Conclusion The detection of micrometastatic disease at initial diagnosis by flow cytometry or RT-PCR is not associated with outcome in newly diagnosed patients with ES. Flow cytometry provides a tool to characterize occult micrometastatic tumor cells for proteins of interest. PMID:26861456
Masks in Imaging Flow Cytometry

PubMed Central

Dominical, Venina; Samsel, Leigh; McCoy, J. Philip

2016-01-01

Data analysis in imaging flow cytometry incorporates elements of flow cytometry together with other aspects of morphological analysis of images. A crucial early step in this analysis is the creation of a mask to distinguish the portion of the image upon which further examination of specified features can be performed. Default masks are provided by the manufacturer of the imaging flow cytometer but additional custom masks can be created by the individual user for specific applications. Flawed or inaccurate masks can have a substantial negative impact on the overall analysis of a sample, thus great care must be taken to ensure the accuracy of masks. Here we discuss various types of masks and cite examples of their use. Furthermore we provide our insight for how to approach selecting and assessing the optimal mask for a specific analysis. PMID:27461256
Clinical Investigation Program. Annual Progress Report. Volume 1

DTIC Science & Technology

1994-01-20

Suport Labs Resch Chemist 13 0644 GS Salata, KF Allergy Microbiologist 12 0403 CS Billups, L Flow Cytom Microbiologist 12 0403 GS Dobek, AS Inf Disease 5...continued to increase laboratory research support to principal investigators throughout the medical center. The DCI Flow Cytometry Laboratory provided...Kalman PhD. Mitogen-Inducible T Suppressor Cell 12 Assay by Flow Cytometry (12/89) * Reference is to page number(s) in Volume II. 30 PROTOCOL NUMBER
An Analysis of the Effects of Chip-groove Geometry on Machining Performance Using Finite Element Methods

NASA Astrophysics Data System (ADS)

Ee, K. C.; Dillon, O. W.; Jawahir, I. S.

2004-06-01

This paper discusses the influence of major chip-groove parameters of a cutting tool on the chip formation process in orthogonal machining using finite element (FE) methods. In the FE formulation, a thermal elastic-viscoplastic material model is used together with a modified Johnson-Cook material law for the flow stress. The chip back-flow angle and the chip up-curl radius are calculated for a range of cutting conditions by varying the chip-groove parameters. The analysis provides greater understanding of the effectiveness of chip-groove configurations and points a way to correlate cutting conditions with tool-wear when machining with a grooved cutting tool.
Microfluidic proportional flow controller

PubMed Central

Prentice-Mott, Harrison; Toner, Mehmet; Irimia, Daniel

2011-01-01

Precise flow control in microfluidic chips is important for many biochemical assays and experiments at microscale. While several technologies for controlling fluid flow have been implemented either on- or off-chip, these can provide either high-speed or high-precision control, but seldom could accomplish both at the same time. Here we describe a new on-chip, pneumatically activated flow controller that allows for fast and precise control of the flow rate through a microfluidic channel. Experimental results show that the new proportional flow controllers exhibited a response time of approximately 250 ms, while our numerical simulations suggest that faster actuation down to approximately 50 ms could be achieved with alternative actuation schemes. PMID:21874096
Waveguide detection of right-angle-scattered light in flow cytometry

DOEpatents

Mariella, Jr., Raymond P.

2000-01-01

A transparent flow cell is used as an index-guided optical waveguide. A detector for the flow cell but not the liquid stream detects the Right-Angle-Scattered (RAS) Light exiting from one end of the flow cell. The detector(s) could view the trapped RAS light from the flow cell either directly or through intermediate optical light guides. If the light exits one end of the flow cell, then the other end of the flow cell can be given a high-reflectivity coating to approximately double the amount of light collected. This system is more robust in its alignment than the traditional flow cytometry systems which use imaging optics, such as microscope objectives.
Immunological Tools: Engaging Students in the Use and Analysis of Flow Cytometry and Enzyme-linked Immunosorbent Assay (ELISA)

ERIC Educational Resources Information Center

Ott, Laura E.; Carson, Susan

2014-01-01

Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and…
Differences in leukocyte differentiation molecule abundances on domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis) neutrophils identified by flow cytometry

USDA-ARS?s Scientific Manuscript database

Although both domestic sheep (DS) and bighorn sheep (BHS) are affected by similar respiratory bacterial pathogens, experimental and field data indicate BHS are more susceptible to pneumonia. Cross-reactive monoclonal antibodies (mAbs) for use in flow cytometry (FC) are valuable reagents for interspe...
Utilization of flow cytometry to identify chimeral sectors in leaf tissue of Lolium multiflorum x L. arundinaceum hybrids

USDA-ARS?s Scientific Manuscript database

We have identified a method whereby Lolium multiflorum (Lm) or L. arundinaceum (Fa) genomes are preferentially eliminated through a mitotic loss behavior in interspecific Lm x Fa F1 hybrids, generating either dihaploid Lm lines or Fa lines. Flow cytometry, a method for rapidly characterizing optical...
A light sheet confocal microscope for image cytometry with a variable linear slit detector

NASA Astrophysics Data System (ADS)

Hutcheson, Joshua A.; Khan, Foysal Z.; Powless, Amy J.; Benson, Devin; Hunter, Courtney; Fritsch, Ingrid; Muldoon, Timothy J.

2016-03-01

We present a light sheet confocal microscope (LSCM) capable of high-resolution imaging of cell suspensions in a microfluidic environment. In lieu of conventional pressure-driven flow or mechanical translation of the samples, we have employed a novel method of fluid transport, redox-magnetohydrodynamics (redox-MHD). This method achieves fluid motion by inducing a small current into the suspension in the presence of a magnetic field via electrodes patterned onto a silicon chip. This on-chip transportation requires no moving parts, and is coupled to the remainder of the imaging system. The microscopy system comprises a 450 nm diode 20 mW laser coupled to a single mode fiber and a cylindrical lens that converges the light sheet into the back aperture of a 10x, 0.3 NA objective lens in an epi-illumination configuration. The emission pathway contains a 150 mm tube lens that focuses the light onto the linear sensor at the conjugate image plane. The linear sensor (ELiiXA+ 8k/4k) has three lateral binning modes which enables variable detection aperture widths between 5, 10, or 20 μm, which can be used to vary axial resolution. We have demonstrated redox-MHD-enabled light sheet microscopy in suspension of fluorescent polystyrene beads. This approach has potential as a high-throughput image cytometer with myriad cellular diagnostic applications.
CytometryML: a data standard which has been designed to interface with other standards

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2007-02-01

Because of the differences in the requirements, needs, and past histories including existing standards of the creating organizations, a single encompassing cytology-pathology standard will not, in the near future, replace the multiple existing or under development standards. Except for DICOM and FCS, these standardization efforts are all based on XML. CytometryML is a collection of XML schemas, which are based on the Digital Imaging and Communications in Medicine (DICOM) and Flow Cytometry Standard (FCS) datatypes. The CytometryML schemas contain attributes that link them to the DICOM standard and FCS. Interoperability with DICOM has been facilitated by, wherever reasonable, limiting the difference between CytometryML and the previous standards to syntax. In order to permit the Resource Description Framework, RDF, to reference the CytometryML datatypes, id attributes have been added to many CytometryML elements. The Laboratory Digital Imaging Project (LDIP) Data Exchange Specification and the Flowcyt standards development effort employ RDF syntax. Documentation from DICOM has been reused in CytometryML. The unity of analytical cytology was demonstrated by deriving a microscope type and a flow cytometer type from a generic cytometry instrument type. The feasibility of incorporating the Flowcyt gating schemas into CytometryML has been demonstrated. CytometryML is being extended to include many of the new DICOM Working Group 26 datatypes, which describe patients, specimens, and analytes. In situations where multiple standards are being created, interoperability can be facilitated by employing datatypes based on a common set of semantics and building in links to standards that employ different syntax.
Optimizing transformations for automated, high throughput analysis of flow cytometry data

PubMed Central

2010-01-01

Background In a high throughput setting, effective flow cytometry data analysis depends heavily on proper data preprocessing. While usual preprocessing steps of quality assessment, outlier removal, normalization, and gating have received considerable scrutiny from the community, the influence of data transformation on the output of high throughput analysis has been largely overlooked. Flow cytometry measurements can vary over several orders of magnitude, cell populations can have variances that depend on their mean fluorescence intensities, and may exhibit heavily-skewed distributions. Consequently, the choice of data transformation can influence the output of automated gating. An appropriate data transformation aids in data visualization and gating of cell populations across the range of data. Experience shows that the choice of transformation is data specific. Our goal here is to compare the performance of different transformations applied to flow cytometry data in the context of automated gating in a high throughput, fully automated setting. We examine the most common transformations used in flow cytometry, including the generalized hyperbolic arcsine, biexponential, linlog, and generalized Box-Cox, all within the BioConductor flowCore framework that is widely used in high throughput, automated flow cytometry data analysis. All of these transformations have adjustable parameters whose effects upon the data are non-intuitive for most users. By making some modelling assumptions about the transformed data, we develop maximum likelihood criteria to optimize parameter choice for these different transformations. Results We compare the performance of parameter-optimized and default-parameter (in flowCore) data transformations on real and simulated data by measuring the variation in the locations of cell populations across samples, discovered via automated gating in both the scatter and fluorescence channels. We find that parameter-optimized transformations improve visualization, reduce variability in the location of discovered cell populations across samples, and decrease the misclassification (mis-gating) of individual events when compared to default-parameter counterparts. Conclusions Our results indicate that the preferred transformation for fluorescence channels is a parameter- optimized biexponential or generalized Box-Cox, in accordance with current best practices. Interestingly, for populations in the scatter channels, we find that the optimized hyperbolic arcsine may be a better choice in a high-throughput setting than current standard practice of no transformation. However, generally speaking, the choice of transformation remains data-dependent. We have implemented our algorithm in the BioConductor package, flowTrans, which is publicly available. PMID:21050468
Optimizing transformations for automated, high throughput analysis of flow cytometry data.

PubMed

Finak, Greg; Perez, Juan-Manuel; Weng, Andrew; Gottardo, Raphael

2010-11-04

In a high throughput setting, effective flow cytometry data analysis depends heavily on proper data preprocessing. While usual preprocessing steps of quality assessment, outlier removal, normalization, and gating have received considerable scrutiny from the community, the influence of data transformation on the output of high throughput analysis has been largely overlooked. Flow cytometry measurements can vary over several orders of magnitude, cell populations can have variances that depend on their mean fluorescence intensities, and may exhibit heavily-skewed distributions. Consequently, the choice of data transformation can influence the output of automated gating. An appropriate data transformation aids in data visualization and gating of cell populations across the range of data. Experience shows that the choice of transformation is data specific. Our goal here is to compare the performance of different transformations applied to flow cytometry data in the context of automated gating in a high throughput, fully automated setting. We examine the most common transformations used in flow cytometry, including the generalized hyperbolic arcsine, biexponential, linlog, and generalized Box-Cox, all within the BioConductor flowCore framework that is widely used in high throughput, automated flow cytometry data analysis. All of these transformations have adjustable parameters whose effects upon the data are non-intuitive for most users. By making some modelling assumptions about the transformed data, we develop maximum likelihood criteria to optimize parameter choice for these different transformations. We compare the performance of parameter-optimized and default-parameter (in flowCore) data transformations on real and simulated data by measuring the variation in the locations of cell populations across samples, discovered via automated gating in both the scatter and fluorescence channels. We find that parameter-optimized transformations improve visualization, reduce variability in the location of discovered cell populations across samples, and decrease the misclassification (mis-gating) of individual events when compared to default-parameter counterparts. Our results indicate that the preferred transformation for fluorescence channels is a parameter- optimized biexponential or generalized Box-Cox, in accordance with current best practices. Interestingly, for populations in the scatter channels, we find that the optimized hyperbolic arcsine may be a better choice in a high-throughput setting than current standard practice of no transformation. However, generally speaking, the choice of transformation remains data-dependent. We have implemented our algorithm in the BioConductor package, flowTrans, which is publicly available.
Experimental study of cassava sun drying

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

Njie, D.N.; Rumsey, T.R.

1997-03-01

Sun drying experiments were performed to compare drying of cassava chips in sheet-metal trays with drying on mesh wire trays. In the sheet-metal trays, there was air flow across the top of the bed chips, while the mesh wire trays permitted air to flow through the bed. Drying rate was faster and more uniform in the trays with through-flow air circulation. Higher temperatures were reached by chips in the sheet-metal trays than those in the mesh trays because of contact heating, but the drying rate was lower because of the reduced air flow.
flowVS: channel-specific variance stabilization in flow cytometry

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

Azad, Ariful; Rajwa, Bartek; Pothen, Alex

Comparing phenotypes of heterogeneous cell populations from multiple biological conditions is at the heart of scientific discovery based on flow cytometry (FC). When the biological signal is measured by the average expression of a biomarker, standard statistical methods require that variance be approximately stabilized in populations to be compared. Since the mean and variance of a cell population are often correlated in fluorescence-based FC measurements, a preprocessing step is needed to stabilize the within-population variances.
flowVS: channel-specific variance stabilization in flow cytometry

DOE PAGES

Azad, Ariful; Rajwa, Bartek; Pothen, Alex

2016-07-28

Comparing phenotypes of heterogeneous cell populations from multiple biological conditions is at the heart of scientific discovery based on flow cytometry (FC). When the biological signal is measured by the average expression of a biomarker, standard statistical methods require that variance be approximately stabilized in populations to be compared. Since the mean and variance of a cell population are often correlated in fluorescence-based FC measurements, a preprocessing step is needed to stabilize the within-population variances.

Time-gated flow cytometry: an ultra-high selectivity method to recover ultra-rare-event μ-targets in high-background biosamples

NASA Astrophysics Data System (ADS)

Jin, Dayong; Piper, James A.; Leif, Robert C.; Yang, Sean; Ferrari, Belinda C.; Yuan, Jingli; Wang, Guilan; Vallarino, Lidia M.; Williams, John W.

2009-03-01

A fundamental problem for rare-event cell analysis is auto-fluorescence from nontarget particles and cells. Time-gated flow cytometry is based on the temporal-domain discrimination of long-lifetime (>1 μs) luminescence-stained cells and can render invisible all nontarget cell and particles. We aim to further evaluate the technique, focusing on detection of ultra-rare-event 5-μm calibration beads in environmental water dirt samples. Europium-labeled 5-μm calibration beads with improved luminescence homogeneity and reduced aggregation were evaluated using the prototype UV LED excited time-gated luminescence (TGL) flow cytometer (FCM). A BD FACSAria flow cytometer was used to sort accurately a very low number of beads (<100 events), which were then spiked into concentrated samples of environmental water. The use of europium-labeled beads permitted the demonstration of specific detection rates of 100%+/-30% and 91%+/-3% with 10 and 100 target beads, respectively, that were mixed with over one million nontarget autofluorescent background particles. Under the same conditions, a conventional FCM was unable to recover rare-event fluorescein isothiocyanate (FITC) calibration beads. Preliminary results on Giardia detection are also reported. We have demonstrated the scientific value of lanthanide-complex biolabels in flow cytometry. This approach may augment the current method that uses multifluorescence-channel flow cytometry gating.
2006 Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: optimal reagents and reporting for the flow cytometric diagnosis of hematopoietic neoplasia.

PubMed

Wood, Brent L; Arroz, Maria; Barnett, David; DiGiuseppe, Joseph; Greig, Bruce; Kussick, Steven J; Oldaker, Teri; Shenkin, Mark; Stone, Elizabeth; Wallace, Paul

2007-01-01

Immunophenotyping by flow cytometry has become standard practice in the evaluation and monitoring of patients with hematopoietic neoplasia. However, despite its widespread use, considerable variability continues to exist in the reagents used for evaluation and the format in which results are reported. As part of the 2006 Bethesda Consensus conference, a committee was formed to attempt to define a consensus set of reagents suitable for general use in the diagnosis and monitoring of hematopoietic neoplasms. The committee included laboratory professionals from private, public, and university hospitals as well as large reference laboratories that routinely operate clinical flow cytometry laboratories with an emphasis on lymphoma and leukemia immunophenotyping. A survey of participants successfully identified the cell lineage(s) to be evaluated for each of a variety of specific medical indications and defined a set of consensus reagents suitable for the initial evaluation of each cell lineage. Elements to be included in the reporting of clinical flow cytometric results for leukemia and lymphoma evaluation were also refined and are comprehensively listed. The 2006 Bethesda Consensus conference represents the first successful attempt to define a set of consensus reagents suitable for the initial evaluation of hematopoietic neoplasia. Copyright 2007 Clinical Cytometry Society.
Non-linear optical flow cytometry using a scanned, Bessel beam light-sheet.

PubMed

Collier, Bradley B; Awasthi, Samir; Lieu, Deborah K; Chan, James W

2015-05-29

Modern flow cytometry instruments have become vital tools for high-throughput analysis of single cells. However, as issues with the cellular labeling techniques often used in flow cytometry have become more of a concern, the development of label-free modalities for cellular analysis is increasingly desired. Non-linear optical phenomena (NLO) are of growing interest for label-free analysis because of the ability to measure the intrinsic optical response of biomolecules found in cells. We demonstrate that a light-sheet consisting of a scanned Bessel beam is an optimal excitation geometry for efficiently generating NLO signals in a microfluidic environment. The balance of photon density and cross-sectional area provided by the light-sheet allowed significantly larger two-photon fluorescence intensities to be measured in a model polystyrene microparticle system compared to measurements made using other excitation focal geometries, including a relaxed Gaussian excitation beam often used in conventional flow cytometers.
High throughput, parallel imaging and biomarker quantification of human spermatozoa by ImageStream flow cytometry.

PubMed

Buckman, Clayton; George, Thaddeus C; Friend, Sherree; Sutovsky, Miriam; Miranda-Vizuete, Antonio; Ozanon, Christophe; Morrissey, Phil; Sutovsky, Peter

2009-12-01

Spermatid specific thioredoxin-3 protein (SPTRX-3) accumulates in the superfluous cytoplasm of defective human spermatozoa. Novel ImageStream technology combining flow cytometry with cell imaging was used for parallel quantification and visualization of SPTRX-3 protein in defective spermatozoa of five men from infertile couples. The majority of the SPTRX-3 containing cells were overwhelmingly spermatozoa with a variety of morphological defects, detectable in the ImageStream recorded images. Quantitative parameters of relative SPTRX-3 induced fluorescence measured by ImageStream correlated closely with conventional flow cytometric measurements of the same sample set and reflected the results of clinical semen evaluation. Image Stream quantification of SPTRX-3 combines and surpasses the informative value of both conventional flow cytometry and light microscopic semen evaluation. The observed patterns of the retention of SPTRX-3 in the sperm samples from infertility patients support the view that SPTRX3 is a biomarker of male infertility.
Characterization and use of a rabbit-anti-mouse VPAC1 antibody by flow cytometry

PubMed Central

Hermann, Rebecca J.; Van der Steen, Travis; Vomhof-DeKrey, Emilie E.; Benton, Keith D.; Failing, Jarrett J.; Haring, Jodie S.; Dorsam, Glenn P.

2011-01-01

Vasoactive intestinal peptide receptor – 1 signaling in lymphocytes has been shown to regulate chemotaxis, proliferation, apoptosis and differentiation. During T cell activation, VPAC1 mRNA is downregulated, but the effect on its protein levels is less clear. A small number of studies have reported measurement of human VPAC1 by flow cytometry, but murine VPAC1 reagents are unavailable. Therefore, we set out to generate a reliable and highly specific α-mouse VPAC1 polyclonal antibody for use with flow cytometry. After successfully generating a rabbit α-VPAC1 polyclonal antibody (α-mVPAC1 pAb), we characterized its cross-reactivity and showed that it does not recognize other family receptors (mouse VPAC2 and PAC1, human VPAC1, VPAC2 and PAC1) by flow cytometry. Partial purification of the rabbit α-VPAC1 sera increased the specific-activity of the α-mVPAC1 pAb by 20-fold, and immunofluorescence microscopy (IF) confirmed a plasma membrane subcellular localization for mouse VPAC1 protein. To test the usefulness of this specific α-mVPAC1 pAb, we showed that primary, resting mouse T cells express detectable levels of VPAC1 protein, with little detectable signal from activated T cells, or CD19 B cells. These data support our previously published data showing a downregulation of VPAC1 mRNA during T cell activation. Collectively, we have established a well-characterized, and highly species specific α-mVPAC1 pAb for VPAC1 surface measurement by IF and flow cytometry. PMID:22079255
Development of a bead-based multiplexed assay for simultaneous quantification of five bovine cytokines by flow cytometry.

PubMed

Rodrigues, Valérie; Baudier, Jean Baptiste; Chantal, Isabelle

2017-09-01

Quantifying cytokines is extremely important in studies of host-pathogen interactions. Multiplex assays are commercially available but only for human and mouse cytokines. Here a method for the simultaneous quantification of five important bovine cytokines IFNγ, IL-4, IL-10, IL-12, and TNFα in cell culture supernatants, using flow cytometry was reported. Functional beads from BD Biosciences expressing specific APC intensity were used. Commercially available antibodies against bovine cytokines were covalently coupled to beads as capture antibodies. Fixed recombinant cytokines were revealed with a second monoclonal antibody coupled with biotin, then revealed with streptavidin-PE. This complex was analyzed using a standard flow cytometer. Experiments were performed to check no cross reactions had occurred. The limits of detection ranged between 0.08 and 0.4 ng/ml depending on the cytokine, and the linearity between the lower and higher limits was remarkable (R 2 > 99.8%). Finally, native cytokines from cell culture supernatants were tested. Results were compared using the standard ELISA test and showed that concentrations of native cytokine in cell culture supernatants were comparable with the two methods, with a wider dynamic range using beads and flow cytometry than with ELISA assays. Bovine IFNγ, IL-4, IL-10, IL-12, and TNFα in culture supernatants can be now simultaneously detected in a single assay, using a standard flow cytometer for both basic and high-throughput analyses. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.
Stability validation of paraformaldehyde-fixed samples for the assessment of the platelet PECAM-1, P-selectin, and PAR-1 thrombin receptor by flow cytometry.

PubMed

Atar, Oliver D; Eisert, Christian; Pokov, Ilya; Serebruany, Victor L

2010-07-01

Sample fixation for storage and/or transportation represents an unsolved challenge for multicenter clinical trials assessing serial changes in platelet activity, or monitoring various antiplatelet regimens. Whole blood flow cytometry represents a major advance in defining platelet function, although special training and expensive equipment is required. We sought to determine how fixation with 2% paraformaldehyde (PFA), and storage of blood samples over 1 week affects the flow cytometry readings for both intact and thrombin-activating four major surface platelet receptors. Whole blood platelet expression of PECAM-1, P-selectin, PAR-1 inactive receptor (SPAN-12), and cleaved (WEDE-15) epitope was assessed immediately after blood draw, after staining with 2% PFA, and at day 1, 3, 5, and 7. The study was performed in 6 volunteers with multiple risk factors for vascular disease, not receiving any antiplatelet agents. Staining with PFA resulted in a slight decrease of fluorescence intensity, especially for PECAM-1, while antigen expression at day 1, 3 and 5 remains consistent, and highly reproducible. At day 7 there was a small but inconsistent trend towards diminished fluorescence intensity. The platelet data were consistent while validated with the isotype-matched irrelevant antibody. These data suggest that there is a 5 day window to perform final flow cytometry readings of whole blood PFA-fixed inactivated platelet samples. In contrast, thrombin activation cause gradual loss of flow cytometry signal, and cannot be recommended for long-term storage. This is critical logistic information for conducting multicenter platelet substudies within the framework of major clinical trials.
Progress on an implementation of MIFlowCyt in XML

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Leif, Stephanie H.

2015-03-01

Introduction: The International Society for Advancement of Cytometry (ISAC) Data Standards Task Force (DSTF) has created a standard for the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt 1.0). The CytometryML schemas, are based in part upon the Flow Cytometry Standard and Digital Imaging and Communication (DICOM) standards. CytometryML has and will be extended and adapted to include MIFlowCyt, as well as to serve as a common standard for flow and image cytometry (digital microscopy). Methods: The MIFlowCyt data-types were created, as is the rest of CytometryML, in the XML Schema Definition Language (XSD1.1). Individual major elements of the MIFlowCyt schema were translated into XML and filled with reasonable data. A small section of the code was formatted with HTML formatting elements. Results: The differences in the amount of detail to be recorded for 1) users of standard techniques including data analysts and 2) others, such as method and device creators, laboratory and other managers, engineers, and regulatory specialists required that separate data-types be created to describe the instrument configuration and components. A very substantial part of the MIFlowCyt element that describes the Experimental Overview part of the MIFlowCyt and substantial parts of several other major elements have been developed. Conclusions: The future use of structured XML tags and web technology should facilitate searching of experimental information, its presentation, and inclusion in structured research, clinical, and regulatory documents, as well as demonstrate in publications adherence to the MIFlowCyt standard. The use of CytometryML together with XML technology should also result in the textual and numeric data being published using web technology without any change in composition. Preliminary testing indicates that CytometryML XML pages can be directly formatted with the combination of HTML and CSS.
Microfluidic "Pouch" Chips for Immunoassays and Nucleic Acid Amplification Tests.

PubMed

Mauk, Michael G; Liu, Changchun; Qiu, Xianbo; Chen, Dafeng; Song, Jinzhao; Bau, Haim H

2017-01-01

Microfluidic cassettes ("chips") for processing and analysis of clinical specimens and other sample types facilitate point-of-care (POC) immunoassays and nucleic acid based amplification tests. These single-use test chips can be self-contained and made amenable to autonomous operation-reducing or eliminating supporting instrumentation-by incorporating laminated, pliable "pouch" and membrane structures for fluid storage, pumping, mixing, and flow control. Materials and methods for integrating flexible pouch compartments and diaphragm valves into hard plastic (e.g., acrylic and polycarbonate) microfluidic "chips" for reagent storage, fluid actuation, and flow control are described. We review several versions of these pouch chips for immunoassay and nucleic acid amplification tests, and describe related fabrication techniques. These protocols thus offer a "toolbox" of methods for storage, pumping, and flow control functions in microfluidic devices.
Multivariate data analysis methods for the interpretation of microbial flow cytometric data.

PubMed

Davey, Hazel M; Davey, Christopher L

2011-01-01

Flow cytometry is an important technique in cell biology and immunology and has been applied by many groups to the analysis of microorganisms. This has been made possible by developments in hardware that is now sensitive enough to be used routinely for analysis of microbes. However, in contrast to advances in the technology that underpin flow cytometry, there has not been concomitant progress in the software tools required to analyse, display and disseminate the data and manual analysis, of individual samples remains a limiting aspect of the technology. We present two new data sets that illustrate common applications of flow cytometry in microbiology and demonstrate the application of manual data analysis, automated visualisation (including the first description of a new piece of software we are developing to facilitate this), genetic programming, principal components analysis and artificial neural nets to these data. The data analysis methods described here are equally applicable to flow cytometric applications with other cell types.
High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry

PubMed Central

Bonar, Micha M.; Tilton, John C.

2017-01-01

Detection of viruses by flow cytometry is complicated by their small size. Here, we characterized the ability of a standard (FACSAria II) and a sub-micron flow cytometer (A50 Micro) to resolve HIV-1 viruses. The A50 was superior at resolving small particles but did not reliably distinguish HIV-1, extracellular vesicles, and laser noise by light scatter properties alone. However, single fluorescent HIV-1 particles could readily be detected by both cytometers. Fluorescent particles were sorted and retained infectivity, permitting further exploration of the functional consequences of HIV-1 heterogeneity. Finally, flow cytometry had a limit of detection of 80 viruses/ml, nearly equal to PCR assays. These studies demonstrate the power of flow cytometry to detect and sort viral particles and provide a critical toolkit to validate methods to label wild-type HIV-1; quantitatively assess integrity and aggregation of viruses and virus-based therapeutics; and efficiently screen drugs inhibiting viral assembly and release. PMID:28235684
High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry.

PubMed

Bonar, Michał M; Tilton, John C

2017-05-01

Detection of viruses by flow cytometry is complicated by their small size. Here, we characterized the ability of a standard (FACSAria II) and a sub-micron flow cytometer (A50 Micro) to resolve HIV-1 viruses. The A50 was superior at resolving small particles but did not reliably distinguish HIV-1, extracellular vesicles, and laser noise by light scatter properties alone. However, single fluorescent HIV-1 particles could readily be detected by both cytometers. Fluorescent particles were sorted and retained infectivity, permitting further exploration of the functional consequences of HIV-1 heterogeneity. Finally, flow cytometry had a limit of detection of 80 viruses/ml, nearly equal to PCR assays. These studies demonstrate the power of flow cytometry to detect and sort viral particles and provide a critical toolkit to validate methods to label wild-type HIV-1; quantitatively assess integrity and aggregation of viruses and virus-based therapeutics; and efficiently screen drugs inhibiting viral assembly and release. Copyright © 2017 Elsevier Inc. All rights reserved.
Use of flow cytometry, fluorescence microscopy, and PCR-based techniques to assess intraspecific and interspecific matings of Armillaria species

Treesearch

Mee-Sook Kim; Ned B. Klopfenstein; Geral I. McDonald; Kathiravetpillai Arumuganathan

2001-01-01

For assessments of intraspecific mating using flow cytometry and fluorescence microscopy, two compatible basidiospore-derived isolates were selected from each of four parental basidiomata of North American Biological Species (NABS) X. The nuclear status in NABS X varied with basidiospore-derived isolates. Nuclei within basidiospore-derived isolates existed as haploids...
Candidiasis and the impact of flow cytometry on antifungal drug discovery.

PubMed

Ku, Tsun Sheng N; Bernardo, Stella; Walraven, Carla J; Lee, Samuel A

2017-11-01

Invasive candidiasis continues to be associated with significant morbidity and mortality as well as substantial health care costs nationally and globally. One of the contributing factors is the development of resistance to antifungal agents that are already in clinical use. Moreover, there are known treatment limitations with all of the available antifungal agents. Since traditional techniques in novel drug discovery are time consuming, high-throughput screening using flow cytometry presents as a potential tool to identify new antifungal agents that would be useful in the management of these patients. Areas covered: In this review, the authors discuss the use of automated high-throughput screening assays based upon flow cytometry to identify potential antifungals from a library comprised of a large number of bioactive compounds. They also review studies that employed the use of this research methodology that has identified compounds with antifungal activity. Expert opinion: High-throughput screening using flow cytometry has substantially decreased the processing time necessary for screening thousands of compounds, and has helped enhance our understanding of fungal pathogenesis. Indeed, the authors see this technology as a powerful tool to help scientists identify new antifungal agents that can be added to the clinician's arsenal in their fight against invasive candidiasis.
Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry.

PubMed

Wigg, Alan J; Phillips, John W; Wheatland, Loretta; Berry, Michael N

2003-06-01

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.
Mouse monoclonal antibodies against human c-Mpl and characterization for flow cytometry applications.

PubMed

Abbott, Christina; Huang, Guo; Ellison, Aaron R; Chen, Ching; Arora, Taruna; Szilvassy, Stephen J; Wei, Ping

2010-04-01

Mouse monoclonal antibodies (MAbs) against human c-Mpl, the cognate receptor for thrombopoietin (TPO), were generated using hybridoma technology and characterized by various assays to demonstrate their specificity and affinity. Two such MAbs, 1.6 and 1.75, were determined to be superior for flow cytometry studies and exhibited double-digit picomolar (pM) affinities to soluble human c-Mpl protein. Both MAbs specifically bound to cells engineered to overexpress human c-Mpl protein, immortalized human hematopoietic cell lines that express endogenous c-Mpl, primary human bone marrow and peripheral blood-derived CD34(+) cells, and purified human platelets. No binding was detected on cell lines that did not express c-Mpl. Receptor competition and siRNA knock-down studies further confirmed the specificity of antibodies 1.6 and 1.75 for human c-Mpl. In contrast to these newly generated MAbs, none of eight commercially available anti-c-Mpl antibodies tested were found to bind specifically to human c-Mpl and were thus shown to be unsuitable for flow cytometry studies. Monoclonal antibodies 1.6 and 1.75 will therefore be useful flow cytometry reagents to detect cell surface c-Mpl expression.
[Flow cytometry in datecting lymph node micrometastasis in colorectal cancer].

PubMed

Sun, Q; Ding, Y; Zhang, J

2001-01-25

To study the methodology and significance of flow cytometry in detecting lymph node micrometastasis of colorectal cancer. One hundred sixty-two cellular suspensions were prepared with lymph nodes which were resected radically on 25 patients with colorectal cancer and in which no cancer cells were found by HE staining. Different concentrations of cultured Lovo colorectal cancer cells were added into the celular suspension prepared from lymph node tissue of persons without colorectal cancer in order to prepare a control model. Dual staining with CK/FTTC and PI was made to the sedimetns from those 2 kinds of suspension. Flow cytometry was used to detect cancer cells. An ideal correlation was obtained between the detection value and the theoretical value of cancer cells in the specimen suspensions and control models (r = 0.097 6) with a sensitivity rate of 10/10(5). Cancer cells were detected from 7 out of the 25 patients and 30 of the 162 cellular suspensions. The detection rate was correlated with the size and infiltrating depth of the cancer. Flow cytometry is a reliable, rapid, and quantitative method for detecting lymph node micrometastasis in colorectal cancer.
Characterization of subpopulations and immune-related parameters of hemocytes in the green-lipped mussel Perna viridis.

PubMed

Wang, Youji; Hu, Menghong; Chiang, M W L; Shin, P K S; Cheung, S G

2012-03-01

The green-lipped mussel Perna viridis is distributed widely in the estuarine and coastal areas of the Indo-Pacific region and extensively cultured as an inexpensive protein source. Morphology and immunological activities of hemocytes of P. viridis were investigated using flow cytometry and light and electron microscopy. Three major types of hemocytes were identified in the hemolymph, including dense-granulocyte, semi-granulocyte (small and large size) and hyalinocyte. Other hemocytes, which occurred in low numbers, included granulocytes with different electron-dense/lucent granules and hemoblast-like cells. Based on flow cytometry, two subpopulations were identified. Granulocytes were larger cells, and the more abundant, containing numerous granules in the cytoplasm, and hyalinocytes were the smaller and less abundant with the fewest granules. Flow cytometry revealed that the granulocytes were more active in cell phagocytosis, contained the higher lysosomal content, and showed higher esterase activity and reactive oxygen species (ROS) generation compared with hyalinocytes. Immune functions assessed by the flow cytometry indicated that the granulocytes were the main hemocytes involved in the cellular defence in P. viridis. Copyright © 2011. Published by Elsevier Ltd.
Verification and characterization of chromosome duplication in haploid maize.

PubMed

de Oliveira Couto, E G; Resende Von Pinho, E V; Von Pinho, R G; Veiga, A D; de Carvalho, M R; de Oliveira Bustamante, F; Nascimento, M S

2015-06-26

Doubled haploid technology has been used by various private companies. However, information regarding chromosome duplication methodologies, particularly those concerning techniques used to identify duplication in cells, is limited. Thus, we analyzed and characterized artificially doubled haploids using microsatellites molecular markers, pollen viability, and flow cytometry techniques. Evaluated material was obtained using two different chromosome duplication protocols in maize seeds considered haploids, resulting from the cross between the haploid inducer line KEMS and 4 hybrids (GNS 3225, GNS 3032, GNS 3264, and DKB 393). Fourteen days after duplication, plant samples were collected and assessed by flow cytometry. Further, the plants were transplanted to a field, and samples were collected for DNA analyses using microsatellite markers. The tassels were collected during anthesis for pollen viability analyses. Haploid, diploid, and mixoploid individuals were detected using flow cytometry, demonstrating that this technique was efficient for identifying doubled haploids. The microsatellites markers were also efficient for confirming the ploidies preselected by flow cytometry and for identifying homozygous individuals. Pollen viability showed a significant difference between the evaluated ploidies when the Alexander and propionic-carmin stains were used. The viability rates between the plodies analyzed show potential for fertilization.
Toward development of a comprehensive external quality assurance program for polyfunctional intracellular cytokine staining assays

PubMed Central

Staats, Janet S.; Enzor, Jennifer H.; Sanchez, Ana M.; Rountree, Wes; Chan, Cliburn; Jaimes, Maria; Chan, Ray Chun-Fai; Gaur, Amitabh; Denny, Thomas N.; Weinhold, Kent J.

2014-01-01

The External Quality Assurance Program Oversight Laboratory (EQAPOL) Flow Cytometry Program assesses the proficiency of NIH/NIAID/DAIDS-supported and potentially other interested research laboratories in performing Intracellular Cytokine Staining (ICS) assays. The goal of the EQAPOL Flow Cytometry External Quality Assurance Program (EQAP) is to provide proficiency testing and remediation for participating sites. The program is not punitive; rather, EQAPOL aims to help sites identify areas for improvement. EQAPOL utilizes a highly standardized ICS assay to minimize variability and readily identify those sites experiencing technical difficulties with their assays. Here, we report the results of External Proficiency 3 (EP3) where participating sites performed a 7-color ICS assay. On average, sites perform well in the Flow Cytometry EQAP (median score is “Good”). The most common technical issues identified by the program involve protocol adherence and data analysis; these areas have been the focus of site remediation. The EQAPOL Flow Cytometry team is now in the process of expanding the program to 8-color ICS assays. Evaluating polyfunctional ICS responses would align the program with assays currently being performed in support of HIV immune monitoring assays. PMID:24968072

Flow cytometry and conventional enumeration of microorganisms in ships' ballast water and marine samples.

PubMed

Joachimsthal, Eva L; Ivanov, Volodymyr; Tay, Joo-Hwa; Tay, Stephen T-L

2003-03-01

Conventional methods for bacteriological testing of water quality take long periods of time to complete. This makes them inappropriate for a shipping industry that is attempting to comply with the International Maritime Organization's anticipated regulations for ballast water discharge. Flow cytometry for the analysis of marine and ship's ballast water is a comparatively fast and accurate method. Compared to a 5% standard error for flow cytometry analysis the standard methods of culturing and epifluorescence analysis have errors of 2-58% and 10-30%, respectively. Also, unlike culturing methods, flow cytometry is capable of detecting both non-viable and viable but non-culturable microorganisms which can still pose health risks. The great variability in both cell concentrations and microbial content for the samples tested is an indication of the difficulties facing microbial monitoring programmes. The concentration of microorganisms in the ballast tank was generally lower than in local seawater. The proportion of aerobic, microaerophilic, and facultative anaerobic microorganisms present appeared to be influenced by conditions in the ballast tank. The gradual creation of anaerobic conditions in a ballast tank could lead to the accumulation of facultative anaerobic microorganisms, which might represent a potential source of pathogenic species.
Review of methods to probe single cell metabolism and bioenergetics

DOE PAGES

Vasdekis, Andreas E.; Stephanopoulos, Gregory

2014-10-31

The sampling and manipulation of cells down to the individual has been of substantial interest since the very beginning of Life Sciences. Herein, our objective is to highlight the most recent developments in single cell manipulation, as well as pioneering ones. First, flow-through methods will be discussed, namely methods in which the single cells flow continuously in an ordered manner during their analysis. This section will be followed by confinement techniques that enable cell isolation and confinement in one, two- or three-dimensions. Flow cytometry and droplet microfluidics are the two most common methods of flow-through analysis. While both are high-throughputmore » techniques, their difference lays in the fact that the droplet encapsulated cells experience a restricted and personal microenvironment, while in flow cytometry cells experience similar nutrient and stimuli initial concentrations. These methods are rather well established; however, they recently enabled immense strides in single cell phenotypic analysis, namely the identification and analysis of metabolically distinct individuals from an isogenic population using both droplet microfluidics and flow cytometry.« less
Flow Cytometry Data Preparation Guidelines for Improved Automated Phenotypic Analysis.

PubMed

Jimenez-Carretero, Daniel; Ligos, José M; Martínez-López, María; Sancho, David; Montoya, María C

2018-05-15

Advances in flow cytometry (FCM) increasingly demand adoption of computational analysis tools to tackle the ever-growing data dimensionality. In this study, we tested different data input modes to evaluate how cytometry acquisition configuration and data compensation procedures affect the performance of unsupervised phenotyping tools. An analysis workflow was set up and tested for the detection of changes in reference bead subsets and in a rare subpopulation of murine lymph node CD103 + dendritic cells acquired by conventional or spectral cytometry. Raw spectral data or pseudospectral data acquired with the full set of available detectors by conventional cytometry consistently outperformed datasets acquired and compensated according to FCM standards. Our results thus challenge the paradigm of one-fluorochrome/one-parameter acquisition in FCM for unsupervised cluster-based analysis. Instead, we propose to configure instrument acquisition to use all available fluorescence detectors and to avoid integration and compensation procedures, thereby using raw spectral or pseudospectral data for improved automated phenotypic analysis. Copyright © 2018 by The American Association of Immunologists, Inc.
Electrogates for stop-and-go control of liquid flow in microfluidics

NASA Astrophysics Data System (ADS)

Arango, Y.; Temiz, Y.; Gökçe, O.; Delamarche, E.

2018-04-01

Diagnostics based on microfluidic devices necessitate specific reagents, flow conditions, and kinetics for optimal performance. Such an optimization is often achieved using assay-specific microfluidic chip designs or systems with external liquid pumps. Here, we present "electrogates" for stop-and-go control of flow of liquids in capillary-driven microfluidic chips by combining liquid pinning and electrowetting. Electrogates are simple to fabricate and efficient: a sample pipetted to a microfluidic chip flows autonomously in 15-μm-deep hydrophilic channels until the liquid meniscus is pinned at the edge of a 1.5-μm-deep trench patterned at the bottom of a rectangular microchannel. The flow can then be resumed by applying a DC voltage between the liquid and the trench via integrated electrodes. Using a trench geometry with a semicircular shape, we show that retention times longer than 30 min are achieved for various aqueous solutions such as biological buffers, artificial urine, and human serum. We studied the activation voltage and activation delay of electrogates using a chip architecture having 6 independent flow paths and experimentally showed that the flow can be resumed in less than 1 s for voltages smaller than 10 V, making this technique compatible with low-power and portable microfluidic systems. Electrogates therefore can make capillary-driven microfluidic chips very versatile by adding flow control in microfluidic channels in a flexible manner.
Minimal Residual Disease Evaluation in Childhood Acute Lymphoblastic Leukemia: An Economic Analysis

PubMed Central

Gajic-Veljanoski, O.; Pham, B.; Pechlivanoglou, P.; Krahn, M.; Higgins, Caroline; Bielecki, Joanna

2016-01-01

Background Minimal residual disease (MRD) testing by higher performance techniques such as flow cytometry and polymerase chain reaction (PCR) can be used to detect the proportion of remaining leukemic cells in bone marrow or peripheral blood during and after the first phases of chemotherapy in children with acute lymphoblastic leukemia (ALL). The results of MRD testing are used to reclassify these patients and guide changes in treatment according to their future risk of relapse. We conducted a systematic review of the economic literature, cost-effectiveness analysis, and budget-impact analysis to ascertain the cost-effectiveness and economic impact of MRD testing by flow cytometry for management of childhood precursor B-cell ALL in Ontario. Methods A systematic literature search (1998–2014) identified studies that examined the incremental cost-effectiveness of MRD testing by either flow cytometry or PCR. We developed a lifetime state-transition (Markov) microsimulation model to quantify the cost-effectiveness of MRD testing followed by risk-directed therapy to no MRD testing and to estimate its marginal effect on health outcomes and on costs. Model input parameters were based on the literature, expert opinion, and data from the Pediatric Oncology Group of Ontario Networked Information System. Using predictions from our Markov model, we estimated the 1-year cost burden of MRD testing versus no testing and forecasted its economic impact over 3 and 5 years. Results In a base-case cost-effectiveness analysis, compared with no testing, MRD testing by flow cytometry at the end of induction and consolidation was associated with an increased discounted survival of 0.0958 quality-adjusted life-years (QALYs) and increased discounted costs of $4,180, yielding an incremental cost-effectiveness ratio (ICER) of $43,613/QALY gained. After accounting for parameter uncertainty, incremental cost-effectiveness of MRD testing was associated with an ICER of $50,249/QALY gained. In the budget-impact analysis, the 1-year cost expenditure for MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL was estimated at $340,760. We forecasted that the province would have to pay approximately $1.3 million over 3 years and $2.4 million over 5 years for MRD testing by flow cytometry in this population. Conclusions Compared with no testing, MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL represents good value for money at commonly used willingness-to-pay thresholds of $50,000/QALY and $100,000/QALY. PMID:27099644
Minimal Residual Disease Evaluation in Childhood Acute Lymphoblastic Leukemia: An Economic Analysis.

PubMed

2016-01-01

Minimal residual disease (MRD) testing by higher performance techniques such as flow cytometry and polymerase chain reaction (PCR) can be used to detect the proportion of remaining leukemic cells in bone marrow or peripheral blood during and after the first phases of chemotherapy in children with acute lymphoblastic leukemia (ALL). The results of MRD testing are used to reclassify these patients and guide changes in treatment according to their future risk of relapse. We conducted a systematic review of the economic literature, cost-effectiveness analysis, and budget-impact analysis to ascertain the cost-effectiveness and economic impact of MRD testing by flow cytometry for management of childhood precursor B-cell ALL in Ontario. A systematic literature search (1998-2014) identified studies that examined the incremental cost-effectiveness of MRD testing by either flow cytometry or PCR. We developed a lifetime state-transition (Markov) microsimulation model to quantify the cost-effectiveness of MRD testing followed by risk-directed therapy to no MRD testing and to estimate its marginal effect on health outcomes and on costs. Model input parameters were based on the literature, expert opinion, and data from the Pediatric Oncology Group of Ontario Networked Information System. Using predictions from our Markov model, we estimated the 1-year cost burden of MRD testing versus no testing and forecasted its economic impact over 3 and 5 years. In a base-case cost-effectiveness analysis, compared with no testing, MRD testing by flow cytometry at the end of induction and consolidation was associated with an increased discounted survival of 0.0958 quality-adjusted life-years (QALYs) and increased discounted costs of $4,180, yielding an incremental cost-effectiveness ratio (ICER) of $43,613/QALY gained. After accounting for parameter uncertainty, incremental cost-effectiveness of MRD testing was associated with an ICER of $50,249/QALY gained. In the budget-impact analysis, the 1-year cost expenditure for MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL was estimated at $340,760. We forecasted that the province would have to pay approximately $1.3 million over 3 years and $2.4 million over 5 years for MRD testing by flow cytometry in this population. Compared with no testing, MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL represents good value for money at commonly used willingness-to-pay thresholds of $50,000/QALY and $100,000/QALY.
Nitrogen removal in wood chip combined substrate baffled subsurface-flow constructed wetlands: impact of matrix arrangement and intermittent aeration.

PubMed

Li, Huai; Chi, Zifang; Yan, Baixing; Cheng, Long; Li, Jianzheng

2017-02-01

In this study, two lab-scale baffled subsurface-flow constructed wetlands (BSFCWs), including gravel-wood chips-slag and gravel-slag-wood chips, were operated at different intermittent aeration to evaluate the effect of artificial aeration and slow-released carbon source on the treatment efficiency of high-strength nitrogen wastewater. Results indicated that gravel-slag-wood chips extended aerobic/anaerobic alternating environment to gravel and slag zones and maintained anaerobic condition in the subsequent wood chip section. The order of gravel-slag-wood chip was more beneficial to pollutant removal. Sufficient carbon source supply resulted from wood-chip-framework substrate simultaneously obtained high removals of COD (97%), NH 4 + -N (95%), and TN (94%) in BSFCWs at 2 h aeration per day. The results suggest that intermittent aeration combined with wood chips could achieve high nitrogen removal in BSFCWs.
An introduction to mass cytometry: fundamentals and applications.

PubMed

Tanner, Scott D; Baranov, Vladimir I; Ornatsky, Olga I; Bandura, Dmitry R; George, Thaddeus C

2013-05-01

Mass cytometry addresses the analytical challenges of polychromatic flow cytometry by using metal atoms as tags rather than fluorophores and atomic mass spectrometry as the detector rather than photon optics. The many available enriched stable isotopes of the transition elements can provide up to 100 distinguishable reporting tags, which can be measured simultaneously because of the essential independence of detection provided by the mass spectrometer. We discuss the adaptation of traditional inductively coupled plasma mass spectrometry to cytometry applications. We focus on the generation of cytometry-compatible data and on approaches to unsupervised multivariate clustering analysis. Finally, we provide a high-level review of some recent benchmark reports that highlight the potential for massively multi-parameter mass cytometry.
Supercontinuum white light lasers for flow cytometry

PubMed Central

Telford, William G.; Subach, Fedor V.; Verkhusha, Vladislav V.

2009-01-01

Excitation of fluorescent probes for flow cytometry has traditionally been limited to a few discrete laser lines, an inherent limitation in our ability to excite the vast array of fluorescent probes available for cellular analysis. In this report, we have used a supercontinuum (SC) white light laser as an excitation source for flow cytometry. By selectively filtering the wavelength of interest, almost any laser wavelength in the visible spectrum can be separated and used for flow cytometric analysis. The white light lasers used in this study were integrated into a commercial flow cytometry platform, and a series of high-transmission bandpass filters used to select wavelength ranges from the blue (~480 nm) to the long red (>700 nm). Cells labeled with a variety of fluorescent probes or expressing fluorescent proteins were then analyzed, in comparison with traditional lasers emitting at wavelengths similar to the filtered SC source. Based on a standard sensitivity metric, the white light laser bandwidths produced similar excitation levels to traditional lasers for a wide variety of fluorescent probes and expressible proteins. Sensitivity assessment using fluorescent bead arrays confirmed that the SC laser and traditional sources resulted in similar levels of detection sensitivity. Supercontinuum white light laser sources therefore have the potential to remove a significant barrier in flow cytometric analysis, namely the limitation of excitation wavelengths. Almost any visible wavelength range can be made available for excitation, allowing access to virtually any fluorescent probe, and permitting “fine-tuning” of excitation wavelength to particular probes. PMID:19072836
Flow cytometry of sputum: assessing inflammation and immune response elements in the bronchial airways

PubMed Central

Lay, John C.; Peden, David B.; Alexis, Neil E.

2012-01-01

Background The evaluation of sputum leukocytes by flow cytometry is an opportunity to assess characteristics of cells residing in the central airways, yet it is hampered by certain inherent properties of sputum including mucus and large amounts of contaminating cells and debris. Objective To develop a gating strategy based on specific antibody panels in combination with light scatter properties for flow cytometric evaluation of sputum cells. Methods Healthy and mild asthmatic volunteers underwent sputum induction. Manually selected mucus “plug” material was treated with dithiothrietol, filtered and total leukocytes acquired. Multicolor flow cytometry was performed using specific gating strategies based on light scatter properties, differential expression of CD45 and cell lineage markers to discriminate leukocytes from squamous epithelial cells and debris. Results The combination of forward scatter and CD45 expression reliably segregated sputum leukocytes from contaminating squamous epithelial cells and debris. Overlap of major leukocyte populations (neutrophils, macrophages/monocytes) required the use of specific antibodies (e.g. CD16, CD64, CD14, HLA-DR) that differentiated granulocytes from monocytes and macrophages. These gating strategies allowed identification of small populations of eosinophils, CD11c+ myeloid dendritic cells, B cells and NK cells. Conclusions Multicolor flow cytometry can be successfully applied to sputum samples to identify and characterize leukocyte populations residing on the surfaces of the central airways. PMID:21639708
Flow cytometry for receptor analysis from ex-vivo brain tissue in adult rat.

PubMed

Benoit, A; Guillamin, M; Aitken, P; Smith, P F; Philoxene, B; Sola, B; Poulain, L; Coquerel, A; Besnard, S

2018-07-01

Flow cytometry allows single-cell analysis of peripheral biological samples and is useful in many fields of research and clinical applications, mainly in hematology, immunology, and oncology. In the neurosciences, the flow cytometry separation method was first applied to stem cell extraction from healthy or cerebral tumour tissue and was more recently tested in order to phenotype brain cells, hippocampal neurogenesis, and to detect prion proteins. However, it remains sparsely applied in quantifying membrane receptors in relation to synaptic plasticity. We aimed to optimize a flow cytometric procedure for receptor quantification in neurons and non-neurons. A neural dissociation process, myelin separation, fixation, and membrane permeability procedures were optimized to maximize cell survival and analysis in hippocampal tissue obtained from adult rodents. We then aimed to quantify membrane muscarinic acetylcholine receptors (mAChRs) in rats with and without bilateral vestibular loss (BVL). mAChR's were quantified for neuronal and non-neuronal cells in the hippocampus and striatum following BVL. At day 30 but not at day 7 following BVL, there was a significant increase (P ≤ 0.05) in the percentage of neurons expressing M 2/4 mAChRs in both the hippocampus and the striatum. Here, we showed that flow cytometry appears to be a reliable method of membrane receptor quantification in ex-vivo brain tissue. Copyright © 2018 Elsevier B.V. All rights reserved.
High throughput, real-time detection of Naegleria lovaniensis in natural river water using LED-illuminated Fountain Flow Cytometry.

PubMed

Johnson, P E; Deromedi, A J; Lebaron, P; Catala, P; Havens, C; Pougnard, C

2007-09-01

To test Fountain Flow Cytometry (FFC) for the rapid and sensitive detection of Naegleria lovaniensis amoebae (an analogue for Naegleria fowleri) in natural river waters. Samples were incubated with one of two fluorescent labels to facilitate detection: ChemChrome V6, a viability indicator, and an R-phycoerytherin (RPE) immunolabel to detect N. lovaniensis specifically. The resulting aqueous sample was passed as a stream in front of a light-emitting diode, which excited the fluorescent labels. The fluorescence was detected with a digital camera as the sample flowed toward the imager. Detections of N. lovaniensis were made in inoculated samples of natural water from eight rivers in France and the United States. FFC enumeration yielded results that are consistent with other counting methods: solid-phase cytometry, flow cytometry, and hemocytometry, down to concentrations of 0.06 amoebae ml(-1), using a flow rate of 15 ml min(-1). This study supports the efficacy of using FFC for the detection of viable protozoa in natural waters and indicates that use of RPE illuminated at 530 nm and detected at 585 nm provides a satisfactory means of attenuating background. Because of the severe global public health issues with drinking water and sanitation, there is an urgent need to develop a technique for the real-time detection of viable pathogens in environmental samples at low concentrations. FFC addresses this need.
Comparison of flow cytometry, fluorescence microscopy and spectrofluorometry for analysis of gene electrotransfer efficiency.

PubMed

Marjanovič, Igor; Kandušer, Maša; Miklavčič, Damijan; Keber, Mateja Manček; Pavlin, Mojca

2014-12-01

In this study, we compared three different methods used for quantification of gene electrotransfer efficiency: fluorescence microscopy, flow cytometry and spectrofluorometry. We used CHO and B16 cells in a suspension and plasmid coding for GFP. The aim of this study was to compare and analyse the results obtained by fluorescence microscopy, flow cytometry and spectrofluorometry and in addition to analyse the applicability of spectrofluorometry for quantifying gene electrotransfer on cells in a suspension. Our results show that all the three methods detected similar critical electric field strength, around 0.55 kV/cm for both cell lines. Moreover, results obtained on CHO cells showed that the total fluorescence intensity and percentage of transfection exhibit similar increase in response to increase electric field strength for all the three methods. For B16 cells, there was a good correlation at low electric field strengths, but at high field strengths, flow cytometer results deviated from results obtained by fluorescence microscope and spectrofluorometer. Our study showed that all the three methods detected similar critical electric field strengths and high correlations of results were obtained except for B16 cells at high electric field strengths. The results also demonstrated that flow cytometry measures higher values of percentage transfection compared to microscopy. Furthermore, we have demonstrated that spectrofluorometry can be used as a simple and consistent method to determine gene electrotransfer efficiency on cells in a suspension.
Imaging Flow Cytometry Analysis to Identify Differences of Survival Motor Neuron Protein Expression in Patients With Spinal Muscular Atrophy.

PubMed

Arakawa, Reiko; Arakawa, Masayuki; Kaneko, Kaori; Otsuki, Noriko; Aoki, Ryoko; Saito, Kayoko

2016-08-01

Spinal muscular atrophy is a neurodegenerative disorder caused by the deficient expression of survival motor neuron protein in motor neurons. A major goal of disease-modifying therapy is to increase survival motor neuron expression. Changes in survival motor neuron protein expression can be monitored via peripheral blood cells in patients; therefore we tested the sensitivity and utility of imaging flow cytometry for this purpose. After the immortalization of peripheral blood lymphocytes from a human healthy control subject and two patients with spinal muscular atrophy type 1 with two and three copies of SMN2 gene, respectively, we used imaging flow cytometry analysis to identify significant differences in survival motor neuron expression. A bright detail intensity analysis was used to investigate differences in the cellular localization of survival motor neuron protein. Survival motor neuron expression was significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. Moreover, survival motor neuron expression correlated with the clinical severity of spinal muscular atrophy according to SMN2 copy number. The cellular accumulation of survival motor neuron protein was also significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. The benefits of imaging flow cytometry for peripheral blood analysis include its capacities for analyzing heterogeneous cell populations; visualizing cell morphology; and evaluating the accumulation, localization, and expression of a target protein. Imaging flow cytometry analysis should be implemented in future studies to optimize its application as a tool for spinal muscular atrophy clinical trials. Copyright © 2016 Elsevier Inc. All rights reserved.
Single cell analysis using surface enhanced Raman scattering (SERS) tags

PubMed Central

Nolan, John P.; Duggan, Erika; Liu, Er; Condello, Danilo; Dave, Isha; Stoner, Samuel A.

2013-01-01

Fluorescence is a mainstay of bioanalytical methods, offering sensitive and quantitative reporting, often in multiplexed or multiparameter assays. Perhaps the best example of the latter is flow cytometry, where instruments equipped with multiple lasers and detectors allow measurement of 15 or more different fluorophores simultaneously, but increases beyond this number are limited by the relatively broad emission spectra. Surface enhanced Raman scattering (SERS) from metal nanoparticles can produce signal intensities that rival fluorescence, but with narrower spectral features that allow a greater degree of multiplexing. We are developing nanoparticle SERS tags as well as Raman flow cytometers for multiparameter single cell analysis of suspension or adherent cells. SERS tags are based on plasmonically active nanoparticles (gold nanorods) whose plasmon resonance can be tuned to give optimal SERS signals at a desired excitation wavelength. Raman resonant compounds are adsorbed on the nanoparticles to confer a unique spectral fingerprint on each SERS tag, which are then encapsulated in a polymer coating for conjugation to antibodies or other targeting molecules. Raman flow cytometry employs a high resolution spectral flow cytometer capable of measuring the complete SERS spectra, as well as conventional flow cytometry measurements, from thousands of individual cells per minute. Automated spectral unmixing algorithms extract the contributions of each SERS tag from each cell to generate high content, multiparameter single cell population data. SERS-based cytometry is a powerful complement to conventional fluorescence-based cytometry. The narrow spectral features of the SERS signal enables more distinct probes to be measured in a smaller region of the optical spectrum with a single laser and detector, allowing for higher levels of multiplexing and multiparameter analysis. PMID:22498143
B-ALL minimal residual disease flow cytometry: an application of a novel method for optimization of a single-tube model.

PubMed

Shaver, Aaron C; Greig, Bruce W; Mosse, Claudio A; Seegmiller, Adam C

2015-05-01

Optimizing a clinical flow cytometry panel can be a subjective process dependent on experience. We develop a quantitative method to make this process more rigorous and apply it to B lymphoblastic leukemia/lymphoma (B-ALL) minimal residual disease (MRD) testing. We retrospectively analyzed our existing three-tube, seven-color B-ALL MRD panel and used our novel method to develop an optimized one-tube, eight-color panel, which was tested prospectively. The optimized one-tube, eight-color panel resulted in greater efficiency of time and resources with no loss in diagnostic power. Constructing a flow cytometry panel using a rigorous, objective, quantitative method permits optimization and avoids problems of interdependence and redundancy in a large, multiantigen panel. Copyright© by the American Society for Clinical Pathology.
A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry.

PubMed

Alves, L P S; Almeida, A T; Cruz, L M; Pedrosa, F O; de Souza, E M; Chubatsu, L S; Müller-Santos, M; Valdameri, G

2017-01-16

The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.
Flow cytometry as a method for the evaluation of raw material, product and process in the dairy industry.

PubMed

Ruszczyńska, A; Szteyn, J; Wiszniewska-Laszczych, A

2007-01-01

Producing dairy products which are safe for consumers requires the constant monitoring of the microbiological quality of raw material, the production process itself and the end product. Traditional methods, still a "gold standard", require a specialized laboratory working on recognized and validated methods. Obtaining results is time- and labor-consuming and do not allow rapid evaluation. Hence, there is a need for a rapid, precise method enabling the real-time monitoring of microbiological quality, and flow cytometry serves this function well. It is based on labeling cells suspended in a solution with fluorescent dyes and pumping them into a measurement zone where they are exposed to a precisely focused laser beam. This paper is aimed at presenting the possibilities of applying flow cytometry in the dairy industry.
Cutting process simulation of flat drill

NASA Astrophysics Data System (ADS)

Tamura, Shoichi; Matsumura, Takashi

2018-05-01

Flat drills at a point angle of 180 deg. have recently been developed for drilling of automobile parts with the inclination of the workpiece surfaces. The paper studies the cutting processes of the flat drills in the analytical simulation. A predictive force model is applied to simulation of the cutting force with the chip flow direction. The chip flow model is piled up with orthogonal cuttings in the plane containing the cutting velocities and the chip flow velocities, in which the chip flow direction is determined to minimize the cutting energy. Then, the cutting force is predicted in the determined in the chip flow model. The typical cutting force of the flat drill is discussed with comparing to that of the standard drill. The typical differences are confirmed in the cutting force change during the tool engagement and disengagement. The cutting force, then, is simulated in drilling for an inclined workpiece with a flat drill. The horizontal components in the cutting forces are simulated with changing the inclination angle of the plate. The horizontal force component in the flat drilling is stable to be controlled in terms of the machining accuracy and the tool breakage.
Line-Focused Optical Excitation of Parallel Acoustic Focused Sample Streams for High Volumetric and Analytical Rate Flow Cytometry.

PubMed

Kalb, Daniel M; Fencl, Frank A; Woods, Travis A; Swanson, August; Maestas, Gian C; Juárez, Jaime J; Edwards, Bruce S; Shreve, Andrew P; Graves, Steven W

2017-09-19

Flow cytometry provides highly sensitive multiparameter analysis of cells and particles but has been largely limited to the use of a single focused sample stream. This limits the analytical rate to ∼50K particles/s and the volumetric rate to ∼250 μL/min. Despite the analytical prowess of flow cytometry, there are applications where these rates are insufficient, such as rare cell analysis in high cellular backgrounds (e.g., circulating tumor cells and fetal cells in maternal blood), detection of cells/particles in large dilute samples (e.g., water quality, urine analysis), or high-throughput screening applications. Here we report a highly parallel acoustic flow cytometer that uses an acoustic standing wave to focus particles into 16 parallel analysis points across a 2.3 mm wide optical flow cell. A line-focused laser and wide-field collection optics are used to excite and collect the fluorescence emission of these parallel streams onto a high-speed camera for analysis. With this instrument format and fluorescent microsphere standards, we obtain analysis rates of 100K/s and flow rates of 10 mL/min, while maintaining optical performance comparable to that of a commercial flow cytometer. The results with our initial prototype instrument demonstrate that the integration of key parallelizable components, including the line-focused laser, particle focusing using multinode acoustic standing waves, and a spatially arrayed detector, can increase analytical and volumetric throughputs by orders of magnitude in a compact, simple, and cost-effective platform. Such instruments will be of great value to applications in need of high-throughput yet sensitive flow cytometry analysis.

Using the developed cross-flow filtration chip for collecting blood plasma under high flow rate condition and applying the immunoglobulin E detection

NASA Astrophysics Data System (ADS)

Yeh, Chia-Hsien; Hung, Chia-Wei; Wu, Chun-Han; Lin, Yu-Cheng

2014-09-01

This paper presents a cross-flow filtration chip for separating blood cells (white blood cells, red blood cells, and platelets) and obtaining blood plasma from human blood. Our strategy is to flow the sample solution in parallel to the membrane, which can generate a parallel shear stress to remove the clogging microparticles on the membrane, so the pure sample solution is obtained in the reservoir. The cross-flow filtration chip includes a cross-flow layer, a Ni-Pd alloy micro-porous membrane, and a reservoir layer. The three layers are packaged in a polymethylmethacrylate (PMMA) frame to create the cross-flow filtration chip. Various dilutions of the blood sample (original, 2 × , 3 × , 5 × , and 10×), pore sizes with different diameters (1 µm, 2 µm, 4 µm, 7 µm, and 10 µm), and different flow rates (1 mL/min, 3 mL/min, 5 mL/min, 7 mL/min, and 10 mL/min) are tested to determine their effects on filtration percentage. The best filtration percentage is 96.2% when the dilution of the blood sample is 10 × , the diameter of pore size of a Ni-Pd alloy micro-porous membrane is 2 µm, and the flow rate is 10 mL/min. Finally, for the clinical tests of the immunoglobulin E (IgE) concentration, the cross-flow filtration chip is used to filter the blood of the allergy patients to obtain the blood plasma. This filtered blood plasma is compared with that obtained using the conventional centrifugation based on the enzyme-linked immunosorbent assay. The results reveal that these two blood separation methods have similar detection trends. The proposed filtration chip has the advantages of low cost, short filtration time, and easy operation and thus can be applied to the separation of microparticles, cells, bacteria, and blood.
Screw-actuated displacement micropumps for thermoplastic microfluidics.

PubMed

Han, J Y; Rahmanian, O D; Kendall, E L; Fleming, N; DeVoe, D L

2016-10-05

The fabrication of on-chip displacement pumps integrated into thermoplastic chips is explored as a simple and low cost method for achieving precise and programmable flow control for disposable microfluidic systems. The displacement pumps consist of stainless steel screws inserted into threaded ports machined into a thermoplastic substrate which also serve as on-chip reagent storage reservoirs. Three different methods for pump sealing are investigated to enable high pressure flows without leakage, and software-defined control of multiple pumps is demonstrated in a self-contained platform using a compact and self-contained microcontroller for operation. Using this system, flow rates ranging from 0.5-40 μl min -1 are demonstrated. The pumps are combined with on-chip burst valves to fully seal multiple reagents into fabricated chips while providing on-demand fluid distribution in a downstream microfluidic network, and demonstrated for the generation of size-tunable water-in-oil emulsions.
Targeting Breast Cancer Vasculature

DTIC Science & Technology

2006-03-01

E., and Hanahan, D. Stage-specific vascular markers revealed by phage display in a mouse model of pancreatic islet tumorigenesis. Cancer Cell 4:393...expressing full-length myc-tagged metadherin, myc-vimen- tin, or myc-pCMV were analyzed by flow cytometry . Anti-myc antibodies were applied to the cells...In 4T1 tumor cell extracts, anti-metadherin(378-440) detectedthen stained with anti-myc antibodies. Using flow cytometry , proteins with apparent
Addressing the malaria drug resistance challenge using flow cytometry to discover new antimalarials.

PubMed

Grimberg, Brian T; Jaworska, Maria M; Hough, Lindsay B; Zimmerman, Peter A; Phillips, James G

2009-09-15

A new flow cytometry method that uses an optimized DNA and RNA staining strategy to monitor the growth and development of the Plasmodium falciparum strain W2mef has been used in a pilot study and has identified Bay 43-9006 1, SU 11274 2, and TMC 125 5 as compounds that exhibit potent (<1 microM) overall and ring stage in vitro antimalarial activity.
Stochastic Individual-Based Modeling of Bacterial Growth and Division Using Flow Cytometry.

PubMed

García, Míriam R; Vázquez, José A; Teixeira, Isabel G; Alonso, Antonio A

2017-01-01

A realistic description of the variability in bacterial growth and division is critical to produce reliable predictions of safety risks along the food chain. Individual-based modeling of bacteria provides the theoretical framework to deal with this variability, but it requires information about the individual behavior of bacteria inside populations. In this work, we overcome this problem by estimating the individual behavior of bacteria from population statistics obtained with flow cytometry. For this objective, a stochastic individual-based modeling framework is defined based on standard assumptions during division and exponential growth. The unknown single-cell parameters required for running the individual-based modeling simulations, such as cell size growth rate, are estimated from the flow cytometry data. Instead of using directly the individual-based model, we make use of a modified Fokker-Plank equation. This only equation simulates the population statistics in function of the unknown single-cell parameters. We test the validity of the approach by modeling the growth and division of Pediococcus acidilactici within the exponential phase. Estimations reveal the statistics of cell growth and division using only data from flow cytometry at a given time. From the relationship between the mother and daughter volumes, we also predict that P. acidilactici divide into two successive parallel planes.
In vivo, label-free, and noninvasive detection of melanoma metastasis by photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Liu, Rongrong; Wang, Cheng; Hu, Cheng; Wang, Xueding; Wei, Xunbin

2014-02-01

Melanoma, a malignant tumor of melanocytes, is the most serious type of skin cancer in the world. It accounts for about 80% of deaths of all skin cancer. For cancer detection, circulating tumor cells (CTCs) serve as a marker for metastasis development, cancer recurrence, and therapeutic efficacy. Melanoma tumor cells have high content of melanin, which has high light absorption and can serve as endogenous biomarker for CTC detection without labeling. Here, we have developed an in vivo photoacoustic flow cytometry (PAFC) to monitor the metastatic process of melanoma cancer by counting CTCs of melanoma tumor bearing mice in vivo. To test in vivo PAFC's capability of detecting melanoma cancer, we have constructed a melanoma tumor model by subcutaneous inoculation of highly metastatic murine melanoma cancer cells, B16F10. In order to effectively distinguish the targeting PA signals from background noise, we have used the algorithm of Wavelet denoising method to reduce the background noise. The in vivo flow cytometry (IVFC) has shown a great potential for detecting circulating tumor cells quantitatively in the blood stream. Compared with fluorescence-based in vivo flow cytometry (IVFC), PAFC technique can be used for in vivo, label-free, and noninvasive detection of circulating tumor cells (CTCs).
Discovering cell types in flow cytometry data with random matrix theory

NASA Astrophysics Data System (ADS)

Shen, Yang; Nussenblatt, Robert; Losert, Wolfgang

Flow cytometry is a widely used experimental technique in immunology research. During the experiments, peripheral blood mononuclear cells (PBMC) from a single patient, labeled with multiple fluorescent stains that bind to different proteins, are illuminated by a laser. The intensity of each stain on a single cell is recorded and reflects the amount of protein expressed by that cell. The data analysis focuses on identifying specific cell types related to a disease. Different cell types can be identified by the type and amount of protein they express. To date, this has most often been done manually by labelling a protein as expressed or not while ignoring the amount of expression. Using a cross correlation matrix of stain intensities, which contains both information on the proteins expressed and their amount, has been largely ignored by researchers as it suffers from measurement noise. Here we present an algorithm to identify cell types in flow cytometry data which uses random matrix theory (RMT) to reduce noise in a cross correlation matrix. We demonstrate our method using a published flow cytometry data set. Compared with previous analysis techniques, we were able to rediscover relevant cell types in an automatic way. Department of Physics, University of Maryland, College Park, MD 20742.
Advantages of flow cytometry immunophenotyping for the diagnosis of central nervous system non-Hodgkin's lymphoma in AIDS patients.

PubMed

Subirá, D; Górgolas, M; Castañón, S; Serrano, C; Román, A; Rivas, F; Tomás, J F

2005-01-01

Neurological disorders are common in HIV-infected patients. Central nervous system (CNS) lymphoma should always be considered because it is an important cause of morbidity and mortality. To investigate the clinical utility of flow cytometry immunophenotyping (FCI) in diagnosing or discarding leptomeningeal involvement in HIV-infected patients and to compare its sensitivity with that of conventional cytological methods. Fifty-six cerebrospinal fluid (CSF) samples from 29 HIV-infected patients were independently evaluated by flow cytometry and cytology. The description of an aberrant immunophenotype was the criterion used to define the malignant nature of any CSF cell population. FCI and cytology gave concordant results for 48 of the 56 CSF samples studied: 37 were negative for malignancy and 11 had evidence of CNS lymphoma. Discordant results were obtained for eight CSF samples, and the accuracy of the FCI findings could be demonstrated for four CSF samples described as positive for malignancy according to the FCI criteria. A high level of agreement was found between the results obtained using the two methods, but FCI gave at least 25% higher sensitivity than conventional cytomorphological methods for the detection of malignant cells. This advantage suggests that, in case of negative flow cytometry results, disorders other than non-Hodgkin's lymphoma should be strongly considered.
Drug testing and flow cytometry analysis on a large number of uniform sized tumor spheroids using a microfluidic device

NASA Astrophysics Data System (ADS)

Patra, Bishnubrata; Peng, Chien-Chung; Liao, Wei-Hao; Lee, Chau-Hwang; Tung, Yi-Chung

2016-02-01

Three-dimensional (3D) tumor spheroid possesses great potential as an in vitro model to improve predictive capacity for pre-clinical drug testing. In this paper, we combine advantages of flow cytometry and microfluidics to perform drug testing and analysis on a large number (5000) of uniform sized tumor spheroids. The spheroids are formed, cultured, and treated with drugs inside a microfluidic device. The spheroids can then be harvested from the device without tedious operation. Due to the ample cell numbers, the spheroids can be dissociated into single cells for flow cytometry analysis. Flow cytometry provides statistical information in single cell resolution that makes it feasible to better investigate drug functions on the cells in more in vivo-like 3D formation. In the experiments, human hepatocellular carcinoma cells (HepG2) are exploited to form tumor spheroids within the microfluidic device, and three anti-cancer drugs: Cisplatin, Resveratrol, and Tirapazamine (TPZ), and their combinations are tested on the tumor spheroids with two different sizes. The experimental results suggest the cell culture format (2D monolayer vs. 3D spheroid) and spheroid size play critical roles in drug responses, and also demonstrate the advantages of bridging the two techniques in pharmaceutical drug screening applications.
Immunological tools: engaging students in the use and analysis of flow cytometry and enzyme-linked immunosorbent assay (ELISA).

PubMed

Ott, Laura E; Carson, Susan

2014-01-01

Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and evaluation of a novel half-semester course that focused on introducing undergraduate and graduate students to advance conceptual and technical skills associated with flow cytometry and ELISA, with emphasis on applications, experimental design, and data analysis. This course was offered in the North Carolina State University Biotechnology Program over three semesters and consisted of weekly lectures and laboratories. Students performed and/or analyzed flow cytometry and ELISA in three separate laboratory exercises: (1) identification of transgenic zebrafish hematopoietic cells, (2) analysis of transfection efficiency, and (3) analysis of cytokine production upon lipopolysaccharide stimulation. Student learning outcomes were achieved as demonstrated by multiple means of assessment, including three laboratory reports, a data analysis laboratory practicum, and a cumulative final exam. Further, anonymous student self-assessment revealed increased student confidence in the knowledge and skill sets defined in the learning outcomes. Copyright © 2014 The International Union of Biochemistry and Molecular Biology.
Integrated potentiometric detector for use in chip-based flow cells

PubMed

Tantra; Manz

2000-07-01

A new kind of potentiometric chip sensor for ion-selective electrodes (ISE) based on a solvent polymeric membrane is described. The chip sensor is designed to trap the organic cocktail inside the chip and to permit sample solution to flow past the membrane. The design allows the sensor to overcome technical problems of ruggedness and would therefore be ideal for industrial processes. The sensor performance for a Ba2+-ISE membrane based on a Vogtle ionophore showed electrochemical behavior similar to that observed in conventional electrodes and microelectrode arrangements.
Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): A Method for Bacterial Small RNA Detection

DTIC Science & Technology

2012-01-10

flow cytometry, locked nucleic acid, sRNA, Vibrio , Date Published: 1/10/2012 This is an open-access article distributed under the terms of the Creative...solubilization process to maintain a 10 mL volume. Aliquot the 60% dextran sulfate solution and store at -20 °C until use. 1. Harvest 1x108 cells of...bioluminescent Vibrio campbellii or your bacteria of interest and transfer them into a 1.5 mL microcentrifuge tube. This quantity of cells provides
Measuring sickle cell morphology in flow using spectrally encoded flow cytometry (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kviatkovsky, Inna; Zeidan, Adel; Yeheskely-Hayon, Daniella; Dann, Eldad J.; Yelin, Dvir

2017-02-01

During a sickle cell crisis in sickle cell anemia patients, deoxygenated red blood cells may change their mechanical properties and block small blood vessels, causing pain, local tissue damage and even organ failure. Measuring these cellular structural and morphological changes is important for understanding the factors contributing to vessel blockage and developing an effective treatment. In this work, we use spectrally encoded flow cytometry for confocal, high-resolution imaging of flowing blood cells from sickle cell anemia patients. A wide variety of cell morphologies were observed by analyzing the interference patterns resulting from reflections from the front and back faces of the cells' membrane. Using numerical simulation for calculating the two-dimensional reflection pattern from the cells, we propose an analytical expression for the three-dimensional shape of a characteristic sickle cell and compare it to a previous from the literature. In vitro spectrally encoded flow cytometry offers new means for analyzing the morphology of sickle cells in stress-free environment, and could provide an effective tool for studying the unique physiological properties of these cells.
Multiplexed and Microparticle-based Analyses: Quantitative Tools for the Large-Scale Analysis of Biological Systems

PubMed Central

Nolan, John P.; Mandy, Francis

2008-01-01

While the term flow cytometry refers to the measurement of cells, the approach of making sensitive multiparameter optical measurements in a flowing sample stream is a very general analytical approach. The past few years have seen an explosion in the application of flow cytometry technology for molecular analysis and measurements using micro-particles as solid supports. While microsphere-based molecular analyses using flow cytometry date back three decades, the need for highly parallel quantitative molecular measurements that has arisen from various genomic and proteomic advances has driven the development in particle encoding technology to enable highly multiplexed assays. Multiplexed particle-based immunoassays are now common place, and new assays to study genes, protein function, and molecular assembly. Numerous efforts are underway to extend the multiplexing capabilities of microparticle-based assays through new approaches to particle encoding and analyte reporting. The impact of these developments will be seen in the basic research and clinical laboratories, as well as in drug development. PMID:16604537
Evaluation of the platelet counting by Abbott CELL-DYN SAPPHIRE haematology analyser compared with flow cytometry.

PubMed

Grimaldi, E; Del Vecchio, L; Scopacasa, F; Lo Pardo, C; Capone, F; Pariante, S; Scalia, G; De Caterina, M

2009-04-01

The Abbot Cell-Dyn Sapphire is a new generation haematology analyser. The system uses optical/fluorescence flow cytometry in combination with electronic impedance to produce a full blood count. Optical and impedance are the default methods for platelet counting while automated CD61-immunoplatelet analysis can be run as selectable test. The aim of this study was to determine the platelet count performance of the three counting methods available on the instrument and to compare the results with those provided by Becton Dickinson FACSCalibur flow cytometer used as reference method. A lipid interference experiment was also performed. Linearity, carryover and precision were good, and satisfactory agreement with reference method was found for the impedance, optical and CD61-immunoplatelet analysis, although this latter provided the closest results in comparison with flow cytometry. In the lipid interference experiment, a moderate inaccuracy of optical and immunoplatelet counts was observed starting from a very high lipid value.
Microflow1, a sheathless fiber-optic flow cytometry biomedical platform: demonstration onboard the international space station.

PubMed

Dubeau-Laramée, Geneviève; Rivière, Christophe; Jean, Isabelle; Mermut, Ozzy; Cohen, Luchino Y

2014-04-01

A fiber-optic based flow cytometry platform was designed to build a portable and robust instrument for space applications. At the core of the Microflow1 is a unique fiber-optic flow cell fitted to a fluidic system and fiber coupled to the source and detection channels. A Microflow1 engineering unit was first tested and benchmarked against a commercial flow cytometer as a reference in a standard laboratory environment. Testing in parabolic flight campaigns was performed to establish Microflow1's performance in weightlessness, before operating the new platform on the International Space Station. Microflow1 had comparable performances to commercial systems, and operated remarkably and robustly in weightlessness (microgravity). Microflow1 supported immunophenotyping as well as microbead-based multiplexed cytokine assays in the space environment and independently of gravity levels. Results presented here provide evidence that this fiber-optic cytometer technology is inherently compatible with the space environment with negligible compromise to analytical performance. © 2013 International Society for Advancement of Cytometry.
Managing Multi-center Flow Cytometry Data for Immune Monitoring

PubMed Central

White, Scott; Laske, Karoline; Welters, Marij JP; Bidmon, Nicole; van der Burg, Sjoerd H; Britten, Cedrik M; Enzor, Jennifer; Staats, Janet; Weinhold, Kent J; Gouttefangeas, Cécile; Chan, Cliburn

2014-01-01

With the recent results of promising cancer vaccines and immunotherapy1–5, immune monitoring has become increasingly relevant for measuring treatment-induced effects on T cells, and an essential tool for shedding light on the mechanisms responsible for a successful treatment. Flow cytometry is the canonical multi-parameter assay for the fine characterization of single cells in solution, and is ubiquitously used in pre-clinical tumor immunology and in cancer immunotherapy trials. Current state-of-the-art polychromatic flow cytometry involves multi-step, multi-reagent assays followed by sample acquisition on sophisticated instruments capable of capturing up to 20 parameters per cell at a rate of tens of thousands of cells per second. Given the complexity of flow cytometry assays, reproducibility is a major concern, especially for multi-center studies. A promising approach for improving reproducibility is the use of automated analysis borrowing from statistics, machine learning and information visualization21–23, as these methods directly address the subjectivity, operator-dependence, labor-intensive and low fidelity of manual analysis. However, it is quite time-consuming to investigate and test new automated analysis techniques on large data sets without some centralized information management system. For large-scale automated analysis to be practical, the presence of consistent and high-quality data linked to the raw FCS files is indispensable. In particular, the use of machine-readable standard vocabularies to characterize channel metadata is essential when constructing analytic pipelines to avoid errors in processing, analysis and interpretation of results. For automation, this high-quality metadata needs to be programmatically accessible, implying the need for a consistent Application Programming Interface (API). In this manuscript, we propose that upfront time spent normalizing flow cytometry data to conform to carefully designed data models enables automated analysis, potentially saving time in the long run. The ReFlow informatics framework was developed to address these data management challenges. PMID:26085786
An inter-laboratory comparison of PNH clone detection by high-sensitivity flow cytometry in a Russian cohort.

PubMed

Sipol, Alexandra A; Babenko, Elena V; Borisov, Vyacheslav I; Naumova, Elena V; Boyakova, Elena V; Yakunin, Dimitry I; Glazanova, Tatyana V; Chubukina, Zhanna V; Pronkina, Natalya V; Popov, Alexander M; Saveliev, Leonid I; Lugovskaya, Svetlana A; Lisukov, Igor A; Kulagin, Alexander D; Illingworth, Andrea J

2015-01-01

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by partial or absolute deficiency of glycophosphatidyl-inositol (GPI) anchor-linked surface proteins on blood cells. A lack of precise diagnostic standards for flow cytometry has hampered useful comparisons of data between laboratories. We report data from the first study evaluating the reproducibility of high-sensitivity flow cytometry for PNH in Russia. PNH clone sizes were determined at diagnosis in PNH patients at a central laboratory and compared with follow-up measurements in six laboratories across the country. Analyses in each laboratory were performed according to recommendations from the International Clinical Cytometry Society (ICCS) and the more recent 'practical guidelines'. Follow-up measurements were compared with each other and with the values determined at diagnosis. PNH clone size measurements were determined in seven diagnosed PNH patients (five females, two males: mean age 37 years); five had a history of aplastic anemia and three (one with and two without aplastic anemia) had severe hemolytic PNH and elevated plasma lactate dehydrogenase. PNH clone sizes at diagnosis were low in patients with less severe clinical symptoms (0.41-9.7% of granulocytes) and high in patients with severe symptoms (58-99%). There were only minimal differences in the follow-up clone size measurement for each patient between the six laboratories, particularly in those with high values at diagnosis. The ICCS-recommended high-sensitivity flow cytometry protocol was effective for detecting major and minor PNH clones in Russian PNH patients, and showed high reproducibility between laboratories.
Development of an unbiased, semi-automated approach for classifying plasma cell immunophenotype following multicolor flow cytometry of bone marrow aspirates.

PubMed

Post, Steven R; Post, Ginell R; Nikolic, Dejan; Owens, Rebecca; Insuasti-Beltran, Giovanni

2018-03-24

Despite increased usage of multiparameter flow cytometry (MFC) to assess diagnosis, prognosis, and therapeutic efficacy (minimal residual disease, MRD) in plasma cell neoplasms (PCNs), standardization of methodology and data analysis is suboptimal. We investigated the utility of using the mean and median fluorescence intensities (FI) obtained from MFC to objectively describe parameters that distinguish plasma cell (PC) phenotypes. In this retrospective study, flow cytometry results from bone marrow aspirate specimens from 570 patients referred to the Myeloma Institute at UAMS were evaluated. Mean and median FI data were obtained from 8-color MFC of non-neoplastic, malignant, and mixed PC populations using antibodies to CD38, CD138, CD19, CD20, CD27, CD45, CD56, and CD81. Of 570 cases, 252 cases showed only non-neoplastic PCs, 168 showed only malignant PCs, and 150 showed mixed PC populations. Statistical analysis of median FI data for each CD marker showed no difference in expression intensity on non-neoplastic and malignant PCs, between pure and mixed PC populations. ROC analysis of the median FI of CD expression in non-neoplastic and malignant PCs was used to develop an algorithm to convert quantitative FI values to qualitative assessments including "negative," "positive," "dim," and "heterogeneous" expression. FI data derived from 8-color MFC can be used to define marker expression on PCs. Translation of FI data from Infinicyt software to an Excel worksheet streamlines workflow and eliminates transcriptional errors when generating flow reports. © 2018 International Clinical Cytometry Society. © 2018 International Clinical Cytometry Society.
Cytobank: providing an analytics platform for community cytometry data analysis and collaboration.

PubMed

Chen, Tiffany J; Kotecha, Nikesh

2014-01-01

Cytometry is used extensively in clinical and laboratory settings to diagnose and track cell subsets in blood and tissue. High-throughput, single-cell approaches leveraging cytometry are developed and applied in the computational and systems biology communities by researchers, who seek to improve the diagnosis of human diseases, map the structures of cell signaling networks, and identify new cell types. Data analysis and management present a bottleneck in the flow of knowledge from bench to clinic. Multi-parameter flow and mass cytometry enable identification of signaling profiles of patient cell samples. Currently, this process is manual, requiring hours of work to summarize multi-dimensional data and translate these data for input into other analysis programs. In addition, the increase in the number and size of collaborative cytometry studies as well as the computational complexity of analytical tools require the ability to assemble sufficient and appropriately configured computing capacity on demand. There is a critical need for platforms that can be used by both clinical and basic researchers who routinely rely on cytometry. Recent advances provide a unique opportunity to facilitate collaboration and analysis and management of cytometry data. Specifically, advances in cloud computing and virtualization are enabling efficient use of large computing resources for analysis and backup. An example is Cytobank, a platform that allows researchers to annotate, analyze, and share results along with the underlying single-cell data.

Modeling of cytometry data in logarithmic space: When is a bimodal distribution not bimodal?

PubMed

Erez, Amir; Vogel, Robert; Mugler, Andrew; Belmonte, Andrew; Altan-Bonnet, Grégoire

2018-02-16

Recent efforts in systems immunology lead researchers to build quantitative models of cell activation and differentiation. One goal is to account for the distributions of proteins from single-cell measurements by flow cytometry or mass cytometry as readout of biological regulation. In that context, large cell-to-cell variability is often observed in biological quantities. We show here that these readouts, viewed in logarithmic scale may result in two easily-distinguishable modes, while the underlying distribution (in linear scale) is unimodal. We introduce a simple mathematical test to highlight this mismatch. We then dissect the flow of influence of cell-to-cell variability proposing a graphical model which motivates higher-dimensional analysis of the data. Finally we show how acquiring additional biological information can be used to reduce uncertainty introduced by cell-to-cell variability, helping to clarify whether the data is uni- or bimodal. This communication has cautionary implications for manual and automatic gating strategies, as well as clustering and modeling of single-cell measurements. © 2018 International Society for Advancement of Cytometry. © 2018 International Society for Advancement of Cytometry.
Chip-based in situ hybridization for identification of bacteria from the human microbiome.

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

Light, Yooli Kim; Meagher, Robert J.; Singh, Anup K.

2010-11-01

The emerging field of metagenomics seeks to assess the genetic diversity of complex mixed populations of bacteria, such as those found at different sites within the human body. A single person's mouth typically harbors up to 100 bacterial species, while surveys of many people have found more than 700 different species, of which {approx}50% have never been cultivated. In typical metagenomics studies, the cells themselves are destroyed in the process of gathering sequence information, and thus the connection between genotype and phenotype is lost. A great deal of sequence information may be generated, but it is impossible to assign anymore » given sequence to a specific cell. We seek non-destructive, culture-independent means of gathering sequence information from selected individual cells from mixed populations. As a first step, we have developed a microfluidic device for concentrating and specifically labeling bacteria from a mixed population. Bacteria are electrophoretically concentrated against a photopolymerized membrane element, and then incubated with a specific fluorescent label, which can include antibodies as well as specific or non-specific nucleic acid stains. Unbound stain is washed away, and the labeled bacteria are released from the membrane. The stained cells can then be observed via epifluorescence microscopy, or counted via flow cytometry. We have tested our device with three representative bacteria from the human microbiome: E. coli (gut, Gram-negative), Lactobacillus acidophilus (mouth, Gram-positive), and Streptococcus mutans (mouth, Gram-positive), with results comparable to off-chip labeling techniques.« less
Electrokinetic Analysis of Cell Translocation in Low-Cost Microfluidic Cytometry for Tumor Cell Detection and Enumeration.

PubMed

Guo, Jinhong; Pui, Tze Sian; Ban, Yong-Ling; Rahman, Abdur Rub Abdur; Kang, Yuejun

2013-12-01

Conventional Coulter counters have been introduced as an important tool in biological cell assays since several decades ago. Recently, the emerging portable Coulter counter has demonstrated its merits in point of care diagnostics, such as on chip detection and enumeration of circulating tumor cells (CTC). The working principle is based on the cell translocation time and amplitude of electrical current change that the cell induces. In this paper, we provide an analysis of a Coulter counter that evaluates the hydrodynamic and electrokinetic properties of polystyrene microparticles in a microfluidic channel. The hydrodynamic force and electrokinetic force are concurrently analyzed to determine the translocation time and the electrical current pulses induced by the particles. Finally, we characterize the chip performance for CTC detection. The experimental results validate the numerical analysis of the microfluidic chip. The presented model can provide critical insight and guidance for developing micro-Coulter counter for point of care prognosis.
Screening of carcinoma metastasis by flow cytometry: A study of 238 cases.

PubMed

Acosta, Maria; Pereira, José; Arroz, Maria

2016-05-01

Malignant epithelial cells may be detected in different specimens, by immunophenotyping using flow cytometry (FCM). CD326 (epithelial-specific antigen, clone Ber-Ep4) was used to identify epithelial cells, CD45 to discriminate between leucocytes (positive for this antigen) and non-hematological cells (negative for this antigen), and CD33 to identify monocytes/macrophages. This combination is particularly useful in effusions to characterize large cells and distinguish between monocyte/macrophages (CD45+ CD33+ CD326-), mesothelial cells (CD45 ± (dim) CD33 - CD326-) and epithelial cells (CD45 - CD33 - CD326 +). We evaluated the efficiency of flow cytometry to detect malignant epithelial cells in 238 fresh samples, including effusions, lymph node biopsies, fine needle aspirates, bone marrow aspirates, cerebrospinal fluid, among others. These are specimens expected to lack epithelial cells. FCM results were then compared to the results of smear and cell block morphology, as well as immunocytochemistry on paraffin wax embedded cell blocks, when available. Final diagnosis was the gold standard and a very good sensitivity (96.7%) and specificity (99.3%) were obtained. We concluded that the detection of CD326 positive cells using FCM is strongly indicative of the presence of carcinoma cells. © 2015 International Clinical Cytometry Society. © 2015 International Clinical Cytometry Society.
Flow karyotyping and sorting of human chromosomes

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

Gray, J.W.; Lucas, J.; Peters, D.

1986-07-16

Flow cytometry and sorting are becoming increasingly useful as tools for chromosome classfication and for the detection of numerical and structural chromosome aberrations. Chromosomes of a single type can be purified with these tools to facilitate gene mapping or production of chromosome specific recombinant DNA libraries. For analysis of chromosomes with flow cytometry, the chromosomes are extracted from mitotic cells, stained with one or more fluorescent dyes and classified one-by-one according to their dye content(s). Thus, the flow approach is fundamentally different than conventional karyotyping where chromosomes are classified within the context of a metaphase spread. Flow sorting allows purificationmore » of chromosomes that can be distinguished flow cytometrically. The authors describe the basic principles of flow cytometric chromosome classification i.e. flow karyotyping, and chromosome sorting and describe several applications. 30 refs., 8 figs.« less
Isomorphic red blood cells using automated urine flow cytometry is a reliable method in diagnosis of bladder cancer.

PubMed

Muto, Satoru; Sugiura, Syo-Ichiro; Nakajima, Akiko; Horiuchi, Akira; Inoue, Masahiro; Saito, Keisuke; Isotani, Shuji; Yamaguchi, Raizo; Ide, Hisamitsu; Horie, Shigeo

2014-10-01

We aimed to identify patients with a chief complaint of hematuria who could safely avoid unnecessary radiation and instrumentation in the diagnosis of bladder cancer (BC), using automated urine flow cytometry to detect isomorphic red blood cells (RBCs) in urine. We acquired urine samples from 134 patients over the age of 35 years with a chief complaint of hematuria and a positive urine occult blood test or microhematuria. The data were analyzed using the UF-1000i (®) (Sysmex Co., Ltd., Kobe, Japan) automated urine flow cytometer to determine RBC morphology, which was classified as isomorphic or dysmorphic. The patients were divided into two groups (BC versus non-BC) for statistical analysis. Multivariate logistic regression analysis was used to determine the predictive value of flow cytometry versus urine cytology, the bladder tumor antigen test, occult blood in urine test, and microhematuria test. BC was confirmed in 26 of 134 patients (19.4 %). The area under the curve for RBC count using the automated urine flow cytometer was 0.94, representing the highest reference value obtained in this study. Isomorphic RBCs were detected in all patients in the BC group. On multivariate logistic regression analysis, only isomorphic RBC morphology was significantly predictive for BC (p < 0.001). Analytical parameters such as sensitivity, specificity, positive predictive value, and negative predictive value of isomorphic RBCs in urine were 100.0, 91.7, 74.3, and 100.0 %, respectively. Detection of urinary isomorphic RBCs using automated urine flow cytometry is a reliable method in the diagnosis of BC with hematuria.
Development of Antibodies Against Novel Cell Surface Proteins In Hormone Refractory Prostate Cancer. Addendum

DTIC Science & Technology

2011-07-01

marker of hormone refractory metastatic prostate cancer. Clin Cancer Res, 2005. 15: 2237- 43 . 3. Tomita K, van Bokhoven A, van Leenders GJ, Ruijter...Santa Cruz Biotechnology), vimentin, Ki-67 (DakoCytomation) and caspase-3 (Cell Signaling Technology). Flow cytometry was performed with N...transduced cells were labeled with N-cadherin–specific antibody and sorted by flow cytometry , gating for a GFP-positive, N-cadherinlow population. The
Label-free in vivo flow cytometry in zebrafish using two-photon autofluorescence imaging.

PubMed

Zeng, Yan; Xu, Jin; Li, Dong; Li, Li; Wen, Zilong; Qu, Jianan Y

2012-07-01

We demonstrate a label-free in vivo flow cytometry in zebrafish blood vessels based on two-photon excited autofluorescence imaging. The major discovery in this work is the strong autofluorescence emission from the plasma in zebrafish blood. The plasma autofluorescence provides excellent contrast for visualizing blood vessels and counting blood cells. In addition, the cellular nicotinamide adenine dinucleotide autofluorescence enables in vivo imaging and counting of white blood cells (neutrophils).
Flow Cytometry Techniques in Radiation Biology

DTIC Science & Technology

1988-06-01

Henidtopoietic stem cells SUMMARY Hematopoietic stem cells ( HSC ) are present in the marrow at a concentration of approximately 2-3 HSC per 1000 nucleated marrow...cells. In the past, only clonogenic assays requiring 8-13 days and ten irradiated recipient rodents were available for assaying HSC . Because of the...importance of HSC in the postirradiation syndrome, we have developed a new rapid method based on flow cytometry not only to assay but also to purify and
Flow cytometry on the stromal-vascular fraction of white adipose tissue.

PubMed

Brake, Danett K; Smith, C Wayne

2008-01-01

Adipose tissue contains cell types other than adipocytes that may contribute to complications linked to obesity. For example, macrophages have been shown to infiltrate adipose tissue in response to a high-fat diet. Isolation of the stromal-vascular fraction of adipose tissue allows one to use flow cytometry to analyze cell surface markers on leukocytes. Here, we present a technical approach to identify subsets of leukocytes that differentially express cell surface markers.
Screening of Compounds Toxicity against Human Monocytic cell line-THP-1 by Flow Cytometry

PubMed Central

Pick, Neora; Cameron, Scott; Arad, Dorit

2004-01-01

The worldwide rapid increase in bacterial resistance to numerous antibiotics requires on-going development of new drugs to enter the market. As the development of new antibiotics is lengthy and costly, early monitoring of compound's toxicity is essential in the development of novel agents. Our interest is in a rapid, simple, high throughput screening method to assess cytotoxicity induced by potential agents. Some intracellular pathogens, such as Mycobacterium tuberculosis primary site of infection is human alveolar macrophages. Thus, evaluation of candidate drugs for macrophage toxicity is crucial. Protocols for high throughput drug toxicity screening of macrophages using flow cytometry are lacking in the literature. For this application we modified a preexisting technique, propidium iodide (PI) exclusion staining and utilized it for rapid toxicity tests. Samples were prepared in 96 well plates and analyzed by flow cytometry, which allowed for rapid, inexpensive and precise assessment of compound's toxicity associated with cell death. PMID:15472722
Tracking by flow cytometry antigen-specific follicular helper T cells in wild-type animals after protein vaccination.

PubMed

Chakarov, Svetoslav; Fazilleau, Nicolas

2015-01-01

Flow cytometry is a valuable technology used in immunology to characterize and enumerate the different cell subpopulations specific for a nonself-antigen in the context of an ongoing immune response. Among them, follicular helper T cells are the cognate regulators of B cells in secondary lymphoid tissues. Thus, tracking them is of high interest especially in the context of protein vaccination. For this purpose, transgenic antigen-receptor mouse models have been largely used. It is now clear that transgenic models are not always the best means to study the dynamics of the immune response since they can modify the response. In this chapter, we describe how to track endogenous antigen-specific follicular helper T cells by flow cytometry after protein vaccination in nonmodified wild-type animals, which ultimately provides a comprehensive way to enumerate, characterize, and isolate these particular cells in vivo.
A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

PubMed Central

Alves, L.P.S.; Almeida, A.T.; Cruz, L.M.; Pedrosa, F.O.; de Souza, E.M.; Chubatsu, L.S.; Müller-Santos, M.; Valdameri, G.

2017-01-01

The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots. PMID:28099582
Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

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

Venkateswaran, K.S.; Nasarabadi, S.; Langlois, R.G.

2000-05-05

Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCRmore » amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.« less
Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening

PubMed Central

Galanzha, Ekaterina I; Tuchin, Valery V; Zharov, Vladimir P

2007-01-01

Using animal mesentery with intravital optical microscopy is a well-established experimental model for studying blood and lymph microcirculation in vivo. Recent advances in cell biology and optical techniques provide the basis for extending this model for new applications, which should generate significantly improved experimental data. This review summarizes the achievements in this specific area, including in vivo label-free blood and lymph photothermal flow cytometry, super-sensitive fluorescence image cytometry, light scattering and speckle flow cytometry, microvessel dynamic microscopy, infrared (IR) angiography, and high-speed imaging of individual cells in fast flow. The capabilities of these techniques, using the rat mesentery model, were demonstrated in various studies; e.g., real-time quantitative detection of circulating and migrating individual blood and cancer cells, studies on vascular dynamics with a focus on lymphatics under normal conditions and under different interventions (e.g. lasers, drugs, nicotine), assessment of lymphatic disturbances from experimental lymphedema, monitoring cell traffic between blood and lymph systems, and high-speed imaging of cell transient deformability in flow. In particular, the obtained results demonstrated that individual cell transportation in living organisms depends on cell type (e.g., normal blood or leukemic cells), the cell’s functional state (e.g., live, apoptotic, or necrotic), and the functional status of the organism. Possible future applications, including in vivo early diagnosis and prevention of disease, monitoring immune response and apoptosis, chemo- and radio-sensitivity tests, and drug screening, are also discussed. PMID:17226898
An improved method for differentiating cell-bound from internalized particles by imaging flow cytometry.

PubMed

Smirnov, Asya; Solga, Michael D; Lannigan, Joanne; Criss, Alison K

2015-08-01

Recognition, binding, internalization, and elimination of pathogens and cell debris are important functions of professional as well as non-professional phagocytes. However, high-throughput methods for quantifying cell-associated particles and discriminating bound from internalized particles have been lacking. Here we describe a protocol for using imaging flow cytometry to quantify the attached and phagocytosed particles that are associated with a population of cells. Cells were exposed to fluorescent particles, fixed, and exposed to an antibody of a different fluorophore that recognizes the particles. The antibody is added without cell permeabilization, such that the antibody only binds extracellular particles. Cells with and without associated particles were identified by imaging flow cytometry. For each cell with associated particles, a spot count algorithm was employed to quantify the number of extracellular (double fluorescent) and intracellular (single fluorescent) particles per cell, from which the percent particle internalization was determined. The spot count algorithm was empirically validated by examining the fluorescence and phase contrast images acquired by the flow cytometer. We used this protocol to measure binding and internalization of the bacterium Neisseria gonorrhoeae by primary human neutrophils, using different bacterial variants and under different cellular conditions. The results acquired using imaging flow cytometry agreed with findings that were previously obtained using conventional immunofluorescence microscopy. This protocol provides a rapid, powerful method for measuring the association and internalization of any particle by any cell type. Copyright © 2015 Elsevier B.V. All rights reserved.
The Transient Receptor Potential Vanilloid 2 Cation Channel Is Abundant in Macrophages Accumulating at the Peri-Infarct Zone and May Enhance Their Migration Capacity towards Injured Cardiomyocytes following Myocardial Infarction

PubMed Central

Goryainov, Pavel; Landa, Natalie; Barshack, Iris; Avivi, Camila; Semo, Jonathan; Keren, Gad

2014-01-01

Purpose A novel family of transient receptor potential (TRP) channels, that may hold a role in calcium homeostasis, has recently been described. By employing a GeneChip array analysis we have demonstrated a clear and specific upregulation of the TRP vanilloid 2 (TRPV2) mRNA in the left ventricles (LV) 3–5 days post-acute myocardial infarction (MI) compared to sham-operated controls, both in rats and in mice. We sought to characterize the cardiac cellular subpopulations in which TRPV2 is overexpressed upon acute MI. Methods Lewis rats underwent an acute MI by ligation of the left anterior descending artery or chest opening only (sham). The animals were terminated at various time points and an immunohistochemical (IHC) and immunofloerescent (IFC) staining of the LV sections as well as a flow cytometry analysis of LV-derived cells were carried out, using anti-TRPV2 and anti-monocyte/macrophage antibodies. Rat alveolar macrophage cells, NR8383, transiently transfected with TRPV2 siRNA were allowed to migrate towards hypoxic conditioned media of the rat cardiac myoblast line H9C2 using a trans-well migration assay. The macrophage cells migrating to the bottom side of the inserts were counted. Results The IHC and IFC staining as well as the flow cytometry data demonstrated a substantial expression of TRPV2 in infiltrating macrophages in the peri-infarct region 3–5 days post-acute MI. The in vitro migration assay data demonstrated that following inhibition of the TRPV2 channel, the number of migrating macrophages towards conditioned medium of hypoxic cardiomyocytes was significantly reduced. Conclusions TRPV2 is highly expressed on the peri-infarct infiltrating macrophages and may play an important role in post-MI phagocytosis. Better characterization of this channel may pave the way for identifying a new target for modulating the dramatic post-MI immune reactions. PMID:25136832
An easy to assemble microfluidic perfusion device with a magnetic clamp

PubMed Central

Tkachenko, Eugene; Gutierrez, Edgar; Ginsberg, Mark H.; Groisman, Alex

2009-01-01

We have built and characterized a magnetic clamp for reversible sealing of PDMS microfluidic chips against cover glasses with cell cultures and a microfluidic chip for experiments on shear stress response of endothelial cells. The magnetic clamp exerts a reproducible uniform pressure on the microfluidic chip, achieving fast and reliable sealing for liquid pressures up to 40 kPa inside the chip with <10% deformations of microchannels and minimal variations of the substrate shear stress in perfusion flow. The microfluidic chip has 8 test regions with the substrate shear stress varying by a factor of 2 between each region, thus covering a 128-fold range from low venous to arterial. The perfusion is driven by differential pressure, which makes it possible to create pulsatile flows mimicking pulsing in the vasculature. The setup is tested by 15 – 40 hours perfusions over endothelial monolayers with shear stress in the range of 0.07 - 9 dyn/cm2. Excellent cell viability at all shear stresses and alignment of cells along the flow at high shear stresses are repeatedly observed. A scratch wound healing assay under a shear flow is demonstrated and cell migration velocities are measured. Transfection of cells with a fluorescent protein is performed, and migrating fluorescent cells are imaged at a high resolution under shear flow in real time. The magnetic clamp can be closed with minimal mechanical perturbation to cells on the substrate and used with a variety of microfluidic chips for experiments with adherent and non-adherent cells. PMID:19350090
Numerical and experimental evaluation of microfluidic sorting devices.

PubMed

Taylor, Jay K; Ren, Carolyn L; Stubley, G D

2008-01-01

The development of lab-on-a-chip devices calls for the isolation or separation of specific bioparticles or cells. The design of a miniaturized cell-sorting device for handheld operation must follow the strict parameters associated with lab-on-a-chip technology. The limitations include applied voltage, high efficiency of cell-separation, reliability, size, flow control, and cost, among others. Currently used designs have achieved successful levels of cell isolation; however, further improvements in the microfluidic chip design are important to incorporate into larger systems. This study evaluates specific design modifications that contribute to the reduction of required applied potential aiming for developing portable devices, improved operation reliability by minimizing induced pressure disturbance when electrokinetic pumping is employed, and improved flow control by incorporating directing streams achieving dynamic sorting and counting. The chip designs fabricated in glass and polymeric materials include asymmetric channel widths for sample focusing, nonuniform channel depth for minimizing induced pressure disturbance, directing streams to assist particle flow control, and online filters for reducing channel blockage. Fluorescence-based visualization experimental results of electrokinetic focusing, flow field phenomena, and dynamic sorting demonstrate the advantages of the chip design. Numerical simulations in COMSOL are validated by the experimental data and used to investigate the effects of channel geometry and fluid properties on the flow field.
Simultaneous Moisture Content and Mass Flow Measurements in Wood Chip Flows Using Coupled Dielectric and Impact Sensors.

PubMed

Pan, Pengmin; McDonald, Timothy; Fulton, John; Via, Brian; Hung, John

2016-12-23

An 8-electrode capacitance tomography (ECT) sensor was built and used to measure moisture content (MC) and mass flow of pine chip flows. The device was capable of directly measuring total water quantity in a sample but was sensitive to both dry matter and moisture, and therefore required a second measurement of mass flow to calculate MC. Two means of calculating the mass flow were used: the first being an impact sensor to measure total mass flow, and the second a volumetric approach based on measuring total area occupied by wood in images generated using the capacitance sensor's tomographic mode. Tests were made on 109 groups of wood chips ranging in moisture content from 14% to 120% (dry basis) and wet weight of 280 to 1100 g. Sixty groups were randomly selected as a calibration set, and the remaining were used for validation of the sensor's performance. For the combined capacitance/force transducer system, root mean square errors of prediction (RMSEP) for wet mass flow and moisture content were 13.42% and 16.61%, respectively. RMSEP using the combined volumetric mass flow/capacitance sensor for dry mass flow and moisture content were 22.89% and 24.16%, respectively. Either of the approaches was concluded to be feasible for prediction of moisture content in pine chip flows, but combining the impact and capacitance sensors was easier to implement. In situations where flows could not be impeded, however, the tomographic approach would likely be more useful.

Analytical study of a microfludic DNA amplification chip using water cooling effect.

PubMed

Chen, Jyh Jian; Shen, Chia Ming; Ko, Yu Wei

2013-04-01

A novel continuous-flow polymerase chain reaction (PCR) chip has been analyzed in our work. Two temperature zones are controlled by two external controllers and the other temperature zone at the chip center is controlled by the flow rate of the fluid inside a channel under the glass chip. By employing a water cooling channel at the chip center, the sequence of denaturation, annealing, and extension can be created due to the forced convection effect. The required annealing temperature of PCR less than 313 K can also be demonstrated in this chip. The Poly(methyl methacrylate) (PMMA) cooling channel with the thin aluminum cover is utilized to enhance the temperature uniformity. The size of this chip is 76 mm × 26 mm × 3 mm. This device represents the first demonstration of water cooling thermocycling within continuous-flow PCR microfluidics. The commercial software CFD-ACE+(TM) is utilized to determine the distances between the heating assemblies within the chip. We investigate the influences of various chip materials, operational parameters of the cooling channel and geometric parameters of the chip on the temperature uniformity on the chip surface. Concerning the temperature uniformity of the working zones and the lowest temperature at the annealing zone, the air gap spacing of 1 mm and the cooling channel thicknesses of 1 mm of the PMMA channel with an aluminum cover are recommended in our design. The hydrophobic surface of the PDMS channel was modified by filling it with 20 % Tween 20 solution and then adding bovine serum albumin (BSA) solution to the PCR mixture. DNA fragments with different lengths (372 bp and 478 bp) are successfully amplified with the device.
Tracking protein aggregation and mislocalization in cells with flow cytometry.

PubMed

Ramdzan, Yasmin M; Polling, Saskia; Chia, Cheryl P Z; Ng, Ivan H W; Ormsby, Angelique R; Croft, Nathan P; Purcell, Anthony W; Bogoyevitch, Marie A; Ng, Dominic C H; Gleeson, Paul A; Hatters, Danny M

2012-03-18

We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.
A Stem Cell-Seeded Nanofibrous Scaffold for Auditory Nerve Replacement

DTIC Science & Technology

2013-10-01

the brightest GFP+ cells by flow cytometry and compared these with GFP- cells (Figure 1A-C). The transfected cells showed robust GFP expression even...al., 2011), but no normative data were provided on SGN loss by cochlear turn and, in contrast to our results, those authors reported no impact on...A) Flow cytometry analysis to identify GFP+ and GFP- cells. The large cluster of cells on the left represent the GFP- cells and exhibited similar
Endothelial Progenitor Cells (EPC) Count by Multicolor Flow Cytometry in Healthy Individuals and Diabetes Mellitus (DM) Patients.

PubMed

Falay, Mesude; Aktas, Server

2016-11-01

The present study aimed to determine circulating Endothelial Progenitor Cell (EPC) counts by multicolor flow cytometry in healthy individuals and diabetic subjects by means of forming an analysis procedure using a combination of monoclonal antibodies (moAbs), which would correctly detect the circulating EPC count. The circulating EPC count was detected in 40 healthy individuals (20 Female, 20 Male; age range: 26 - 50 years) and 30 Diabetes Mellitus (DM) patients (15 Female, 15 Male; age range: 42 - 55) by multicolor flow cytometry (FCM) in a single-tube panel consisting of 5 CD45/CD31/CD34/CD309/ SYTO® and 16 monoclonal antibodies. Circulating EPC count was 11.33 (7.89 - 15.25) cells/µL in the healthy control group and 4.80 (0.70 - 10.85) cells/µL in the DM group. EPC counts were significantly lower in DM cases that developed coronary artery disease (53.3%) as compared to those that did not (p < 0.001). In the present study, we describe a method that identifies circulating EPC counts by multicolor flow cytometry in a single tube and determines the circulating EPC count in healthy individuals. This is the first study conducted on EPC count in Turkish population. We think that the EPC count found in the present study will be a guide for future studies.
A comparison of methods to assess the antimicrobial activity of nanoparticle combinations on bacterial cells.

PubMed

Bankier, Claire; Cheong, Yuen; Mahalingam, Suntharavathanan; Edirisinghe, Mohan; Ren, Guogang; Cloutman-Green, Elaine; Ciric, Lena

2018-01-01

Bacterial cell quantification after exposure to antimicrobial compounds varies widely throughout industry and healthcare. Numerous methods are employed to quantify these antimicrobial effects. With increasing demand for new preventative methods for disease control, we aimed to compare and assess common analytical methods used to determine antimicrobial effects of novel nanoparticle combinations on two different pathogens. Plate counts of total viable cells, flow cytometry (LIVE/DEAD BacLight viability assay) and qPCR (viability qPCR) were used to assess the antimicrobial activity of engineered nanoparticle combinations (NPCs) on Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria at different concentrations (0.05, 0.10 and 0.25 w/v%). Results were analysed using linear models to assess the effectiveness of different treatments. Strong antimicrobial effects of the three NPCs (AMNP0-2) on both pathogens could be quantified using the plate count method and flow cytometry. The plate count method showed a high log reduction (>8-log) for bacteria exposed to high NPC concentrations. We found similar antimicrobial results using the flow cytometry live/dead assay. Viability qPCR analysis of antimicrobial activity could not be quantified due to interference of NPCs with qPCR amplification. Flow cytometry was determined to be the best method to measure antimicrobial activity of the novel NPCs due to high-throughput, rapid and quantifiable results.
Monoclonal Antibody L1Mab-13 Detected Human PD-L1 in Lung Cancers.

PubMed

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Chang, Yao-Wen; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2018-04-01

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L 1 Mab-13 (IgG 1 , kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses. L 1 Mab-13 specifically reacted hPD-L1 of hPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (human fibroblast) in flow cytometry and Western blot. Furthermore, L 1 Mab-13 reacted with lung cancer cell lines (EBC-1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L 1 Mab-13, is very useful for detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.
Controlled and tunable polymer particles' production using a single microfluidic device

NASA Astrophysics Data System (ADS)

Amoyav, Benzion; Benny, Ofra

2018-04-01

Microfluidics technology offers a new platform to control liquids under flow in small volumes. The advantage of using small-scale reactions for droplet generation along with the capacity to control the preparation parameters, making microfluidic chips an attractive technology for optimizing encapsulation formulations. However, one of the drawback in this methodology is the ability to obtain a wide range of droplet sizes, from sub-micron to microns using a single chip design. In fact, typically, droplet chips are used for micron-dimension particles, while nanoparticles' synthesis requires complex chips design (i.e., microreactors and staggered herringbone micromixer). Here, we introduce the development of a highly tunable and controlled encapsulation technique, using two polymer compositions, for generating particles ranging from microns to nano-size using the same simple single microfluidic chip design. Poly(lactic-co-glycolic acid) (PLGA 50:50) or PLGA/polyethylene glycol polymeric particles were prepared with focused-flow chip, yielding monodisperse particle batches. We show that by varying flow rate, solvent, surfactant and polymer composition, we were able to optimize particles' size and decrease polydispersity index, using simple chip designs with no further related adjustments or costs. Utilizing this platform, which offers tight tuning of particle properties, could offer an important tool for formulation development and can potentially pave the way towards a better precision nanomedicine.
Size, shape and flow characterization of ground wood chip and ground wood pellet particles

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

Rezaei, Hamid; Lim, C. Jim; Lau, Anthony

Size, shape and density of biomass particles influence their transportation, fluidization, rates of drying and thermal decomposition. Pelleting wood particles increases the particle density and reduces the variability of physical properties among biomass particles. In this study, pine chips prepared for pulping and commercially produced pine pellets were ground in a hammer mill using grinder screens of 3.2, 6.3, 12.7 and 25.4mmperforations. Pellets consumed about 7 times lower specific grinding energy than chips to produce the same size of particles. Grinding pellets produced the smaller particles with narrower size distribution than grinding chips. Derived shape factors in digital image analysismore » showed that chip particles were rectangular and had the aspect ratios about one third of pellet particles. Pellet particles were more circular shape. The mechanical sieving underestimated the actual particle size and did not represent the size of particles correctly. Instead, digital imaging is preferred. Angle of repose and compressibility tests represented the flow properties of ground particles. Pellet particles made a less compacted bulk, had lower cohesion and did flow easier in a pile of particles. In conclusion, particle shape affected the flow properties more than particle size« less
Size, shape and flow characterization of ground wood chip and ground wood pellet particles

DOE PAGES

Rezaei, Hamid; Lim, C. Jim; Lau, Anthony; ...

2016-07-11

Size, shape and density of biomass particles influence their transportation, fluidization, rates of drying and thermal decomposition. Pelleting wood particles increases the particle density and reduces the variability of physical properties among biomass particles. In this study, pine chips prepared for pulping and commercially produced pine pellets were ground in a hammer mill using grinder screens of 3.2, 6.3, 12.7 and 25.4mmperforations. Pellets consumed about 7 times lower specific grinding energy than chips to produce the same size of particles. Grinding pellets produced the smaller particles with narrower size distribution than grinding chips. Derived shape factors in digital image analysismore » showed that chip particles were rectangular and had the aspect ratios about one third of pellet particles. Pellet particles were more circular shape. The mechanical sieving underestimated the actual particle size and did not represent the size of particles correctly. Instead, digital imaging is preferred. Angle of repose and compressibility tests represented the flow properties of ground particles. Pellet particles made a less compacted bulk, had lower cohesion and did flow easier in a pile of particles. In conclusion, particle shape affected the flow properties more than particle size« less
Imaging flow cytometry assays for quantifying pigment grade titanium dioxide particle internalization and interactions with immune cells in whole blood

PubMed Central

Vis, Bradley; Pele, Laetitia C.; Faria, Nuno; Powell, Jonathan J.

2017-01-01

Abstract Pigment grade titanium dioxide is composed of sub‐micron sized particles, including a nanofraction, and is widely utilized in food, cosmetic, pharmaceutical, and biomedical industries. Oral exposure to pigment grade titanium dioxide results in at least some material entering the circulation in humans, although subsequent interactions with blood immune cells are unknown. Pigment grade titanium dioxide is employed for its strong light scattering properties, and this work exploited that attribute to determine whether single cell–particle associations could be determined in immune cells of human whole blood at “real life” concentrations. In vitro assays, initially using isolated peripheral blood mononuclear cells, identified titanium dioxide associated with the surface of, and within, immune cells by darkfield reflectance in imaging flow cytometry. This was confirmed at the population level by side scatter measurements using conventional flow cytometry. Next, it was demonstrated that imaging flow cytometry could quantify titanium dioxide particle‐bearing cells, within the immune cell populations of fresh whole blood, down to titanium dioxide levels of 10 parts per billion, which is in the range anticipated for human blood following titanium dioxide ingestion. Moreover, surface association and internal localization of titanium dioxide particles could be discriminated in the assays. Overall, results showed that in addition to the anticipated activity of blood monocytes internalizing titanium dioxide particles, neutrophil internalization and cell membrane adhesion also occurred, the latter for both phagocytic and nonphagocytic cell types. What happens in vivo and whether this contributes to activation of one or more of these different cells types in blood merits further attention. © 2017 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC. PMID:28941170
In vitro flow cytometry-based screening platform for cellulase engineering

PubMed Central

Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

2016-01-01

Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 107 events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298
Study of low speed flow cytometry for diffraction imaging with different chamber and nozzle designs.

PubMed

Sa, Yu; Feng, Yuanming; Jacobs, Kenneth M; Yang, Jun; Pan, Ran; Gkigkitzis, Ioannis; Lu, Jun Q; Hu, Xin-Hua

2013-11-01

Achieving effective hydrodynamic focusing and flow stability at low speed presents a challenging design task in flow cytometry for studying phenomena such as cell adhesion and diffraction imaging of cells with low-cost cameras. We have developed different designs of flow chamber and sheath nozzle to accomplish the above goal. A 3D computational model of the chambers has been established to simulate the fluid dynamics in different chamber designs and measurements have been performed to determine the velocity and size distributions of the core fluid from the nozzle. Comparison of the simulation data with experimental results shows good agreement. With the computational model significant insights were gained for optimization of the chamber design and improvement of the cell positioning accuracy for study of slow moving cells. The benefit of low flow speed has been demonstrated also by reduced blurring in the diffraction images of single cells. Based on these results, we concluded that the new designs of chamber and sheath nozzle produce stable hydrodynamic focusing of the core fluid at low speed and allow detailed study of cellular morphology under various rheological conditions using the diffraction imaging method. © 2013 International Society for Advancement of Cytometry.
Human bone perivascular niche-on-a-chip for studying metastatic colonization.

PubMed

Marturano-Kruik, Alessandro; Nava, Michele Maria; Yeager, Keith; Chramiec, Alan; Hao, Luke; Robinson, Samuel; Guo, Edward; Raimondi, Manuela Teresa; Vunjak-Novakovic, Gordana

2018-02-06

Eight out of 10 breast cancer patients die within 5 years after the primary tumor has spread to the bones. Tumor cells disseminated from the breast roam the vasculature, colonizing perivascular niches around blood capillaries. Slow flows support the niche maintenance by driving the oxygen, nutrients, and signaling factors from the blood into the interstitial tissue, while extracellular matrix, endothelial cells, and mesenchymal stem cells regulate metastatic homing. Here, we show the feasibility of developing a perfused bone perivascular niche-on-a-chip to investigate the progression and drug resistance of breast cancer cells colonizing the bone. The model is a functional human triculture with stable vascular networks within a 3D native bone matrix cultured on a microfluidic chip. Providing the niche-on-a-chip with controlled flow velocities, shear stresses, and oxygen gradients, we established a long-lasting, self-assembled vascular network without supplementation of angiogenic factors. We further show that human bone marrow-derived mesenchymal stem cells, which have undergone phenotypical transition toward perivascular cell lineages, support the formation of capillary-like structures lining the vascular lumen. Finally, breast cancer cells exposed to interstitial flow within the bone perivascular niche-on-a-chip persist in a slow-proliferative state associated with increased drug resistance. We propose that the bone perivascular niche-on-a-chip with interstitial flow promotes the formation of stable vasculature and mediates cancer cell colonization.
Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry.

PubMed

Tang, Vera A; Renner, Tyler M; Fritzsche, Anna K; Burger, Dylan; Langlois, Marc-André

2017-12-19

Retroviruses and small EVs overlap in size, buoyant densities, refractive indices and share many cell-derived surface markers making them virtually indistinguishable by standard biochemical methods. This poses a significant challenge when purifying retroviruses for downstream analyses or for phenotypic characterization studies of markers on individual virions given that EVs are a major contaminant of retroviral preparations. Nanoscale flow cytometry (NFC), also called flow virometry, is an adaptation of flow cytometry technology for the analysis of individual nanoparticles such as extracellular vesicles (EVs) and retroviruses. In this study we systematically optimized NFC parameters for the detection of retroviral particles in the range of 115-130 nm, including viral production, sample labeling, laser power and voltage settings. By using the retroviral envelope glycoprotein as a selection marker, and evaluating a number of fluorescent dyes and labeling methods, we demonstrate that it is possible to confidently distinguish retroviruses from small EVs by NFC. Our findings make it now possible to individually phenotype genetically modified retroviral particles that express a fluorescent envelope glycoprotein without removing EV contaminants from the sample.
Cellular vacuolation and mitochondrial-associated factors induced by Clostridium perfringens epsilon toxin detected using acoustic flow cytometry.

PubMed

Ferrarezi, Marina C; Curci, Vera C L M; Cardoso, Tereza C

2013-12-01

Epsilon toxin (ETX) produced by Clostridium perfringens types B and D is a potent toxin that is responsible for fatal enterotoxaemia. In vitro, ETX, which is considered as a pore-forming toxin, forms a heptamer in Madin-Darby canine kidney (MDCK) cell membranes, which is considered to be a pre-pore stage. After binding of the ETX, vacuoles inside cell cytoplasm are produced. ETX causes decreased levels of essential coenzymes required for host cell energy. Here, we optimized and applied acoustic flow cytometry analysis in order to gain further insight into ETX-pathogenesis. Using acoustic flow cytometer analysis, which considered highly sensitive, ETX-exposed MDCK cells revealed mitochondrial membrane decreases followed by 25.48% and 45.45% of the exposed cells expressing the Bax and BCL-2 proteins at a pre-pore stage, respectively. These results together with high cytotoxicity and visualization of cell vacuoles, demonstrates that acoustic flow cytometry analysis potentially represents an effective tool to study ETX pathogenesis. Copyright © 2013. Published by Elsevier Ltd.
A general method for bead-enhanced quantitation by flow cytometry

PubMed Central

Montes, Martin; Jaensson, Elin A.; Orozco, Aaron F.; Lewis, Dorothy E.; Corry, David B.

2009-01-01

Flow cytometry provides accurate relative cellular quantitation (percent abundance) of cells from diverse samples, but technical limitations of most flow cytometers preclude accurate absolute quantitation. Several quantitation standards are now commercially available which, when added to samples, permit absolute quantitation of CD4+ T cells. However, these reagents are limited by their cost, technical complexity, requirement for additional software and/or limited applicability. Moreover, few studies have validated the use of such reagents in complex biological samples, especially for quantitation of non-T cells. Here we show that addition to samples of known quantities of polystyrene fluorescence standardization beads permits accurate quantitation of CD4+ T cells from complex cell samples. This procedure, here termed single bead-enhanced cytofluorimetry (SBEC), was equally capable of enumerating eosinophils as well as subcellular fragments of apoptotic cells, moieties with very different optical and fluorescent characteristics. Relative to other proprietary products, SBEC is simple, inexpensive and requires no special software, suggesting that the method is suitable for the routine quantitation of most cells and other particles by flow cytometry. PMID:17067632
Novel Quantitative Autophagy Analysis by Organelle Flow Cytometry after Cell Sonication

PubMed Central

Degtyarev, Michael; Reichelt, Mike; Lin, Kui

2014-01-01

Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication), takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis. PMID:24489953
Open-access and multi-directional electroosmotic flow chip for positioning heterotypic cells.

PubMed

Terao, Kyohei; Kitazawa, Yuko; Yokokawa, Ryuji; Okonogi, Atsuhito; Kotera, Hidetoshi

2011-04-21

We propose a novel method of cell positioning using electroosmotic flow (EOF) to analyze cell-cell interactions. The EOF chip has an open-to-air configuration, is equipped with four electrodes to induce multi-directional EOF, and allows access of tools for liquid handling and of physical probes for cell measurements. Evaluation of the flow within this chip indicated that it controlled hydrodynamic transport of cells, in terms of both speed and direction. We also evaluated cell viability after EOF application and determined appropriate conditions for cell positioning. Two cells were successively positioned in pocket-like microstructures, one in each micropocket, by controlling the EOF direction. As an experimental demonstration, we observed contact interactions between two individual cells through gap junction channels. The EOF chip should provide ways to elucidate various cell-cell interactions between heterotypic cells.
A new "Logicle" display method avoids deceptive effects of logarithmic scaling for low signals and compensated data.

PubMed

Parks, David R; Roederer, Mario; Moore, Wayne A

2006-06-01

In immunofluorescence measurements and most other flow cytometry applications, fluorescence signals of interest can range down to essentially zero. After fluorescence compensation, some cell populations will have low means and include events with negative data values. Logarithmic presentation has been very useful in providing informative displays of wide-ranging flow cytometry data, but it fails to adequately display cell populations with low means and high variances and, in particular, offers no way to include negative data values. This has led to a great deal of difficulty in interpreting and understanding flow cytometry data, has often resulted in incorrect delineation of cell populations, and has led many people to question the correctness of compensation computations that were, in fact, correct. We identified a set of criteria for creating data visualization methods that accommodate the scaling difficulties presented by flow cytometry data. On the basis of these, we developed a new data visualization method that provides important advantages over linear or logarithmic scaling for display of flow cytometry data, a scaling we refer to as "Logicle" scaling. Logicle functions represent a particular generalization of the hyperbolic sine function with one more adjustable parameter than linear or logarithmic functions. Finally, we developed methods for objectively and automatically selecting an appropriate value for this parameter. The Logicle display method provides more complete, appropriate, and readily interpretable representations of data that includes populations with low-to-zero means, including distributions resulting from fluorescence compensation procedures, than can be produced using either logarithmic or linear displays. The method includes a specific algorithm for evaluating actual data distributions and deriving parameters of the Logicle scaling function appropriate for optimal display of that data. It is critical to note that Logicle visualization does not change the data values or the descriptive statistics computed from them. Copyright 2006 International Society for Analytical Cytology.
Accuracy of Automated Flow Cytometry-Based Leukocyte Counts To Rule Out Urinary Tract Infection in Febrile Children: a Prospective Cross-Sectional Study

PubMed Central

Duong, Hong Phuoc; Wissing, Karl Martin; Tram, Nathalie; Mascart, Georges; Lepage, Philippe

2016-01-01

Automated flow cytometry of urine remains an incompletely validated method to rule out urinary tract infection (UTI) in children. This cross-sectional analytical study was performed to compare the predictive values of flow cytometry and a dipstick test as initial diagnostic tests for UTI in febrile children and prospectively included 1,106 children (1,247 episodes). Urine culture was used as the gold standard test for diagnosing UTI. The performance of screening tests to diagnose UTI were established using receiver operating characteristic (ROC) analysis. Among these 1,247 febrile episodes, 221 UTIs were diagnosed (17.7% [95% confidence interval {CI}, 15.6 to 19.8%]). The area under the ROC curve for flow cytometry white blood cell (WBC) counts (0.99 [95% CI, 0.98 to 0.99]) was significantly superior to that for red blood cell (0.74 [95% CI, 0.70 to 0.78]) and bacterial counts (0.89 [95% CI, 0.87 to 0.92]) (P < 0.001). Urinary WBC counts also had a significantly higher area under the ROC curve than that of the leukocyte esterase (LE) dipstick (0.92 [95% CI, 0.90 to 0.94]), nitrite dipstick (0.83 [95% CI, 0.80 to 0.87]), or the combination of positive LE and/or nitrite dipstick (0.91 [95% CI, 0.89 to 0.93]) test (P < 0.001). The presence of ≥35 WBC/μl of urine was the best cutoff point, yielding both a high sensitivity (99.5% [95% CI, 99 to 100%]) and an acceptable specificity (80.6% [95% CI, 78 to 83%]). Using this cutoff point would have reduced the number of samples sent to the laboratory for culture by 67%. In conclusion, the determination of urinary WBC counts by flow cytometry provides optimal performance as an initial diagnostic test for UTI in febrile children. PMID:27682127

Distinction between asymptomatic monoclonal B-cell lymphocytosis with cyclin D1 overexpression and mantle cell lymphoma: from molecular profiling to flow cytometry.

PubMed

Espinet, Blanca; Ferrer, Ana; Bellosillo, Beatriz; Nonell, Lara; Salar, Antonio; Fernández-Rodríguez, Concepción; Puigdecanet, Eulàlia; Gimeno, Javier; Garcia-Garcia, Mar; Vela, Maria Carmen; Luño, Elisa; Collado, Rosa; Navarro, José Tomás; de la Banda, Esmeralda; Abrisqueta, Pau; Arenillas, Leonor; Serrano, Cristina; Lloreta, Josep; Miñana, Belén; Cerutti, Andrea; Florensa, Lourdes; Orfao, Alberto; Sanz, Ferran; Solé, Francesc; Dominguez-Sola, David; Serrano, Sergio

2014-02-15

According to current diagnostic criteria, mantle cell lymphoma (MCL) encompasses the usual, aggressive variants and rare, nonnodal cases with monoclonal asymptomatic lymphocytosis, cyclin D1-positive (MALD1). We aimed to understand the biology behind this clinical heterogeneity and to identify markers for adequate identification of MALD1 cases. We compared 17 typical MCL cases with a homogeneous group of 13 untreated MALD1 cases (median follow-up, 71 months). We conducted gene expression profiling with functional analysis in five MCL and five MALD1. Results were validated in 12 MCL and 8 MALD1 additional cases by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in 24 MCL and 13 MALD1 cases by flow cytometry. Classification and regression trees strategy was used to generate an algorithm based on CD38 and CD200 expression by flow cytometry. We found 171 differentially expressed genes with enrichment of neoplastic behavior and cell proliferation signatures in MCL. Conversely, MALD1 was enriched in gene sets related to immune activation and inflammatory responses. CD38 and CD200 were differentially expressed between MCL and MALD1 and confirmed by flow cytometry (median CD38, 89% vs. 14%; median CD200, 0% vs. 24%, respectively). Assessment of both proteins allowed classifying 85% (11 of 13) of MALD1 cases whereas 15% remained unclassified. SOX11 expression by qRT-PCR was significantly different between MCL and MALD1 groups but did not improve the classification. We show for the first time that MALD1, in contrast to MCL, is characterized by immune activation and driven by inflammatory cues. Assessment of CD38/CD200 by flow cytometry is useful to distinguish most cases of MALD1 from MCL in the clinical setting. MALD1 should be identified and segregated from the current MCL category to avoid overdiagnosis and unnecessary treatment. ©2013 AACR
Distinction between Asymptomatic Monoclonal B-cell Lymphocytosis with Cyclin D1 Overexpression and Mantle Cell Lymphoma: From Molecular Profiling to Flow Cytometry

PubMed Central

Espinet, Blanca; Ferrer, Ana; Bellosillo, Beatriz; Nonell, Lara; Salar, Antonio; Fernández-Rodríguez, Concepción; Puigdecanet, Eulàlia; Gimeno, Javier; Garcia-Garcia, Mar; Carmen Vela, Maria; Luño, Elisa; Collado, Rosa; Navarro, José Tomás; de la Banda, Esmeralda; Abrisqueta, Pau; Arenillas, Leonor; Serrano, Cristina; Lloreta, Josep; Miñana, Belén; Cerutti, Andrea; Florensa, Lourdes; Orfao, Alberto; Sanz, Ferran; Solé, Francesc; Dominguez-Sola, David; Serrano, Sergio

2015-01-01

Purpose According to current diagnostic criteria, mantle cell lymphoma (MCL) encompasses the usual, aggressive variants and rare, nonnodal cases with monoclonal asymptomatic lymphocytosis, cyclin D1–positive (MALD1). We aimed to understand the biology behind this clinical heterogeneity and to identify markers for adequate identification of MALD1 cases. Experimental Design We compared 17 typical MCL cases with a homogeneous group of 13 untreated MALD1 cases (median follow-up, 71 months). We conducted gene expression profiling with functional analysis in five MCL and five MALD1. Results were validated in 12 MCL and 8 MALD1 additional cases by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in 24 MCL and 13 MALD1 cases by flow cytometry. Classification and regression trees strategy was used to generate an algorithm based on CD38 and CD200 expression by flow cytometry. Results We found 171 differentially expressed genes with enrichment of neoplastic behavior and cell proliferation signatures in MCL. Conversely, MALD1 was enriched in gene sets related to immune activation and inflammatory responses. CD38 and CD200 were differentially expressed between MCL and MALD1 and confirmed by flow cytometry (median CD38, 89% vs. 14%; median CD200, 0% vs. 24%, respectively). Assessment of both proteins allowed classifying 85% (11 of 13) of MALD1 cases whereas 15% remained unclassified. SOX11 expression by qRT-PCR was significantly different between MCL and MALD1 groups but did not improve the classification. Conclusion We show for the first time that MALD1, in contrast to MCL, is characterized by immune activation and driven by inflammatory cues. Assessment of CD38/CD200 by flow cytometry is useful to distinguish most cases of MALD1 from MCL in the clinical setting. MALD1 should be identified and segregated from the current MCL category to avoid overdiagnosis and unnecessary treatment. PMID:24352646
Micropumps, microvalves, and micromixers within PCR microfluidic chips: Advances and trends.

PubMed

Zhang, Chunsun; Xing, Da; Li, Yuyuan

2007-01-01

This review surveys the advances of microvalves, micropumps, and micromixers within PCR microfluidic chips over the past ten years. First, the types of microvalves in PCR chips are discussed, including active and passive microvalves. The active microvalves are subdivided into mechanical (thermopneumatic and shape memory alloy), non-mechanical (hydrogel, sol-gel, paraffin, and ice), and external (modular built-in, pneumatic, and non-pneumatic) microvalves. The passive microvalves also include mechanical (in-line polymerized gel and passive plug) and non-mechanical (hydrophobic) microvalves. The review then discusses mechanical (piezoelectric, pneumatic, and thermopneumatic) and non-mechanical (electrokinetic, magnetohydrodynamic, electrochemical, acoustic-wave, surface tension and capillary, and ferrofluidic magnetic) micropumps in PCR chips. Next, different micromixers within PCR chips are presented, including passive (Y/T-type flow, recirculation flow, and drop) and active (electrokinetically-driven, acoustically-driven, magnetohydrodynamical-driven, microvalves/pumps) micromixers. Finally, general discussions on microvalves, micropumps, and micromixers for PCR chips are given. The microvalve/micropump/micromixers allow high levels of PCR chip integration and analytical throughput.
CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

NASA Astrophysics Data System (ADS)

Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor

2011-07-01

Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with ~ 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of ~ 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in ~ 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of ~ 12 nm retained bright fluorescence over an extended duration of ~ a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of ~ 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of ~ 8.2% in human peripheral blood cells (PBMCs) which are CD33low. The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.
Vortex nozzle for segmenting and transporting metal chips from turning operations

DOEpatents

Bieg, L.F.

1993-04-20

Apparatus for collecting, segmenting and conveying metal chips from machining operations utilizes a compressed gas driven vortex nozzle for receiving the chip and twisting it to cause the chip to segment through the application of torsional forces to the chip. The vortex nozzle is open ended and generally tubular in shape with a converging inlet end, a constant diameter throat section and a diverging exhaust end. Compressed gas is discharged through angled vortex ports in the nozzle throat section to create vortex flow in the nozzle and through an annular inlet at the entrance to the converging inlet end to create suction at the nozzle inlet and cause ambient air to enter the nozzle. The vortex flow in the nozzle causes the metal chip to segment and the segments thus formed to pass out of the discharge end of the nozzle where they are collected, cleaned and compacted as needed.
[Design and Optimization of Microfluidic Chips Used for Mixing Cryoprotectants].

PubMed

Zhou, Xinli; Yi, Xingyue; Zhou, Nanfeng; Yang, Yun

2016-06-01

Microfluidic chips can be used to realize continuous cryoprotectants(CPA)loading/unloading for oocytes,reducing osmotic damage and chemical toxicity of CPA.In this study,five different Y-shape microfluidic chips were fabricated to realize the continuous CPA loading/unloading.The effects of flow rate,entrance angle,aspect ratio and turning radius of microchannels on the mixing efficiency of microfluidic chips were analyzed quantitatively.The experimental results showed that with the decrease of flow rates,the increase of aspect ratios and the decrease of turning raradius of microchannel,the mixing length decreased and the mixing velocity was promoted,while the entrance angle had little effect on the mixing efficiency.However,the operating conditions and structural parameters of the chips in practical application should be determined based on an overall consideration of CPA loading/unloading time and machining accuracy.These results would provide a reference to the application of microfluidic chip in CPA mixing.
Interfacing Lab-on-a-Chip Embryo Technology with High-Definition Imaging Cytometry.

PubMed

Zhu, Feng; Hall, Christopher J; Crosier, Philip S; Wlodkowic, Donald

2015-08-01

To spearhead deployment of zebrafish embryo biotests in large-scale drug discovery studies, automated platforms are needed to integrate embryo in-test positioning and immobilization (suitable for high-content imaging) with fluidic modules for continuous drug and medium delivery under microperfusion to developing embryos. In this work, we present an innovative design of a high-throughput three-dimensional (3D) microfluidic chip-based device for automated immobilization and culture and time-lapse imaging of developing zebrafish embryos under continuous microperfusion. The 3D Lab-on-a-Chip array was fabricated in poly(methyl methacrylate) (PMMA) transparent thermoplastic using infrared laser micromachining, while the off-chip interfaces were fabricated using additive manufacturing processes (fused deposition modelling and stereolithography). The system's design facilitated rapid loading and immobilization of a large number of embryos in predefined clusters of traps during continuous microperfusion of drugs/toxins. It was conceptually designed to seamlessly interface with both upright and inverted fluorescent imaging systems and also to directly interface with conventional microtiter plate readers that accept 96-well plates. Compared with the conventional Petri dish assays, the chip-based bioassay was much more convenient and efficient as only small amounts of drug solutions were required for the whole perfusion system running continuously over 72 h. Embryos were spatially separated in the traps that assisted tracing single embryos, preventing interembryo contamination and improving imaging accessibility.
An SOI CMOS-Based Multi-Sensor MEMS Chip for Fluidic Applications.

PubMed

Mansoor, Mohtashim; Haneef, Ibraheem; Akhtar, Suhail; Rafiq, Muhammad Aftab; De Luca, Andrea; Ali, Syed Zeeshan; Udrea, Florin

2016-11-04

An SOI CMOS multi-sensor MEMS chip, which can simultaneously measure temperature, pressure and flow rate, has been reported. The multi-sensor chip has been designed keeping in view the requirements of researchers interested in experimental fluid dynamics. The chip contains ten thermodiodes (temperature sensors), a piezoresistive-type pressure sensor and nine hot film-based flow rate sensors fabricated within the oxide layer of the SOI wafers. The silicon dioxide layers with embedded sensors are relieved from the substrate as membranes with the help of a single DRIE step after chip fabrication from a commercial CMOS foundry. Very dense sensor packing per unit area of the chip has been enabled by using technologies/processes like SOI, CMOS and DRIE. Independent apparatuses were used for the characterization of each sensor. With a drive current of 10 µA-0.1 µA, the thermodiodes exhibited sensitivities of 1.41 mV/°C-1.79 mV/°C in the range 20-300 °C. The sensitivity of the pressure sensor was 0.0686 mV/(V excit kPa) with a non-linearity of 0.25% between 0 and 69 kPa above ambient pressure. Packaged in a micro-channel, the flow rate sensor has a linearized sensitivity of 17.3 mV/(L/min) -0.1 in the tested range of 0-4.7 L/min. The multi-sensor chip can be used for simultaneous measurement of fluid pressure, temperature and flow rate in fluidic experiments and aerospace/automotive/biomedical/process industries.
An SOI CMOS-Based Multi-Sensor MEMS Chip for Fluidic Applications †

PubMed Central

Mansoor, Mohtashim; Haneef, Ibraheem; Akhtar, Suhail; Rafiq, Muhammad Aftab; De Luca, Andrea; Ali, Syed Zeeshan; Udrea, Florin

2016-01-01

An SOI CMOS multi-sensor MEMS chip, which can simultaneously measure temperature, pressure and flow rate, has been reported. The multi-sensor chip has been designed keeping in view the requirements of researchers interested in experimental fluid dynamics. The chip contains ten thermodiodes (temperature sensors), a piezoresistive-type pressure sensor and nine hot film-based flow rate sensors fabricated within the oxide layer of the SOI wafers. The silicon dioxide layers with embedded sensors are relieved from the substrate as membranes with the help of a single DRIE step after chip fabrication from a commercial CMOS foundry. Very dense sensor packing per unit area of the chip has been enabled by using technologies/processes like SOI, CMOS and DRIE. Independent apparatuses were used for the characterization of each sensor. With a drive current of 10 µA–0.1 µA, the thermodiodes exhibited sensitivities of 1.41 mV/°C–1.79 mV/°C in the range 20–300 °C. The sensitivity of the pressure sensor was 0.0686 mV/(Vexcit kPa) with a non-linearity of 0.25% between 0 and 69 kPa above ambient pressure. Packaged in a micro-channel, the flow rate sensor has a linearized sensitivity of 17.3 mV/(L/min)−0.1 in the tested range of 0–4.7 L/min. The multi-sensor chip can be used for simultaneous measurement of fluid pressure, temperature and flow rate in fluidic experiments and aerospace/automotive/biomedical/process industries. PMID:27827904
Droplet-based micro oscillating-flow PCR chip

NASA Astrophysics Data System (ADS)

Wang, Wei; Li, Zhi-Xin; Luo, Rong; Lü, Shu-Hai; Xu, Ai-Dong; Yang, Yong-Jun

2005-08-01

Polymerase chain reactions (PCR), thermally activated chemical reactions which are widely used for nucleic acid amplification, have recently received much attention in microelectromechanical systems and micro total analysis systems because a wide variety of DNA/RNA molecules can be enriched by PCR for further analyses. In the present work, a droplet-based micro oscillating-flow PCR chip was designed and fabricated by the silicon microfabrication technique. Three different temperature zones, which were stable at denaturation, extension and annealing temperatures and isolated from each other by a thin-wall linkage, were integrated with a single, simple and straight microchannel to form the chip's basic functional structure. The PCR mixture was injected into the chip as a single droplet and flowed through the three temperature zones in the main microchannel in an oscillating manner to achieve the temperature maintenance and transitions. The chip's thermal performance was theoretically analyzed and numerically simulated. The results indicated that the time needed for the temperature of the droplet to change to the target value is less than 1 s, and the root mean square error of temperature is less than 0.2 °C. A droplet of 1 µl PCR mixture with standard HPV (Human Papilloma Virus)-DNA sample inside was amplified by the present chip and the results were analyzed by slab gel electrophoresis with separation of DNA markers in parallel. The electrophoresis results demonstrated that the micro oscillating-flow PCR chip successfully amplified the HPV-DNA, with a processing time of about 15 min which is significantly reduced compared to that for the conventional PCR instrument.
A CLIPS expert system for clinical flow cytometry data analysis

NASA Technical Reports Server (NTRS)

Salzman, G. C.; Duque, R. E.; Braylan, R. C.; Stewart, C. C.

1990-01-01

An expert system is being developed using CLIPS to assist clinicians in the analysis of multivariate flow cytometry data from cancer patients. Cluster analysis is used to find subpopulations representing various cell types in multiple datasets each consisting of four to five measurements on each of 5000 cells. CLIPS facts are derived from results of the clustering. CLIPS rules are based on the expertise of Drs. Stewart, Duque, and Braylan. The rules incorporate certainty factors based on case histories.
Microfabrication and Test of a Three-Dimensional Polymer Hydro-focusing Unit for Flow Cytometry Applications

NASA Technical Reports Server (NTRS)

Yang, Ren; Feeback, Daniel L.; Wang, Wanjun

2004-01-01

This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was microfabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, microfabricated, and tested. Three-dimensional hydro-focusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily microfabricated and integrated with other polymer microfluidic structures.
Microfabrication and Test of a Three-Dimensional Polymer Hydro-Focusing Unit for Flow Cytometry Applications

NASA Technical Reports Server (NTRS)

Yang, Ren; Feedback, Daniel L.; Wang, Wanjun

2004-01-01

This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was micro-fabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, micro-fabricated, and tested. Three-dimensional hydrofocusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily micro-fabricated and integrated with other polymer microfluidic structures.
Multiparameter Flow Cytometry For Clinical Applications

NASA Astrophysics Data System (ADS)

Stewart, Carleton C.

1989-06-01

Flow Cytometry facilities are well established and provide immunophenotyping and DNA content measurement services. The application of immunophenotyping has been primarily in monitoring therapy and in providing further information to aid in the definitive diagnosis of immunological and neoplastic disease such as: immunodeficiency disease, auto immune disease, organ transplantation, and leukemia and lymphoma. DNA content measurements have been particularly important in determining the fraction of cycling cells and presence of aneuploid cells in neoplasia. This information has been useful in the management of patients with solid tumors.
Rapid Identification of Staphylococcus aureus and Methicillin Resistance by Flow Cytometry Using a Peptide Nucleic Acid Probe ▿

PubMed Central

Shrestha, Nabin K.; Scalera, Nikole M.; Wilson, Deborah A.; Brehm-Stecher, Byron; Procop, Gary W.

2011-01-01

A total of 56 Staphylococcus aureus isolates incubated for 2 h in the presence or absence of oxacillin were analyzed by flow cytometry after labeling with an S. aureus-specific peptide nucleic acid (PNA) probe. Two defined ratios, the paired signal count ratio (PSCR) and the gate signal count ratio (GSCR), differentiated methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) with sensitivities of 100% each and specificities of 96% and 100%, respectively. PMID:21795508
Rapid identification of Staphylococcus aureus and methicillin resistance by flow cytometry using a peptide nucleic acid probe.

PubMed

Shrestha, Nabin K; Scalera, Nikole M; Wilson, Deborah A; Brehm-Stecher, Byron; Procop, Gary W

2011-09-01

A total of 56 Staphylococcus aureus isolates incubated for 2 h in the presence or absence of oxacillin were analyzed by flow cytometry after labeling with an S. aureus-specific peptide nucleic acid (PNA) probe. Two defined ratios, the paired signal count ratio (PSCR) and the gate signal count ratio (GSCR), differentiated methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) with sensitivities of 100% each and specificities of 96% and 100%, respectively.
Application of the flow cytometry for determination of the amount of DNA in Yersinia pestis cells under the influence of serotonin (5-hydroxytryptamine)

NASA Astrophysics Data System (ADS)

Korsukov, Vladimir N.; Shchukovskaya, Tatyana N.; Kravtsov, Alexander L.; Popov, Youri A.

2002-07-01

Using flow cytometry a low DNA content in inoculated Yersinia pestis EV cells have been shown at the beginning of culture in Hottinger broth pH 7.2. The dependence serotonin action of its concentration on DNA content have been demonstrated. Serotonin accelerated Yersinia pestis culture growth during cultivation in Hottinger broth pH 7.2 both at 28 degrees C and 37 degrees C at concentration of 10-5 M.
A novel flow cytometry-based method of analyzing Heinz bodies.

PubMed

Palasuwan, D; Palasuwan, A; Charoensappakit, A; Noulsri, E

2017-02-01

Heinz bodies are important to diagnosing and managing patients. However, microscopic examination of Heinz bodies has several disadvantages, demonstrating the need for a better method. We explored the potential use of flow cytometry to examine Heinz bodies. Whole-blood samples were collected from patients deficient in G6PD and healthy volunteers. Acetylphenylhydrazine was used to induce formation of Heinz bodies in red blood cells (RBCs). Then, RBCs positive for Heinz bodies were examined using a FACSCanto II cytometer. RBCs treated with acetylphenylhydrazine formed Heinz bodies and emitted a broad spectrum of fluorescence that could be detected by flow cytometry. The maximum emission of fluorescence was observed at 45 min after the incubation with acetylphenylhydrazine. In addition, the fluorescence emitted was stable for at least 72 h. The flow cytometer could detect the RBCs positive for Heinz bodies even if they made up as little as 0.1% of the total RBC population. Furthermore, the percentage and number, respectively, of RBCs positive for Heinz bodies in G6PD-deficient patients and normal donors exhibited a mean ± standard deviation (SD) of 68.9 ± 27.5 vs. 50.9 ± 28.6 and 96 014 ±35 732 cells/μL vs. 74 688 ± 36 514 cells/μL. Heinz bodies induced by acetylphenylhydrazine emit fluorescence, and this fluorescence could be examined using flow cytometry. Our study suggests the potential use of the developed method to investigate the formation of Heinz bodies in clinical samples. © 2016 John Wiley & Sons Ltd.
Technologies for Single-Cell Isolation

PubMed Central

Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

2015-01-01

The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field. PMID:26213926
Multi-parameter analysis using photovoltaic cell-based optofluidic cytometer

PubMed Central

Yan, Chien-Shun; Wang, Yao-Nan

2016-01-01

A multi-parameter optofluidic cytometer based on two low-cost commercial photovoltaic cells and an avalanche photodetector is proposed. The optofluidic cytometer is fabricated on a polydimethylsiloxane (PDMS) substrate and is capable of detecting side scattered (SSC), extinction (EXT) and fluorescence (FL) signals simultaneously using a free-space light transmission technique without the need for on-chip optical waveguides. The feasibility of the proposed device is demonstrated by detecting fluorescent-labeled polystyrene beads with sizes of 3 μm, 5 μm and 10 μm, respectively, and label-free beads with a size of 7.26 μm. The detection experiments are performed using both single-bead population samples and mixed-bead population samples. The detection results obtained using the SSC/EXT, EXT/FL and SSC/FL signals are compared with those obtained using a commercial flow cytometer. It is shown that the optofluidic cytometer achieves a high detection accuracy for both single-bead population samples and mixed-bead population samples. Consequently, the proposed device provides a versatile, straightforward and low-cost solution for a wide variety of point-of-care (PoC) cytometry applications. PMID:27699122

Technologies for Single-Cell Isolation.

PubMed

Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

2015-07-24

The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.
Micro- and nanotechnologies in plankton research

NASA Astrophysics Data System (ADS)

Mohammed, Javeed Shaikh

2015-05-01

A better understanding of the vast range of plankton and their interactions with the marine environment would allow prediction of their large-scale impact on the marine ecosystem, and provide in-depth knowledge on pollution and climate change. Numerous technologies, especially lab-on-a-chip microsystems, are being used to this end. Marine biofouling is a global issue with significant economic consequences. Ecofriendly polymer nanotechnologies are being developed to combat marine biofouling. Furthermore, nanomaterials hold great potential for bioremediation and biofuel production. Excellent reviews covering focused topics in plankton research exist, with only a handful discussing both micro- and nanotechnologies. This work reviews both micro- and nanotechnologies applied to broad-ranging plankton research topics including flow cytometry, chemotaxis/toxicity assays, biofilm formation, marine antifouling/fouling-release surfaces and coatings, green energy, green nanomaterials, microalgae immobilization, and bioremediation. It is anticipated that developments in plankton research will see engineered exploitation of micro- and nanotechnologies. The current review is therefore intended to promote micro-/nanotechnology researchers to team up with limnologists/oceanographers, and develop novel strategies for understanding and green exploitation of the complex marine ecosystem.
Simultaneous moisture content and mass flow measurements in wood chip flows using coupled dielectric and impact sensors

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

Pan, Pengmin; McDonald, Timothy; Fulton, John

An 8-electrode capacitance tomography (ECT) sensor was built and used to measure moisture content (MC) and mass flow of pine chip flows. The device was capable of directly measuring total water quantity in a sample but was sensitive to both dry matter and moisture, and therefore required a second measurement of mass flow to calculate MC. Two means of calculating the mass flow were used: the first being an impact sensor to measure total mass flow, and the second a volumetric approach based on measuring total area occupied by wood in images generated using the capacitance sensor’s tomographic mode. Testsmore » were made on 109 groups of wood chips ranging in moisture content from 14% to 120% (dry basis) and wet weight of 280 to 1100 g. Sixty groups were randomly selected as a calibration set, and the remaining were used for validation of the sensor’s performance. For the combined capacitance/force transducer system, root mean square errors of prediction (RMSEP) for wet mass flow and moisture content were 13.42% and 16.61%, respectively. RMSEP using the combined volumetric mass flow/capacitance sensor for dry mass flow and moisture content were 22.89% and 24.16%, respectively. Either of the approaches was concluded to be feasible for prediction of moisture content in pine chip flows, but combining the impact and capacitance sensors was easier to implement. As a result, in situations where flows could not be impeded, however, the tomographic approach would likely be more useful.« less
Simultaneous moisture content and mass flow measurements in wood chip flows using coupled dielectric and impact sensors

DOE PAGES

Pan, Pengmin; McDonald, Timothy; Fulton, John; ...

2016-12-23

An 8-electrode capacitance tomography (ECT) sensor was built and used to measure moisture content (MC) and mass flow of pine chip flows. The device was capable of directly measuring total water quantity in a sample but was sensitive to both dry matter and moisture, and therefore required a second measurement of mass flow to calculate MC. Two means of calculating the mass flow were used: the first being an impact sensor to measure total mass flow, and the second a volumetric approach based on measuring total area occupied by wood in images generated using the capacitance sensor’s tomographic mode. Testsmore » were made on 109 groups of wood chips ranging in moisture content from 14% to 120% (dry basis) and wet weight of 280 to 1100 g. Sixty groups were randomly selected as a calibration set, and the remaining were used for validation of the sensor’s performance. For the combined capacitance/force transducer system, root mean square errors of prediction (RMSEP) for wet mass flow and moisture content were 13.42% and 16.61%, respectively. RMSEP using the combined volumetric mass flow/capacitance sensor for dry mass flow and moisture content were 22.89% and 24.16%, respectively. Either of the approaches was concluded to be feasible for prediction of moisture content in pine chip flows, but combining the impact and capacitance sensors was easier to implement. As a result, in situations where flows could not be impeded, however, the tomographic approach would likely be more useful.« less
Simultaneous Moisture Content and Mass Flow Measurements in Wood Chip Flows Using Coupled Dielectric and Impact Sensors

PubMed Central

Pan, Pengmin; McDonald, Timothy; Fulton, John; Via, Brian; Hung, John

2016-01-01

An 8-electrode capacitance tomography (ECT) sensor was built and used to measure moisture content (MC) and mass flow of pine chip flows. The device was capable of directly measuring total water quantity in a sample but was sensitive to both dry matter and moisture, and therefore required a second measurement of mass flow to calculate MC. Two means of calculating the mass flow were used: the first being an impact sensor to measure total mass flow, and the second a volumetric approach based on measuring total area occupied by wood in images generated using the capacitance sensor’s tomographic mode. Tests were made on 109 groups of wood chips ranging in moisture content from 14% to 120% (dry basis) and wet weight of 280 to 1100 g. Sixty groups were randomly selected as a calibration set, and the remaining were used for validation of the sensor’s performance. For the combined capacitance/force transducer system, root mean square errors of prediction (RMSEP) for wet mass flow and moisture content were 13.42% and 16.61%, respectively. RMSEP using the combined volumetric mass flow/capacitance sensor for dry mass flow and moisture content were 22.89% and 24.16%, respectively. Either of the approaches was concluded to be feasible for prediction of moisture content in pine chip flows, but combining the impact and capacitance sensors was easier to implement. In situations where flows could not be impeded, however, the tomographic approach would likely be more useful. PMID:28025536
Identifying the Presence of Prostate Cancer in Individuals with PSA Levels <20 ng ml-1 Using Computational Data Extraction Analysis of High Dimensional Peripheral Blood Flow Cytometric Phenotyping Data.

PubMed

Cosma, Georgina; McArdle, Stéphanie E; Reeder, Stephen; Foulds, Gemma A; Hood, Simon; Khan, Masood; Pockley, A Graham

2017-01-01

Determining whether an asymptomatic individual with Prostate-Specific Antigen (PSA) levels below 20 ng ml -1 has prostate cancer in the absence of definitive, biopsy-based evidence continues to present a significant challenge to clinicians who must decide whether such individuals with low PSA values have prostate cancer. Herein, we present an advanced computational data extraction approach which can identify the presence of prostate cancer in men with PSA levels <20 ng ml -1 on the basis of peripheral blood immune cell profiles that have been generated using multi-parameter flow cytometry. Statistical analysis of immune phenotyping datasets relating to the presence and prevalence of key leukocyte populations in the peripheral blood, as generated from individuals undergoing routine tests for prostate cancer (including tissue biopsy) using multi-parametric flow cytometric analysis, was unable to identify significant relationships between leukocyte population profiles and the presence of benign disease (no prostate cancer) or prostate cancer. By contrast, a Genetic Algorithm computational approach identified a subset of five flow cytometry features ( CD 8 + CD 45 RA - CD 27 - CD 28 - ( CD 8 + Effector Memory cells); CD 4 + CD 45 RA - CD 27 - CD 28 - ( CD 4 + Terminally Differentiated Effector Memory Cells re-expressing CD45RA); CD 3 - CD 19 + (B cells); CD 3 + CD 56 + CD 8 + CD 4 + (NKT cells)) from a set of twenty features, which could potentially discriminate between benign disease and prostate cancer. These features were used to construct a prostate cancer prediction model using the k-Nearest-Neighbor classification algorithm. The proposed model, which takes as input the set of flow cytometry features, outperformed the predictive model which takes PSA values as input. Specifically, the flow cytometry-based model achieved Accuracy = 83.33%, AUC = 83.40%, and optimal ROC points of FPR = 16.13%, TPR = 82.93%, whereas the PSA-based model achieved Accuracy = 77.78%, AUC = 76.95%, and optimal ROC points of FPR = 29.03%, TPR = 82.93%. Combining PSA and flow cytometry predictors achieved Accuracy = 79.17%, AUC = 78.17% and optimal ROC points of FPR = 29.03%, TPR = 85.37%. The results demonstrate the value of computational intelligence-based approaches for interrogating immunophenotyping datasets and that combining peripheral blood phenotypic profiling with PSA levels improves diagnostic accuracy compared to using PSA test alone. These studies also demonstrate that the presence of cancer is reflected in changes in the peripheral blood immune phenotype profile which can be identified using computational analysis and interpretation of complex flow cytometry datasets.
Hyperspectral cytometry.

PubMed

Grégori, Gérald; Rajwa, Bartek; Patsekin, Valery; Jones, James; Furuki, Motohiro; Yamamoto, Masanobu; Paul Robinson, J

2014-01-01

Hyperspectral cytometry is an emerging technology for single-cell analysis that combines ultrafast optical spectroscopy and flow cytometry. Spectral cytometry systems utilize diffraction gratings or prism-based monochromators to disperse fluorescence signals from multiple labels (organic dyes, nanoparticles, or fluorescent proteins) present in each analyzed bioparticle onto linear detector arrays such as multianode photomultipliers or charge-coupled device sensors. The resultant data, consisting of a series of characterizing every analyzed cell, are not compensated by employing the traditional cytometry approach, but rather are spectrally unmixed utilizing algorithms such as constrained Poisson regression or non-negative matrix factorization. Although implementations of spectral cytometry were envisioned as early as the 1980s, only recently has the development of highly sensitive photomultiplier tube arrays led to design and construction of functional prototypes and subsequently to introduction of commercially available systems. This chapter summarizes the historical efforts and work in the field of spectral cytometry performed at Purdue University Cytometry Laboratories and describes the technology developed by Sony Corporation that resulted in release of the first commercial spectral cytometry system-the Sony SP6800. A brief introduction to spectral data analysis is also provided, with emphasis on the differences between traditional polychromatic and spectral cytometry approaches.
Flow cytometry beads rather than the antihuman globulin method should be used to detect HLA Class I IgG antibody (PRA) in cadaveric renal regraft candidates.

PubMed

Bryan, Christopher F; McDonald, Scott B; Baier, Karen A; Luger, Alan M; Aeder, Mark I; Murillo, Daniel; Muruve, Nicolas A; Nelson, Paul W; Shield, Charles F; Warady, Bradley A

2002-01-01

HLA Class I antibody screening can be performed by flow cytometry using a mixture of 30 distinct bead populations each coated with the Class I antigen phenotype derived from different cell lines. In this study we compared the efficacy of Class I antibody screens done by flow cytometry beads with the antihuman globulin (AHG) method for patients awaiting cadaveric renal retransplantation. Class I panel reactive antibody (PRA) screening by flow cytometric beads of 21 regraft serum samples that had all been found to be negative by AHG DTT Class I PRA, revealed that 57.1% (12 of 21) had a flow Class I PRA of > or = 10%. Furthermore, when five regraft sera with an intermediate PRA were screened (mean AHG DTT PRA = 33.2 +/- 13%) the mean flow Class I PRA almost doubled (mean flow PRA = 72.4 +/- 10.2%) (p < 0.01). When active UNOS waiting list regraft candidates, after several months of screening the Class I PRA by flow beads, were divided into the three PRA categories based on their peak flow Class I PRA value (0-20%, 21-79% and > or = 80%), the incidence of a positive flow cross-match was 0%, 72% and 85% and the incidence of retransplantation was 60%, 22% and 10%, in each of these groups, respectively. These data provided our histocompatibility laboratory with the rationale to stop performing the AHG PRA and perform only the flow Class I PRA method for regraft candidates.
SPROC: A multiple-processor DSP IC

NASA Technical Reports Server (NTRS)

Davis, R.

1991-01-01

A large, single-chip, multiple-processor, digital signal processing (DSP) integrated circuit (IC) fabricated in HP-Cmos34 is presented. The innovative architecture is best suited for analog and real-time systems characterized by both parallel signal data flows and concurrent logic processing. The IC is supported by a powerful development system that transforms graphical signal flow graphs into production-ready systems in minutes. Automatic compiler partitioning of tasks among four on-chip processors gives the IC the signal processing power of several conventional DSP chips.
Elastomeric Capture Microparticles (ECmuPs) and Their use with Acoustophoresis to Perform Affinity Capture Assays

NASA Astrophysics Data System (ADS)

Cushing, Kevin Wallace

This dissertation describes the development of elastomeric capture microparticles (ECmicroPs) and their use with acoustophoresis to perform affinity capture assays. ECμPs that function as negative acoustic contrast particles were developed by crosslinking emulsion-based droplets composed of commercially available silicone precursors followed by functionalization with avidin/biotin reagents. The size distribution of the ECμPs was very broad or narrow depending on the emulsion system that was used during the synthesis process. Elastomeric particles exhibited a very broad size distribution when a bulk-emulsion process was used; however, when microfluidic systems were utilized, their size distribution became comparatively narrow. The functionalization of elastomeric particles was accomplished by the non-specific adsorption of avidin protein followed by bovine serum albumin (BSA) blocking and bio-specific adsorption of a biotinylated-capture antibody. Polydisperse ECμPs were functionalized to bind prostate specific antigen (PSA) or IgG-phycoerythrin (PE) in aqueous media (buffer, plasma, blood); whereas monodisperse ECμPs were functionalized to bind a high density lipoprotein in the aqueous media. Polydisperse ECμPs functionalized to bind PSA in a physiological buffer (PBS pH 7.4) demonstrated nanomolar detection using flow cytometry analysis; whereas ECμPs functionalized to bind IgG-PE demonstrated picomolar detection in 10% porcine plasma. ECμPs have a specific density of ~1.03 and are more compressible than their surrounding aqueous media; which allowed the ECμPs to exhibit negative acoustic contrast properties under an ultrasonic acoustic standing wave field. The negative acoustic contrast property of ECμPs was advantageously utilized in an IgG-PE assay conducted in 0.1% whole porcine blood. The ligand-bound ECμPs suspended in the diluted blood sample were flowed through an acoustofluidic device where the application of an ultrasonic acoustic standing wave field focused the ligand-bound ECμPs to pressure antinodes and the positive acoustic contrast blood cells to the central pressure node of the microchannel. As a result of laminar flow, focused ligand-bound ECμPs and blood cells were flowed into properly aligned outlet channels at the downstream trifurcation, where they where collected separately off-chip. The cell-free fraction containing ligand-bound ECμPs was analyzed using flow cytometry; where the detection of IgG-PE was in the picomolar range. This approach has potential applications in the development of rapid assays that detect the presence of low concentrations of biomarkers in a number of biological sample types.
ZAP-70 staining in chronic lymphocytic leukemia.

PubMed

Villamor, Neus

2005-05-01

Chronic lymphocytic leukemia (CLL) is the most common chronic leukemia in Western countries. The disease has an extremely variable clinical course, and several prognostic features have been identified to assess individual risk. The configuration of the immunoglobulin variable heavy-chain gene (IgV(H)) is a strong predictor of the outcome. CLL patients with unmutated IgV(H) status have an aggressive clinical course and a short survival. Unfortunately, analysis of IgV(H) gene configuration is not available in most clinical laboratories. A small number of genes are differentially expressed between unmutated IgV(H) and mutated IgV(H) clinical forms of CLL. One of these genes is ZAP-70, which is detected in leukemic cells from patients with the unmutated IgV(H) form of CLL. Flow cytometry presents advantages over other methods to detect ZAP-70, and its quantification by flow cytometry has proved its predictive value. This unit focuses on protocols to quantify ZAP-70 by flow cytometry in CLL.
Principles and applications of flow cytometry and cell sorting in companion animal medicine.

PubMed

Wilkerson, Melinda J

2012-01-01

Flow cytometry measures multiple characteristic of single cells using light scatter properties and fluorescence properties of fluorescent probes with specificity to cellular constituents. The use of flow cytometry in the veterinary clinical laboratory has become more routine in veterinary diagnostic laboratories and institutions (http://www.vet.k-state.edu/depts/dmp/service/immunology/index.htm), and reference laboratories. The most common applications in small animal medicine includes quantitation of erythrocytes and leukocytes in automated hematology instruments, detection of antibodies to erythrocytes and platelets in cases of immune-mediated diseases, immunophenotyping of leukocytes and lymphocytes in immunodeficiency syndromes, or leukemias and lymphomas. DNA content analysis to identify aneuploidy or replicating cells in tumor preparations has not gained routine acceptance because of the variability of prognostic results. Other applications including cell sorting and multiplexing using microspheres are potential assays of the future once they become validated and the instrumentation footprint becomes more and more compact, less expensive, and easier to use.
Rapid Flow Cytometry-Based Test for the Diagnosis of Lipopolysaccharide Responsive Beige-Like Anchor (LRBA) Deficiency.

PubMed

Gámez-Díaz, Laura; Sigmund, Elena C; Reiser, Veronika; Vach, Werner; Jung, Sophie; Grimbacher, Bodo

2018-01-01

The diagnosis of lipopolysaccharide-responsive beige-like-anchor-protein (LRBA) deficiency currently relies on gene sequencing approaches that do not support a timely diagnosis and clinical management. We developed a rapid and sensitive test for clinical implementation based on the detection of LRBA protein by flow cytometry in peripheral blood cells after stimulation. LRBA protein was assessed in a prospective cohort of 54 healthy donors and 57 patients suspected of LRBA deficiency. Receiver operating characteristics analysis suggested an LRBA:MFI ratio cutoff point of 2.6 to identify LRBA-deficient patients by FACS with 94% sensitivity and 80% specificity and to discriminate them from patients with a similar clinical picture but other disease-causing mutations. This easy flow cytometry-based assay allows a fast screening of patients with suspicion of LRBA deficiency reducing therefore the number of patients requiring LRBA sequencing and accelerating the treatment implementation. Detection of biallelic mutations in LRBA is however required for a definitive diagnosis.
Recombinant scFv antibodies against infectious pancreatic necrosis virus isolated by flow cytometry.

PubMed

Xu, Li-Ming; Zhao, Jing-Zhuang; Liu, Miao; Cao, Yong-Sheng; Yin, Jia-Sheng; Liu, Hong-Bai; Lu, Tongyan

2016-11-01

Infectious pancreatic necrosis is a significant disease of farmed salmonids in China. In this study, a single chain variable fragment (scFv) antibody library derived from rainbow trout (Oncorhynchus mykiss) and viral protein VP2 of a Chinese infectious pancreatic necrosis virus (IPNV) isolate ChRtm213 were co-expressed by a bacterial display technology. The library was subjected to three rounds of screening by flow cytometry (FCM) to select IPNV specific antibodies. Six antibody clones with different mean fluorescence intensities (MFI) were obtained by picking colonies at random. The antibody clones were expressed and purified. The purified IPNV-specific scFv antibodies were used successfully in Western blotting, enzyme linked immunosorbent assay (ELISA) and an immunofluorescence antibody test (IFAT). This method provides a high throughput means to screen an antibody library by flow cytometry, and isolate a panel of antibody that can be used as potential reagents for the detection and study of IPNV that are prevalent in China. Copyright © 2016 Elsevier B.V. All rights reserved.
Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134.

PubMed

Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Kato, Yukinari

2018-07-01

The epidermal growth factor receptor (EGFR) is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb), which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7%) to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375-394 amino acids of EGFR) neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377- RGDSFTHTPP -386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments.
Determination of critical epitope of PcMab-47 against human podocalyxin.

PubMed

Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

2018-07-01

Podocalyxin (PODXL) is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb) which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.
Detection of antibody to Purpureocillium lilacinum by immunofluorescent assay and flow cytometry in serum of infected C57BL/6 mice.

PubMed

de Sequeira, Danielly C M; Peixoto, Mariana L P; De Luca, Paula M; Oliveira-Ferreira, Joseli; Antas, Paulo R Z; Borba, Cintia M

2013-10-31

Purpureocillium lilacinum is an emerging pathogenic fungus that can cause different clinical manifestations ranging from cutaneous and sub-cutaneous infections to severe oculomycosis. In this study, using both conventional indirect immunofluorescence and non-conventional flow cytometry approaches, IgG antibodies were readily detected in both C57BL/6 immunocompetent and immunosuppressed mice after i.v. infection with P. lilacinum. The humoral immune response was specific, since virtually no antibodies were detected in the serum of control mice. Flow cytometry assays also showed both quantitative and qualitative differences in total IgG and its isotypes in sera of immunocompetent and immunosupressed infected mice. Although a good starting point, it is clear that the effectiveness of serological assays for P. lilacinum hyalohyphomycosis identification in clinical studies still requires further standardization. Upon further validation in humans, these techniques have the potential to be suitable to detect P. lilacinum infection in patients, thereby avoiding current laborious and time-consuming culture techniques. © 2013.
Improved signal recovery for flow cytometry based on ‘spatially modulated emission’

NASA Astrophysics Data System (ADS)

Quint, S.; Wittek, J.; Spang, P.; Levanon, N.; Walther, T.; Baßler, M.

2017-09-01

Recently, the technique of ‘spatially modulated emission’ has been introduced (Baßler et al 2008 US Patent 0080181827A1; Kiesel et al 2009 Appl. Phys. Lett. 94 041107; Kiesel et al 2011 Cytometry A 79A 317-24) improving the signal-to-noise ratio (SNR) for detecting bio-particles in the field of flow cytometry. Based on this concept, we developed two advanced signal processing methods which further enhance the SNR and selectivity for cell detection. The improvements are achieved by adapting digital filtering methods from RADAR technology and mainly address inherent offset elimination, increased signal dynamics and moreover reduction of erroneous detections due to processing artifacts. We present a comprehensive theory on SNR gain and provide experimental results of our concepts.
Molded underfill (MUF) encapsulation for flip-chip package: A numerical investigation

NASA Astrophysics Data System (ADS)

Azmi, M. A.; Abdullah, M. K.; Abdullah, M. Z.; Ariff, Z. M.; Saad, Abdullah Aziz; Hamid, M. F.; Ismail, M. A.

2017-07-01

This paper presents the numerical simulation of epoxy molding compound (EMC) filling in multi flip-chip packages during encapsulation process. The empty and a group flip chip packages were considered in the mold cavity in order to study the flow profile of the EMC. SOLIDWORKS software was used for three-dimensional modeling and it was incorporated into fluid analysis software namely as ANSYS FLUENT. The volume of fluid (VOF) technique was used for capturing the flow front profiles and Power Law model was applied for its rheology model. The numerical result are compared and discussed with previous experimental and it was shown a good conformity for model validation. The prediction of flow front was observed and analyzed at different filling time. The possibility and visual of void formation in the package is captured and the number of flip-chip is one factor that contributed to the void formation.
Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening.

PubMed

Doucette, Jaimee; Zhao, Ziyan; Geyer, Rory J; Barra, Melanie M; Balunas, Marcy J; Zweifach, Adam

2016-07-01

Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections. © 2016 Society for Laboratory Automation and Screening.

Capture of Fluorescence Decay Times by Flow Cytometry

PubMed Central

Naivar, Mark A.; Jenkins, Patrick; Freyer, James P.

2012-01-01

In flow cytometry, the fluorescence decay time of an excitable species has been largely underutilized and is not likely found as a standard parameter on any imaging cytometer, sorting, or analyzing system. Most cytometers lack fluorescence lifetime hardware mainly owing to two central issues. Foremost, research and development with lifetime techniques has lacked proper exploitation of modern laser systems, data acquisition boards, and signal processing techniques. Secondly, a lack of enthusiasm for fluorescence lifetime applications in cells and with bead-based assays has persisted among the greater cytometry community. In this unit, we describe new approaches that address these issues and demonstrate the simplicity of digitally acquiring fluorescence relaxation rates in flow. The unit is divided into protocol and commentary sections in order to provide a most comprehensive discourse on acquiring the fluorescence lifetime with frequency-domain methods. The unit covers (i) standard fluorescence lifetime acquisition (protocol-based) with frequency-modulated laser excitation, (ii) digital frequency-domain cytometry analyses, and (iii) interfacing fluorescence lifetime measurements onto sorting systems. Within the unit is also a discussion on how digital methods are used for aliasing in order to harness higher frequency ranges. Also, a final discussion is provided on heterodyning and processing of waveforms for multi-exponential decay extraction. PMID:25419263
Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry

NASA Astrophysics Data System (ADS)

Li, Wenyan; Houston, Kevin D.; Houston, Jessica P.

2017-01-01

Phase-sensitive flow cytometry (PSFC) is a technique in which fluorescence excited state decay times are measured as fluorescently labeled cells rapidly transit a finely focused, frequency-modulated laser beam. With PSFC the fluorescence lifetime is taken as a cytometric parameter to differentiate intracellular events that are challenging to distinguish with standard flow cytometry. For example PSFC can report changes in protein conformation, expression, interactions, and movement, as well as differences in intracellular microenvironments. This contribution focuses on the latter case by taking PSFC measurements of macrophage cells when inoculated with enhanced green fluorescent protein (EGFP)-expressing E. coli. During progressive internalization of EGFP-E. coli, fluorescence lifetimes were acquired and compared to control groups. It was hypothesized that fluorescence lifetimes would correlate well with phagocytosis because phagosomes become acidified and the average fluorescence lifetime of EGFP is known to be affected by pH. We confirmed that average EGFP lifetimes consistently decreased (3 to 2 ns) with inoculation time. The broad significance of this work is the demonstration of how high-throughput fluorescence lifetime measurements correlate well to changes that are not easily tracked by intensity-only cytometry, which is affected by heterogeneous protein expression, cell-to-cell differences in phagosome formation, and number of bacterium engulfed.
Absolute counting of neutrophils in whole blood using flow cytometry.

PubMed

Brunck, Marion E G; Andersen, Stacey B; Timmins, Nicholas E; Osborne, Geoffrey W; Nielsen, Lars K

2014-12-01

Absolute neutrophil count (ANC) is used clinically to monitor physiological dysfunctions such as myelosuppression or infection. In the research laboratory, ANC is a valuable measure to monitor the evolution of a wide range of disease states in disease models. Flow cytometry (FCM) is a fast, widely used approach to confidently identify thousands of cells within minutes. FCM can be optimised for absolute counting using spiked-in beads or by measuring the sample volume analysed. Here we combine the 1A8 antibody, specific for the mouse granulocyte protein Ly6G, with flow cytometric counting in straightforward FCM assays for mouse ANC, easily implementable in the research laboratory. Volumetric and Trucount™ bead assays were optimized for mouse neutrophils, and ANC values obtained with these protocols were compared to ANC measured by a dual-platform assay using the Orphee Mythic 18 veterinary haematology analyser. The single platform assays were more precise with decreased intra-assay variability compared with ANC obtained using the dual protocol. Defining ANC based on Ly6G expression produces a 15% higher estimate than the dual protocol. Allowing for this difference in ANC definition, the flow cytometry counting assays using Ly6G can be used reliably in the research laboratory to quantify mouse ANC from a small volume of blood. We demonstrate the utility of the volumetric protocol in a time-course study of chemotherapy induced neutropenia using four drug regimens. © 2014 International Society for Advancement of Cytometry.
Flow cytometry immunophenotyping in integrated diagnostics of patients with newly diagnosed cytopenia: one tube 10-color 14-antibody screening panel and 3-tube extensive panel for detection of MDS-related features.

PubMed

Porwit, A; Rajab, A

2015-05-01

Acute leukemia, myelodysplastic syndromes (MDS), myeloproliferative neoplasms and lymphomas are the most prevalent diagnoses in adults presenting with new onset cytopenia. Here, we describe two 10-color panels of surface markers (screening and comprehensive panel) applied at the Flow Cytometry Laboratory, University Health Network, Toronto, ON, Canada. A 10-color flow cytometry is applied using the stain-lyse-wash sample preparation method. In patients with <10% blasts and no clear involvement by hematological malignancy based on cytomorphological evaluation of bone marrow (BM) smear, the recently published one-tube 10-color 14-antibody screening panel is applied. This panel allows detection of major B- and T-cell abnormalities, enumeration of cells in blast region (CD45 dim), and gives insight into myeloid BM compartment, including calculation of four-parameter score for MDS-related abnormalities. In patients who present with ≥10 - <20% blasts in blood or BM smears, a comprehensive three-tube panel of surface markers is used up front. The analysis is focused on the detection of abnormal antigen expression patterns not seen in normal/reactive BM, according to the guidelines developed by International/European LeukemiaNet Working Group for Flow Cytometry in MDS. In patients with ≥20% blasts, an additional tube is added to allow the detection of cytoplasmic markers necessary to diagnose mixed phenotype acute leukemia. © 2015 John Wiley & Sons Ltd.
Utility of peripheral blood immunophenotyping by flow cytometry in the diagnosis of pediatric acute leukemia.

PubMed

Metrock, Laura K; Summers, Ryan J; Park, Sunita; Gillespie, Scott; Castellino, Sharon; Lew, Glen; Keller, Frank G

2017-10-01

Childhood acute leukemia is traditionally diagnosed from a bone marrow aspirate (BMA). New-onset acute leukemia patients do not always have visible circulating blasts in the peripheral blood (PB) at diagnosis. While the role of bone marrow flow cytometry for the diagnosis of acute leukemia is well established, the utility of PB flow cytometry (PBFC) is unknown. We performed a single-institution retrospective analysis to compare PBFC versus BMA in establishing or excluding a diagnosis of childhood acute leukemia. We retrospectively identified 485 PBFC samples with concurrent BMA from 2008 to 2013. Results of four-color flow cytometry for immunophenotypic characterization of leukemic versus nonclonal disease were characterized. Sensitivity and specificity were calculated among patients without a known diagnosis or prior therapy. Among 485 samples eligible for analysis, 120 had negative PBFC and BMA, 359 had positive PBFC and BMA, 3 had negative PBFC and positive BMA, and 3 had positive PBFC and negative BMA. There were small but significant differences in sensitivity (100 vs. 93.8%; P = 0.002) and positive predictive value (100 vs. 93.8%; P = 0.002) favoring BMA over PBFC among those demonstrating absence of circulating morphologic blasts. PBFC has high sensitivity and specificity for the diagnosis of childhood acute leukemia. The predictive value of PBFC remains high for patients without visible circulating blasts and may enhance the diagnostic process for determining the indications for marrow testing. © 2017 Wiley Periodicals, Inc.
Flow cytometry analysis of inflammatory cells isolated from the sciatic nerve and DRG after chronic constriction injury in mice.

PubMed

Liu, Liping; Yin, Yan; Li, Fei; Malhotra, Charvi; Cheng, Jianguo

2017-06-01

Cellular responses to nerve injury play a central role in the pathogenesis of neuropathic pain. However, the analysis of site specific cellular responses to nerve injury and neuropathic pain is limited to immunohistochemistry staining with numerous limitations. We proposed to apply flow cytometry to overcome some of the limitations and developed two protocols for isolation of cells from small specimens of the sciatic nerve and dorsal root ganglion (DRG) in mice. RESULTS AND COMPARASION WITH EXISTING: methods We found that both the non-enzymatic and enzymatic approaches were highly effective in harvesting a sufficient number of cells for flow cytometry analysis in normal and pathological conditions. The total number of cells in the injury site of the sciatic and its DRGs increased significantly 14days after chronic constriction injury (CCI) of the sciatic nerve, compared to sham surgery control or the contralateral control. The enzymatic approach yielded a significantly higher total number of cells and CD45 negative cells, suggesting that this approach allows for harvest of more resident cells, compared to the non-enzymatic method. The percentage of CD45 + /CD11b + cells was significantly increased in the sciatic nerve but not in the DRG. These results were consistent with both protocols. We thus offer two simple and effective protocols that allow for application of flow cytometry to the investigation of cellular and molecular mechanisms of neuropathic pain. Copyright © 2017 Elsevier B.V. All rights reserved.
Detection of site specific glycosylation in proteins using flow cytometry†

PubMed Central

Jayakumar, Deepak; Marathe, Dhananjay D.; Neelamegham, Sriram

2009-01-01

We tested the possibility that it is possible to express unique peptide probes on cell surfaces and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can be released from these cells by protease, and the resulting peptide can be readily captured and detected using cytometry-bead assays. Using this strategy, the peptide was immunoprecipitated onto beads bearing mAbs against either the poly-histidine sequence or the human PSGL-1. The carbohydrate epitope associated with the released peptide was detected using HECA-452 and CSLEX-1, monoclonal antibodies that recognize the sialyl Lewis-X epitope. Finally, the peptide released from cells could be separated and enriched using nickel chelate beads. Overall, such an approach that combines recombinant protein expression with flow cytometry, may be useful to quantify changes in site-specific glycosylation for basic science and clinical applications. PMID:19735085
[3-channels data acquisition system based on single-chip-microcomputer used in the measurement of coronary sinus blood flow].

PubMed

Li, Z; Fan, Y; Chen, G

1999-07-01

The coronary sinus blood flow can be figured out, which based on the principle of thermodilution, so long as gets the temperature of blood, indicator and mixture of blood and indicator respectively. This system is a smart slave module with single-chip-microcomputer. The structure and principles of hardware and the flow chart of software are described in detail.
EPIFLUORESCENCE MICROSCOPY AND SOLID PHASE CYTOMETRY AS CONFIRMATORY METHODS FOR THE ENUMERATION OF PROTOZOA BY FLOW CYTOMETRY

EPA Science Inventory

The detection of infective protozoan parasites contained in large volume environmental samples represents a unique challenge in environmental parasitology. Compounding this problem is the fact that infective stages of many protozoan parasites do not readily replicate in media or ...
Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry.

PubMed

Wang, Meiyao; Misakian, Martin; He, Hua-Jun; Bajcsy, Peter; Abbasi, Fatima; Davis, Jeffrey M; Cole, Kenneth D; Turko, Illarion V; Wang, Lili

2014-01-01

In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells. Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry. The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10(5) and (0.85 ± 0.11) × 10(5), respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC. Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.
Advanced Flip Chips in Extreme Temperature Environments

NASA Technical Reports Server (NTRS)

Ramesham, Rajeshuni

2010-01-01

The use of underfill materials is necessary with flip-chip interconnect technology to redistribute stresses due to mismatching coefficients of thermal expansion (CTEs) between dissimilar materials in the overall assembly. Underfills are formulated using organic polymers and possibly inorganic filler materials. There are a few ways to apply the underfills with flip-chip technology. Traditional capillary-flow underfill materials now possess high flow speed and reduced time to cure, but they still require additional processing steps beyond the typical surface-mount technology (SMT) assembly process. Studies were conducted using underfills in a temperature range of -190 to 85 C, which resulted in an increase of reliability by one to two orders of magnitude. Thermal shock of the flip-chip test articles was designed to induce failures at the interconnect sites (-40 to 100 C). The study on the reliability of flip chips using underfills in the extreme temperature region is of significant value for space applications. This technology is considered as an enabling technology for future space missions. Flip-chip interconnect technology is an advanced electrical interconnection approach where the silicon die or chip is electrically connected, face down, to the substrate by reflowing solder bumps on area-array metallized terminals on the die to matching footprints of solder-wettable pads on the chosen substrate. This advanced flip-chip interconnect technology will significantly improve the performance of high-speed systems, productivity enhancement over manual wire bonding, self-alignment during die joining, low lead inductances, and reduced need for attachment of precious metals. The use of commercially developed no-flow fluxing underfills provides a means of reducing the processing steps employed in the traditional capillary flow methods to enhance SMT compatibility. Reliability of flip chips may be significantly increased by matching/tailoring the CTEs of the substrate material and the silicon die or chip, and also the underfill materials. Advanced packaging interconnects technology such as flip-chip interconnect test boards have been subjected to various extreme temperature ranges that cover military specifications and extreme Mars and asteroid environments. The eventual goal of each process step and the entire process is to produce components with 100 percent interconnect and satisfy the reliability requirements. Underfill materials, in general, may possibly meet demanding end use requirements such as low warpage, low stress, fine pitch, high reliability, and high adhesion.
European Seminar on Neural Computing

DTIC Science & Technology

1988-08-31

elements can be fabricated on a single chip . Two specific oriented language (for example, SMALLTALK or cellular arrays, namely, the programmable systolic... chip POOL) the basic concepts are: objects are viewed as (Fisher, 1983) and the connection machine (Treleaven, active, they may contain state, and...flow computer the availability of 1. Programmable Systolic Chip . Programmable Sys- input operands triggers the execution of the instruction tolic Chips
In vivo photoacoustic flow cytometry for early malaria diagnosis.

PubMed

Cai, Chengzhong; Carey, Kai A; Nedosekin, Dmitry A; Menyaev, Yulian A; Sarimollaoglu, Mustafa; Galanzha, Ekaterina I; Stumhofer, Jason S; Zharov, Vladimir P

2016-06-01

In vivo photoacoustic (PA) flow cytometry (PAFC) has already demonstrated a great potential for the diagnosis of deadly diseases through ultrasensitive detection of rare disease-associated circulating markers in whole blood volume. Here, we demonstrate the first application of this powerful technique for early diagnosis of malaria through label-free detection of malaria parasite-produced hemozoin in infected red blood cells (iRBCs) as high-contrast PA agent. The existing malaria tests using blood smears can detect the disease at 0.001-0.1% of parasitemia. On the contrary, linear PAFC showed a potential for noninvasive malaria diagnosis at an extremely low level of parasitemia of 0.0000001%, which is ∼10(3) times better than the existing tests. Multicolor time-of-flight PAFC with high-pulse repetition rate lasers at wavelengths of 532, 671, and 820 nm demonstrated rapid spectral and spatial identification and quantitative enumeration of individual iRBCs. Integration of PAFC with fluorescence flow cytometry (FFC) provided real-time simultaneous detection of single iRBCs and parasites expressing green fluorescence proteins, respectively. A combination of linear and nonlinear nanobubble-based multicolor PAFC showed capability to real-time control therapy efficiency by counting of iRBCs before, during, and after treatment. Our results suggest that high-sensitivity, high-resolution ultrafast PAFC-FFC platform represents a powerful research tool to provide the insight on malaria progression through dynamic study of parasite-cell interactions directly in bloodstream, whereas portable hand-worn PAFC device could be broadly used in humans for early malaria diagnosis. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.
Photoacoustic-fluorescence in vitro flow cytometry for quantification of absorption, scattering and fluorescence properties of the cells

NASA Astrophysics Data System (ADS)

Nedosekin, D. A.; Sarimollaoglu, M.; Foster, S.; Galanzha, E. I.; Zharov, V. P.

2013-03-01

Fluorescence flow cytometry is a well-established analytical tool that provides quantification of multiple biological parameters of cells at molecular levels, including their functional states, morphology, composition, proliferation, and protein expression. However, only the fluorescence and scattering parameters of the cells or labels are available for detection. Cell pigmentation, presence of non-fluorescent dyes or nanoparticles cannot be reliably quantified. Herewith, we present a novel photoacoustic (PA) flow cytometry design for simple integration of absorbance measurements into schematics of conventional in vitro flow cytometers. The integrated system allow simultaneous measurements of light absorbance, scattering and of multicolor fluorescence from single cells in the flow at rates up to 2 m/s. We compared various combinations of excitation laser sources for multicolor detection, including simultaneous excitation of PA and fluorescence using a single 500 kHz pulsed nanosecond laser. Multichannel detection scheme allows simultaneous detection of up to 8 labels, including 4 fluorescent tags and 4 PA colors. In vitro PA-fluorescence flow cytometer was used for studies of nanoparticles uptake and for the analysis of cell line pigmentation, including genetically encoded melanin expression in breast cancer cell line. We demonstrate that this system can be used for direct nanotoxicity studies with simultaneous quantification of nanoparticles content and assessment of cell viability using a conventional fluorescent apoptosis assays.
Label-free high-throughput imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mahjoubfar, A.; Chen, C.; Niazi, K. R.; Rabizadeh, S.; Jalali, B.

2014-03-01

Flow cytometry is an optical method for studying cells based on their individual physical and chemical characteristics. It is widely used in clinical diagnosis, medical research, and biotechnology for analysis of blood cells and other cells in suspension. Conventional flow cytometers aim a laser beam at a stream of cells and measure the elastic scattering of light at forward and side angles. They also perform single-point measurements of fluorescent emissions from labeled cells. However, many reagents used in cell labeling reduce cellular viability or change the behavior of the target cells through the activation of undesired cellular processes or inhibition of normal cellular activity. Therefore, labeled cells are not completely representative of their unaltered form nor are they fully reliable for downstream studies. To remove the requirement of cell labeling in flow cytometry, while still meeting the classification sensitivity and specificity goals, measurement of additional biophysical parameters is essential. Here, we introduce an interferometric imaging flow cytometer based on the world's fastest continuous-time camera. Our system simultaneously measures cellular size, scattering, and protein concentration as supplementary biophysical parameters for label-free cell classification. It exploits the wide bandwidth of ultrafast laser pulses to perform blur-free quantitative phase and intensity imaging at flow speeds as high as 10 meters per second and achieves nanometer-scale optical path length resolution for precise measurements of cellular protein concentration.
Quantification of proteins by flow cytometry: Quantification of human hepatic transporter P-gp and OATP1B1 using flow cytometry and mass spectrometry.

PubMed

Hogg, Karen; Thomas, Jerry; Ashford, David; Cartwright, Jared; Coldwell, Ruth; Weston, Daniel J; Pillmoor, John; Surry, Dominic; O'Toole, Peter

2015-07-01

Flow cytometry is a powerful tool for the quantitation of fluorescence and is proven to be able to correlate the fluorescence intensity to the number of protein on cells surface. Mass spectroscopy can also be used to determine the number of proteins per cell. Here we have developed two methods, using flow cytometry and mass spectroscopy to quantify number of transporters in human cells. These two approaches were then used to analyse the same samples so that a direct comparison could be made. Transporters have a major impact on the behaviour of a diverse number of drugs in human systems. While active uptake studies by transmembrane protein transporters using model substrates are routinely undertaken in human cell lines and hepatocytes as part of drug discovery and development, the interpretation of these results is currently limited by the inability to quantify the number of transporters present in the test samples. Here we provide a flow cytometric method for accurate quantification of transporter levels both on the cell surface and within the cell, and compare this to a quantitative mass spectrometric approach. Two transporters were selected for the study: OATP1B1 (also known as SLCO1B1, LST-1, OATP-C, OATP2) due to its important role in hepatic drug uptake and elimination; P-gp (also known as P-glycoprotein, MDR1, ABCB1) as a well characterised system and due to its potential impact on oral bioavailability, biliary and renal clearance, and brain penetration of drugs that are substrates for this transporter. In all cases the mass spectrometric method gave higher levels than the flow cytometry method. However, the two methods showed very similar trends in the relative ratios of both transporters in the hepatocyte samples investigated. The P-gp antibody allowed quantitative discrimination between externally facing transporters located in the cytoplasmic membrane and the total number of transporters on and in the cell. The proportion of externally facing transporter varied considerably in the four hepatocyte samples analysed, ranging from only 6% to 35% of intact and viable cells. The sample with only 6% externally facing transporter was further analysed by confocal microscopy which qualitatively confirmed the low level of transporter in the membrane and the large internal population. Here we prove that flow cytometry is an important tool for future protein analysis as it can not only quantify the number of proteins that a cell express but also identify the number of proteins on the surface and it is easy to apply for routine assays. Copyright © 2015 Elsevier Inc. All rights reserved.
A novel fluorescent sensor for measurement of CFTR function by flow cytometry.

PubMed

Vijftigschild, Lodewijk A W; van der Ent, Cornelis K; Beekman, Jeffrey M

2013-06-01

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis. CFTR-dependent iodide transport measured by fluorescent quenching of ectopically expressed halide-sensitive yellow fluorescent protein (YFP) is widely being used to study CFTR function by microscopy or plate readers. Since YFP fluorescence in these systems is dependent on YFP expression levels and iodide concentration, differences in sensor expression level between experimental units are normalized at the start of each experiment. To allow accurate measurement of CFTR function by flow cytometry, we reasoned that co-expression of an iodide insensitive fluorescent protein would allow for normalization of sensor expression levels and more accurate quantification of CFTR function. Our data indicated that dsRed and mKate fluorescence are iodide insensitive, and we determined an optimal format for co-expression of these fluorescent proteins with halide-sensitive YFP. We showed using microscopy that ratiometric measurement (YFP/mKate) corrects for differences in sensor expression levels. Ratiometric measurements were essential to accurately measure CFTR function by flow cytometry that we here describe for the first time. Mixing of wild type or mutant CFTR expressing cells indicated that addition of approximately 10% of wild type CFTR expressing cells could be distinguished by ratiometric YFP quenching. Flow cytometric ratiometric YFP quenching also allowed us to study CFTR mutants associated with differential residual function upon ectopic expression. Compared with conventional plate-bound CFTR function assays, the flow cytometric approach described here can be used to study CFTR function in suspension cells. It may be further adapted to study CFTR function in heterologous cell populations using cell surface markers and selection of cells that display high CFTR function by cell sorting. Copyright © 2013 International Society for Advancement of Cytometry.
Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods

PubMed Central

Friedrich, Ralf P; Janko, Christina; Poettler, Marina; Tripal, Philipp; Zaloga, Jan; Cicha, Iwona; Dürr, Stephan; Nowak, Johannes; Odenbach, Stefan; Slabu, Ioana; Liebl, Maik; Trahms, Lutz; Stapf, Marcus; Hilger, Ingrid; Lyer, Stefan; Alexiou, Christoph

2015-01-01

Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEONLA) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEONLA with an additional protein corona formed by bovine serum albumin (SEONLA-BSA) and commercially available Rienso® particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products. PMID:26170658
Flow Cytometry of Spinach Chloroplasts 1

PubMed Central

Schröder, Wolfgang P.; Petit, Patrice X.

1992-01-01

Intact spinach (Spinacia oleracea) chloroplasts, thylakoid membranes, and inside-out or right-side-out thylakoid vesicles have been characterized by flow cytometry with respect to forward angle light scatter, right angle light scatter, and chlorophyll fluorescence. Analysis of intact chloroplasts with respect to forward light scatter and the chlorophyll fluorescence parameter revealed the presence of truly “intact” and “disrupted” chloroplasts. The forward light scatter parameter, normally considered to reflect object size, was instead found to reflect the particle density. One essential advantage of flow cytometry is that additional parameters such as Ricinus communis agglutinin (linked to fluorescein isothiocyanate) fluorescence can be determined through logical conditions placed on bit-maps, amounting to an analytical purification procedure. In the present case, chloroplast subpopulations with fully preserved envelopes, thylakoid membrane, and inside-out or right-side-out thylakoid membranes vesicles can be distinguished. Flow cytometry is also a useful tool to address the question of availability of glycosyl moities on the membrane surfaces if one keeps in mind that organelle-to-organelle interactions could be partially mediated through a recognition process. A high specific binding of R. communis agglutinin and peanut lectin to the chloroplast envelope was detected. This showed that galactose residues were exposed and accessible to specific lectins on the chloroplast surface. No exposed glucose, fucose, or mannose residues could be detected by the appropriate lectins. Ricin binding to the intact chloroplasts caused a strong aggregation. Disruption of these aggregates by resuspension or during passage in the flow cytometer induced partial breakage of the chloroplasts. Only minor binding of R. communis agglutinin and peanut lectin to the purified thylakoid membranes was detected; the binding was found to be low for both inside-out and right-side-out vesicles of the thylakoid membranes. Images Figure 1 Figure 1 Figure 1 PMID:16653090
Improved panels for clinical immune phenotyping: Utilization of the violet laser.

PubMed

Ryherd, Mark; Plassmeyer, Matthew; Alexander, Connor; Eugenio, Ines; Kleschenko, Yuliya; Badger, Ariel; Gupta, Raavi; Alpan, Oral; Sønder, Søren Ulrik

2017-05-10

Clinical diagnostic laboratories are subject to numerous regulations imposed by government agencies. Laboratory developed tests for flow cytometry panels are essentially restricted to the use of analyte-specific reagents (ASR) antibodies. With the advances in clinical flow cytometry systems, there is a trend toward the utilization of blue/red/violet laser flow systems and 8 to 10-color panels. Currently, the selection of commercially available ASR antibodies for the violet laser is very limited. The market is dominated by Brilliant Violet 421 (BV421) manufactured by BD Biosciences and Pacific Blue (PB) manufactured by Beckman Coulter. In this study, we compare BV421 and PB conjugated ASR antibodies. Whole blood was stained and acquired on a Gallios flow cytometer system. For single color staining, the stain index (SI) was calculated. For the two panels, the compensation matrix was calculated and the performance of the antibody cocktails analyzed in FCS Express. The results show that five out of six tested BV421 conjugated antibodies have significantly higher SI than their PB counterparts. Furthermore, BV421 antibodies require less compensation for spillover than PB. Finally, BV421 conjugated antibodies give better separation between negative and positive populations in the context of an 8 and 10 color panel without affecting the intensity of the other dyes. Overall, using BV421 conjugated antibodies results in better separation between populations compared to PB conjugated antibodies without negatively affecting other fluorochromes in our panels. We conclude that the BV421 conjugated ASR antibodies are currently the better available option for clinical flow panels. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

Hydrostatic Pressure Enhances Vital Staining with Carboxyfluorescein or Carboxydichlorofluorescein in Saccharomyces cerevisiae: Efficient Detection of Labeled Yeasts by Flow Cytometry

PubMed Central

Abe, Fumiyoshi

1998-01-01

The extent of intracellular accumulation of the fluorescent dye carboxyfluorescein or carboxydichlorofluorescein (CDCF) in Saccharomyces cerevisiae was found to be increased 5- to 10-fold under a nonlethal hydrostatic pressure of 30 to 50 MPa. This observation was confirmed by analysis of individual labeled cells by flow cytometry. The pressure-induced enhancement of staining with CDCF required d-glucose and was markedly inhibited by 2-deoxy-d-glucose, suggesting that glucose metabolism has a role in the process. PMID:9501452
Augmenting Trastuzumab Therapy Against Breast Cancer Through Selective Activation of NK Cells

DTIC Science & Technology

2013-10-01

selection and assessed for purity (>90% purity as defined by CD3-CD56+ flow cytometry ) and activation (>50% expression of CD137). Breast cancer cell lines...at a ratio of 1:1. After 24 hours, NK cells were isolated by negative selection and assessed for purity (>90% purity as defined by CD3-CD56+ flow ... cytometry ) and activation (>50% expression of CD137). Chromium-labeled breast cancer cell lines including MCF7 (A), BT474M1 (B), HER18 (C), and SKBR3
Treatment-Induced Autophagy Associated with Tumor Dormancy and Relapse

DTIC Science & Technology

2017-07-01

immunogenic apoptosis, autophagy and senescence in MMC and SKBR3 tumor cell lines. We determined that ADR, but not radiation , induced what appears to...hours after WT and BPTF KD 4T1 cells were treated with 5 Gy γ- radiation exposure. (D) γH2AX was measured by flow cytometry 30 minutes after WT and...BPTF KD 4T1 cells were treated with 6Gy γ- radiation . (E) Flow cytometry measurement of 1 ug/ml ADR accumulation after 24 hours exposure to WT and BPTF
Preparation and flow cytometry of uniform silica-fluorescent dye microspheres.

PubMed

Bele, Marjan; Siiman, Olavi; Matijević, Egon

2002-10-15

Uniform fluorescent silica-dye microspheres have been prepared by coating preformed monodispersed silica particles with silica layers containing rhodamine 6G or acridine orange. The resulting dispersions exhibit intense fluorescent emission between 500 and 600 nm, over a broad excitation wavelength range of 460 to 550 nm, even with exceedingly small amounts of dyes incorporated into the silica particles (10-30 ppm, expressed as weight of dye relative to weight of dry particles). The fluorescent particles can be prepared in micrometer diameters suitable for analyses using flow cytometry with 488-nm laser excitation.
A Rare Case of Pure Erythroid Sarcoma in a Pediatric Patient: Case Report and Literature Review

PubMed Central

Tarín, Fabián; Niveiro, María; Tasso, María; Alda, Olga; Verdú, José J.; De Paz, Francisco; López, Silvia; Del Cañizo, María; Such, Esperanza; Barragán, Eva; Martirena, Fernanda

2017-01-01

We describe an exceptional case of erythroid sarcoma in a pediatric patient as a growing orbital mass with no evidence of morphologic bone marrow involvement, who was finally diagnosed of pure erythroid sarcoma based on histopathology and flow cytometry criteria. We discuss the contribution of standardized eight-color flow cytometry as a rapid and reliable diagnostic method. The use of normal bone marrow databases allowed us to identify small aberrant populations in bone marrow and later confirm the diagnosis in the neoplastic tissue. PMID:29261159
A Rare Case of Pure Erythroid Sarcoma in a Pediatric Patient: Case Report and Literature Review.

PubMed

Manresa, Pablo; Tarín, Fabián; Niveiro, María; Tasso, María; Alda, Olga; López, Francisco; Sarmiento, Héctor; Verdú, José J; De Paz, Francisco; López, Silvia; Del Cañizo, María; Such, Esperanza; Barragán, Eva; Martirena, Fernanda

2017-12-20

We describe an exceptional case of erythroid sarcoma in a pediatric patient as a growing orbital mass with no evidence of morphologic bone marrow involvement, who was finally diagnosed of pure erythroid sarcoma based on histopathology and flow cytometry criteria. We discuss the contribution of standardized eight-color flow cytometry as a rapid and reliable diagnostic method. The use of normal bone marrow databases allowed us to identify small aberrant populations in bone marrow and later confirm the diagnosis in the neoplastic tissue.
Microfabrication and Test of a Three-Dimensional Polymer Hydro-focusing Unit for Flow Cytometry Applications

NASA Technical Reports Server (NTRS)

Yang, Ren; Feeback, Daniel L.; Wang, Wan-Jun

2005-01-01

This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was microfabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, microfabricated, and tested. Three-dimensional hydrofocusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily microfabricated and integrated with other polymer microfluidic structures. Keywords: SU-8, three-dimensional hydro-focusing, microfluidic, microchannel, cytometer
Streak Imaging Flow Cytometer for Rare Cell Analysis.

PubMed

Balsam, Joshua; Bruck, Hugh Alan; Ossandon, Miguel; Prickril, Ben; Rasooly, Avraham

2017-01-01

There is a need for simple and affordable techniques for cytology for clinical applications, especially for point-of-care (POC) medical diagnostics in resource-poor settings. However, this often requires adapting expensive and complex laboratory-based techniques that often require significant power and are too massive to transport easily. One such technique is flow cytometry, which has great potential for modification due to the simplicity of the principle of optical tracking of cells. However, it is limited in that regard due to the flow focusing technique used to isolate cells for optical detection. This technique inherently reduces the flow rate and is therefore unsuitable for rapid detection of rare cells which require large volume for analysis.To address these limitations, we developed a low-cost, mobile flow cytometer based on streak imaging. In our new configuration we utilize a simple webcam for optical detection over a large area associated with a wide-field flow cell. The new flow cell is capable of larger volume and higher throughput fluorescence detection of rare cells than the flow cells with hydrodynamic focusing used in conventional flow cytometry. The webcam is an inexpensive, commercially available system, and for fluorescence analysis we use a 1 W 450 nm blue laser to excite Syto-9 stained cells with emission at 535 nm. We were able to detect low concentrations of stained cells at high flow rates of 10 mL/min, which is suitable for rapidly analyzing larger specimen volumes to detect rare cells at appropriate concentration levels. The new rapid detection capabilities, combined with the simplicity and low cost of this device, suggest a potential for clinical POC flow cytometry in resource-poor settings associated with global health.
Leptomeningeal metastases: a RANO proposal for response criteria

PubMed Central

Junck, Larry; Brandsma, Dieta; Soffietti, Riccardo; Rudà, Roberta; Raizer, Jeffrey; Boogerd, Willem; Taillibert, Sophie; Groves, Morris D.; Rhun, Emilie Le; Walker, Julie; van den Bent, Martin; Wen, Patrick Y.; Jaeckle, Kurt A.

2017-01-01

Abstract Leptomeningeal metastases (LM) currently lack standardization with respect to response assessment. A Response Assessment in Neuro-Oncology (RANO) working group with expertise in LM developed a consensus proposal for evaluating patients treated for this disease. Three basic elements in assessing response in LM are proposed: a standardized neurological examination, cerebral spinal fluid (CSF) cytology or flow cytometry, and radiographic evaluation. The group recommends that all patients enrolling in clinical trials undergo CSF analysis (cytology in all cancers; flow cytometry in hematologic cancers), complete contrast-enhanced neuraxis MRI, and in instances of planned intra-CSF therapy, radioisotope CSF flow studies. In conjunction with the RANO Neurological Assessment working group, a standardized instrument was created for assessing the neurological exam in patients with LM. Considering that most lesions in LM are nonmeasurable and that assessment of neuroimaging in LM is subjective, neuroimaging is graded as stable, progressive, or improved using a novel radiological LM response scorecard. Radiographic disease progression in isolation (ie, negative CSF cytology/flow cytometry and stable neurological assessment) would be defined as LM disease progression. The RANO LM working group has proposed a method of response evaluation for patients with LM that will require further testing, validation, and likely refinement with use. PMID:28039364
Dynamic thermoregulation of the sample in flow cytometry.

PubMed

Graves, Steven W; Habbersett, Robert C; Nolan, John P

2002-05-01

Fine control of temperature is an important capability for any analytical platform. A circulating water bath has been the traditional means of maintaining constant temperature in the sample chamber of a flow cytometer, but this approach does not permit rapid changes in sample temperature. This unit explains the use of Peltier modules for regulation of sample temperature. The heat pumping generated by the passage of current through properly matched semiconductors, known as the Peltier effect, makes it possible for these thermoelectric modules to both heat and cool. The authors describe the construction of a Peltier module based thermoregulation unit in step-by-step detail and present a demonstration of flow cytometry measurements as a function of temperature.
Assessment of amphotericin B susceptibility in Leishmania infantum promastigotes by flow cytometric membrane potential assay.

PubMed

Azas, N; Di Giorgio, C; Delmas, F; Gasquet, M; Timon-David, P

1997-06-01

Flow cytometry was used for measuring the effects of amphotericin B on the membrane of Leishmania infantum strains. The technique was adapted from the rapid flow cytometric membrane potential assay developed by Ordonez and Wehman (Cytometry 22:154-157, 1995) for evaluating antibiotic-susceptibility of Candida species. The study consisted of measuring membrane potential changes induced by amphotericin B in 3 initial strains and 12 laboratory-generated variants adapted to grow with amphotericin B. Results showed that, after 3 h of incubation, amphotericin B induced a dose-related decrease of membrane potential that reached its maximal level at the same concentrations that inhibited parasite growth. These results suggest that the flow cytometric membrane potential assay could be used to assess the susceptibility of Leishmania promastigotes to amphotericin B.
Pumpless Microflow Cytometry Enabled by Viscosity Modulation and Immunobead Labeling.

PubMed

Kim, Byeongyeon; Oh, Sein; Shin, Suyeon; Yim, Sang-Gu; Yang, Seung Yun; Hahn, Young Ki; Choi, Sungyoung

2018-06-19

Major challenges of miniaturizing flow cytometry include obviating the need for bulky, expensive, and complex pump-based fluidic and laser-based optical systems while retaining the ability to detect target cells based on their unique surface receptors. We addressed these critical challenges by (i) using a viscous liquid additive to control flow rate passively, without external pumping equipment, and (ii) adopting an immunobead assay that can be quantified with a portable fluorescence cell counter based on a blue light-emitting diode. Such novel features enable pumpless microflow cytometry (pFC) analysis by simply dropping a sample solution onto the inlet reservoir of a disposable cell-counting chamber. With our pFC platform, we achieved reliable cell counting over a dynamic range of 9-298 cells/μL. We demonstrated the practical utility of the platform by identifying a type of cancer cell based on CD326, the epithelial cell adhesion molecule. This portable microflow cytometry platform can be applied generally to a range of cell types using immunobeads labeled with specific antibodies, thus making it valuable for cell-based and point-of-care diagnostics.
DAFi: A directed recursive data filtering and clustering approach for improving and interpreting data clustering identification of cell populations from polychromatic flow cytometry data.

PubMed

Lee, Alexandra J; Chang, Ivan; Burel, Julie G; Lindestam Arlehamn, Cecilia S; Mandava, Aishwarya; Weiskopf, Daniela; Peters, Bjoern; Sette, Alessandro; Scheuermann, Richard H; Qian, Yu

2018-04-17

Computational methods for identification of cell populations from polychromatic flow cytometry data are changing the paradigm of cytometry bioinformatics. Data clustering is the most common computational approach to unsupervised identification of cell populations from multidimensional cytometry data. However, interpretation of the identified data clusters is labor-intensive. Certain types of user-defined cell populations are also difficult to identify by fully automated data clustering analysis. Both are roadblocks before a cytometry lab can adopt the data clustering approach for cell population identification in routine use. We found that combining recursive data filtering and clustering with constraints converted from the user manual gating strategy can effectively address these two issues. We named this new approach DAFi: Directed Automated Filtering and Identification of cell populations. Design of DAFi preserves the data-driven characteristics of unsupervised clustering for identifying novel cell subsets, but also makes the results interpretable to experimental scientists through mapping and merging the multidimensional data clusters into the user-defined two-dimensional gating hierarchy. The recursive data filtering process in DAFi helped identify small data clusters which are otherwise difficult to resolve by a single run of the data clustering method due to the statistical interference of the irrelevant major clusters. Our experiment results showed that the proportions of the cell populations identified by DAFi, while being consistent with those by expert centralized manual gating, have smaller technical variances across samples than those from individual manual gating analysis and the nonrecursive data clustering analysis. Compared with manual gating segregation, DAFi-identified cell populations avoided the abrupt cut-offs on the boundaries. DAFi has been implemented to be used with multiple data clustering methods including K-means, FLOCK, FlowSOM, and the ClusterR package. For cell population identification, DAFi supports multiple options including clustering, bisecting, slope-based gating, and reversed filtering to meet various autogating needs from different scientific use cases. © 2018 International Society for Advancement of Cytometry. © 2018 International Society for Advancement of Cytometry.
Sensor chip and apparatus for tactile and/or flow sensing

NASA Technical Reports Server (NTRS)

Liu, Chang (Inventor); Chen, Jack (Inventor); Engel, Jonathan (Inventor)

2008-01-01

A sensor chip, comprising a flexible, polymer-based substrate, and at least one microfabricated sensor disposed on the substrate and including a conductive element. The at least one sensor comprises at least one of a tactile sensor and a flow sensor. Other embodiments of the present invention include sensors and/or multi-modal sensor nodes.
Sensor chip and apparatus for tactile and/or flow sensing

NASA Technical Reports Server (NTRS)

Liu, Chang (Inventor); Chen, Jack (Inventor); Engel, Jonathan (Inventor)

2009-01-01

A sensor chip, comprising a flexible, polymer-based substrate, and at least one microfabricated sensor disposed on the substrate and including a conductive element. The at least one sensor comprises at least one of a tactile sensor and a flow sensor. Other embodiments of the present invention include sensors and/or multi-modal sensor nodes.
Ultraviolet 320 nm laser excitation for flow cytometry.

PubMed

Telford, William; Stickland, Lynn; Koschorreck, Marco

2017-04-01

Although multiple lasers and high-dimensional analysis capability are now standard on advanced flow cytometers, ultraviolet (UV) lasers (usually 325-365 nm) remain an uncommon excitation source for cytometry. This is primarily due to their cost, and the small number of applications that require this wavelength. The development of the Brilliant Ultraviolet (BUV fluorochromes, however, has increased the importance of this formerly niche excitation wavelength. Historically, UV excitation was usually provided by water-cooled argon- and krypton-ion lasers. Modern flow cytometers primary rely on diode pumped solid state lasers emitting at 355 nm. While useful for all UV-excited applications, DPSS UV lasers are still large by modern solid state laser standards, and remain very expensive. Smaller and cheaper near UV laser diodes (NUVLDs) emitting at 375 nm make adequate substitutes for 355 nm sources in many situations, but do not work as well with very short wavelength probes like the fluorescent calcium chelator indo-1. In this study, we evaluate a newly available UV 320 nm laser for flow cytometry. While shorter in wavelength that conventional UV lasers, 320 is close to the 325 nm helium-cadmium wavelength used in the past on early benchtop cytometers. A UV 320 nm laser was found to excite almost all Brilliant Ultraviolet dyes to nearly the same level as 355 nm sources. Both 320 nm and 355 nm sources worked equally well for Hoechst and DyeCycle Violet side population analysis of stem cells in mouse hematopoetic tissue. The shorter wavelength UV source also showed excellent excitation of indo-1, a probe that is not compatible with NUVLD 375 nm sources. In summary, a 320 nm laser module made a suitable substitute for conventional 355 nm sources. This laser technology is available in a smaller form factor than current 355 nm units, making it useful for small cytometers with space constraints. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.
Bubble-free on-chip continuous-flow polymerase chain reaction: concept and application.

PubMed

Wu, Wenming; Kang, Kyung-Tae; Lee, Nae Yoon

2011-06-07

Bubble formation inside a microscale channel is a significant problem in general microfluidic experiments. The problem becomes especially crucial when performing a polymerase chain reaction (PCR) on a chip which is subject to repetitive temperature changes. In this paper, we propose a bubble-free sample injection scheme applicable for continuous-flow PCR inside a glass/PDMS hybrid microfluidic chip, and attempt to provide a theoretical basis concerning bubble formation and elimination. Highly viscous paraffin oil plugs are employed in both the anterior and posterior ends of a sample plug, completely encapsulating the sample and eliminating possible nucleation sites for bubbles. In this way, internal channel pressure is increased, and vaporization of the sample is prevented, suppressing bubble formation. Use of an oil plug in the posterior end of the sample plug aids in maintaining a stable flow of a sample at a constant rate inside a heated microchannel throughout the entire reaction, as compared to using an air plug. By adopting the proposed sample injection scheme, we demonstrate various practical applications. On-chip continuous-flow PCR is performed employing genomic DNA extracted from a clinical single hair root sample, and its D1S80 locus is successfully amplified. Also, chip reusability is assessed using a plasmid vector. A single chip is used up to 10 times repeatedly without being destroyed, maintaining almost equal intensities of the resulting amplicons after each run, ensuring the reliability and reproducibility of the proposed sample injection scheme. In addition, the use of a commercially-available and highly cost-effective hot plate as a potential candidate for the heating source is investigated.
Annexin-V/quantum dot probes for multimodal apoptosis monitoring in living cells: improving bioanalysis using electrochemistry

NASA Astrophysics Data System (ADS)

Montón, Helena; Parolo, Claudio; Aranda-Ramos, Antonio; Merkoçi, Arben; Nogués, Carme

2015-02-01

There is a great demand to develop novel techniques that allow useful and complete monitoring of apoptosis, which is a key factor of several diseases and a target for drug development. Here, we present the use of a novel dual electrochemical/optical label for the detection and study of apoptosis. We combined the specificity of Annexin-V for phosphatidylserine, a phospholipid expressed in the outer membrane of apoptotic cells, with the optical and electrochemical properties of quantum dots to create a more efficient label. Using this conjugate we addressed three important issues: (i) we made the labeling of apoptotic cells faster (30 min) and easier; (ii) we fully characterized the samples by common cell biological techniques (confocal laser scanning microscopy, scanning electron microscopy and flow cytometry); and (iii) we developed a fast, cheap and quantitative electrochemical detection method for apoptotic cells with results in full agreement with those obtained by flow cytometry.There is a great demand to develop novel techniques that allow useful and complete monitoring of apoptosis, which is a key factor of several diseases and a target for drug development. Here, we present the use of a novel dual electrochemical/optical label for the detection and study of apoptosis. We combined the specificity of Annexin-V for phosphatidylserine, a phospholipid expressed in the outer membrane of apoptotic cells, with the optical and electrochemical properties of quantum dots to create a more efficient label. Using this conjugate we addressed three important issues: (i) we made the labeling of apoptotic cells faster (30 min) and easier; (ii) we fully characterized the samples by common cell biological techniques (confocal laser scanning microscopy, scanning electron microscopy and flow cytometry); and (iii) we developed a fast, cheap and quantitative electrochemical detection method for apoptotic cells with results in full agreement with those obtained by flow cytometry. Electronic supplementary information (ESI) available: Optical microscopy images of apoptotic induced cell cultures at different times and negative control of flow cytometry. See DOI: 10.1039/c4nr07191c
Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

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

Koopman, R P; Langlois, R G; Nasarabadi, S

2002-04-17

This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flowmore » cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.« less
Microfluidic Mixing Technology for a Universal Health Sensor

NASA Technical Reports Server (NTRS)

Chan, Eugene Y.; Bae, Candice

2009-01-01

A highly efficient means of microfluidic mixing has been created for use with the rHEALTH sensor an elliptical mixer and passive curvilinear mixing patterns. The rHEALTH sensor provides rapid, handheld, complete blood count, cell differential counts, electrolyte measurements, and other lab tests based on a reusable, flow-based microfluidic platform. These geometries allow for cleaning in a reusable manner, and also allow for complete mixing of fluid streams. The microfluidic mixing is performed by flowing two streams of fluid into an elliptical or curvilinear design that allows the combination of the flows into one channel. The mixing is accomplished by either chaotic advection around micro - fluidic loops. All components of the microfluidic chip are flow-through, meaning that cleaning solution can be introduced into the chip to flush out cells, plasma proteins, and dye. Tests were performed on multiple chip geometries to show that cleaning is efficient in any flowthrough design. The conclusion from these experiments is that the chip can indeed be flushed out with microliter volumes of solution and biological samples are cleaned readily from the chip with minimal effort. The technology can be applied in real-time health monitoring at patient s bedside or in a doctor s office, and real-time clinical intervention in acute situations. It also can be used for daily measurement of hematocrit for patients on anticoagulant drugs, or to detect acute myocardial damage outside a hospital.

Flow Cytometry in Diagnosis of Myelomatous Pleural Effusion: A Case Report.

PubMed

Arora, Parul; Gupta, Sanjeev Kumar; Mallik, Nabhajit; Mittal, Reena; Sharma, Om Dutt; Kumar, Lalit

2016-06-01

Plasma cell myeloma is a multifocal plasma cell neoplasm associated with increased monoclonal protein in serum and/or urine. Pleural effusions in patients with myeloma are uncommon (6 %). However, effusions due to direct infiltration of the pleura by plasma cells (myelomatous pleural effusion) are extremely rare (<1 %) and usually seen with IgA myeloma. The diagnosis of such cases requires pleural fluid cytology, electrophoresis or pleural biopsy. We present a case of myelomatous pleural effusion diagnosed using flow cytometry immunophenotyping in addition to the pleural fluid cytology. A 45 year old female was diagnosed as plasma cell myeloma (IgG kappa) in 2007. She received multiple lines of therapy during the course of her treatment including thalidomide, dexamethasone, lenalidomide, bortezomib, and doxorubicin based regimens. However, the patient had progressive extramedullary disease and developed pleural effusion in 2014. Cytological examination of the pleural fluid showed degenerative changes. Few preserved areas showed mononuclear cells including morphologically abnormal plasma cells. Immunophenotyping of these cells by flow cytometry revealed a pattern indicating neoplastic plasma cells. There was expression of CD38, CD138, and CD56, with absence of CD19, CD10 and CD45. This confirmed the diagnosis of myelomatous pleural effusion. Subsequently, the patient was offered a dexamethasone, cyclophosphamide, etoposide and cisplatin based regimen but, she declined further treatment and succumbed to her disease 3 months later. Myelomatous pleural effusion is a rare complication of plasma cell myeloma. Flow cytometry can be used as an adjunctive technique in its diagnosis particularly in cases with equivocal cytology and electrophoresis findings.
Expanding the potential of standard flow cytometry by extracting fluorescence lifetimes from cytometric pulse shifts

PubMed Central

Cao, Ruofan; Naivar, Mark A; Wilder, Mark; Houston, Jessica P

2014-01-01

Fluorescence lifetime measurements provide information about the fluorescence relaxation, or intensity decay, of organic fluorophores, fluorescent proteins, and other inorganic molecules that fluoresce. The fluorescence lifetime is emerging in flow cytometry and is helpful in a variety of multiparametric, single cell measurements because it is not impacted by nonlinearity that can occur with fluorescence intensity measurements. Yet time-resolved cytometry systems rely on major hardware modifications making the methodology difficult to reproduce. The motivation of this work is, by taking advantage of the dynamic nature of flow cytometry sample detection and applying digital signal processing methods, to measure fluorescence lifetimes using an unmodified flow cytometer. We collect a new lifetime-dependent parameter, referred to herein as the fluorescence-pulse-delay (FPD), and prove it is a valid representation of the average fluorescence lifetime. To verify we generated cytometric pulses in simulation, with light emitting diode (LED) pulsation, and with true fluorescence measurements of cells and microspheres. Each pulse is digitized and used in algorithms to extract an average fluorescence lifetime inherent in the signal. A range of fluorescence lifetimes is measurable with this approach including standard organic fluorophore lifetimes (∼1 to 22 ns) as well as small, simulated shifts (0.1 ns) under standard conditions (reported herein). This contribution demonstrates how digital data acquisition and signal processing can reveal time-dependent information foreshadowing the exploitation of full waveform analysis for quantification of similar photo-physical events within single cells. © 2014 The Authors. Published by Wiley Periodicals, Inc. PMID:25274073
RNA Flow Cytometry Using the Branched DNA Technique.

PubMed

Soh, Kah Teong; Wallace, Paul K

2018-01-01

The systematic modulation of mRNA and proteins governs the complicated and intermingled biological functions of our cells. Traditionally, transcriptomic technologies such as DNA microarray and RNA-Seq have been used to identify, characterize, and profile gene expression data. These are, however, considered bulk methods as they are unable to measure gene expression at the single-cell level, unless the cells are pre-sorted. Branched DNA is a flow cytometry-based detection platform that has been developed recently to measure mRNA at the single-cell level. Originally adapted from microscopy, the current system has been modified to achieve compatibility with the detection of surface and intracellular antigens using monoclonal antibodies conjugated to fluorochromes, thus permitting simultaneous detection of mRNAs and proteins. The Branched DNA method offers a variety of advantages when compared to traditional or standard methods used for the quantification of mRNA, such as (a) the detection of specific mRNA on a per cell basis, (b) an alternate detection tool when the measurement of a protein is technically infeasible (i.e., no quality antibody exists) or the epitope is not assessable, and (c) correlate the analysis of mRNA with protein. Compared to earlier attempts at measuring nucleic acid by flow cytometry, the hybridization temperature applied in the Branched DNA assay is much lower, thus preserving the integrity of cellular structures for further characterization. It also has greatly increased specificity and sensitivity. Here, we provide detailed instruction for performing the Branched DNA method using it in a model system to correlate the expression of CD8 mRNA and CD8 protein by flow cytometry.
Comparison of cell counting methods in rodent pulmonary toxicity studies: automated and manual protocols and considerations for experimental design

PubMed Central

Zeidler-Erdely, Patti C.; Antonini, James M.; Meighan, Terence G.; Young, Shih-Houng; Eye, Tracy J.; Hammer, Mary Ann; Erdely, Aaron

2016-01-01

Pulmonary toxicity studies often use bronchoalveolar lavage (BAL) to investigate potential adverse lung responses to a particulate exposure. The BAL cellular fraction is counted, using automated (i.e. Coulter Counter®), flow cytometry or manual (i.e. hemocytometer) methods, to determine inflammatory cell influx. The goal of the study was to compare the different counting methods to determine which is optimal for examining BAL cell influx after exposure by inhalation or intratracheal instillation (ITI) to different particles with varying inherent pulmonary toxicities in both rat and mouse models. General findings indicate that total BAL cell counts using the automated and manual methods tended to agree after inhalation or ITI exposure to particle samples that are relatively nontoxic or at later time points after exposure to a pneumotoxic particle when the response resolves. However, when the initial lung inflammation and cytotoxicity was high after exposure to a pneumotoxic particle, significant differences were observed when comparing cell counts from the automated, flow cytometry and manual methods. When using total BAL cell count for differential calculations from the automated method, depending on the cell diameter size range cutoff, the data suggest that the number of lung polymorphonuclear leukocytes (PMN) varies. Importantly, the automated counts, regardless of the size cutoff, still indicated a greater number of total lung PMN when compared with the manual method, which agreed more closely with flow cytometry. The results suggest that either the manual method or flow cytometry would be better suited for BAL studies where cytotoxicity is an unknown variable. PMID:27251196
Evaluation of flow cytometric HIT assays in relation to an IgG-Specific immunoassay and clinical outcome.

PubMed

Kerényi, Adrienne; Beke Debreceni, Ildikó; Oláh, Zsolt; Ilonczai, Péter; Bereczky, Zsuzsanna; Nagy, Béla; Muszbek, László; Kappelmayer, János

2017-09-01

Heparin-induced thrombocytopenia (HIT) is a severe side effect of heparin treatment caused by platelet activating IgG antibodies generated against the platelet factor 4 (PF4)-heparin complex. Thrombocytopenia and thrombosis are the leading clinical symptoms of HIT. The clinical pretest probability of HIT was evaluated by the 4T score system. Laboratory testing of HIT was performed by immunological detection of antibodies against PF4-heparin complex (EIA) and two functional assays. Heparin-dependent activation of donor platelets by patient plasma was detected by flow cytometry. Increased binding of Annexin-V to platelets and elevated number of platelet-derived microparticles (PMP) were the indicators of platelet activation. EIA for IgG isotype HIT antibodies was performed in 405 suspected HIT patients. Based on negative EIA results, HIT was excluded in 365 (90%) of cases. In 40 patients with positive EIA test result functional tests were performed. Platelet activating antibodies were detected in 17 cases by Annexin V binding. PMP count analysis provided nearly identical results. The probability of a positive flow cytometric assay result was higher in patients with elevated antibody titer. 71% of patients with positive EIA and functional assay had thrombosis. EIA is an important first line laboratory test in the diagnosis of HIT; however, HIT must be confirmed by a functional test. Annexin V binding and PMP assays using flow cytometry are functional HIT tests convenient in a clinical diagnostic laboratory. The positive results of functional assays may predict the onset of thrombosis. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.
Comparison of cell counting methods in rodent pulmonary toxicity studies: automated and manual protocols and considerations for experimental design.

PubMed

Zeidler-Erdely, Patti C; Antonini, James M; Meighan, Terence G; Young, Shih-Houng; Eye, Tracy J; Hammer, Mary Ann; Erdely, Aaron

2016-08-01

Pulmonary toxicity studies often use bronchoalveolar lavage (BAL) to investigate potential adverse lung responses to a particulate exposure. The BAL cellular fraction is counted, using automated (i.e. Coulter Counter®), flow cytometry or manual (i.e. hemocytometer) methods, to determine inflammatory cell influx. The goal of the study was to compare the different counting methods to determine which is optimal for examining BAL cell influx after exposure by inhalation or intratracheal instillation (ITI) to different particles with varying inherent pulmonary toxicities in both rat and mouse models. General findings indicate that total BAL cell counts using the automated and manual methods tended to agree after inhalation or ITI exposure to particle samples that are relatively nontoxic or at later time points after exposure to a pneumotoxic particle when the response resolves. However, when the initial lung inflammation and cytotoxicity was high after exposure to a pneumotoxic particle, significant differences were observed when comparing cell counts from the automated, flow cytometry and manual methods. When using total BAL cell count for differential calculations from the automated method, depending on the cell diameter size range cutoff, the data suggest that the number of lung polymorphonuclear leukocytes (PMN) varies. Importantly, the automated counts, regardless of the size cutoff, still indicated a greater number of total lung PMN when compared with the manual method, which agreed more closely with flow cytometry. The results suggest that either the manual method or flow cytometry would be better suited for BAL studies where cytotoxicity is an unknown variable.
Identification of contact and respiratory sensitizers using flow cytometry

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

Goutet, Michele; Pepin, Elsa; Langonne, Isabelle

2005-06-15

Identification of the chemicals responsible for respiratory and contact allergies in the industrial area is an important occupational safety issue. This study was conducted in mice to determine whether flow cytometry is an appropriate method to analyze and differentiate the specific immune responses to the respiratory sensitizer trimellitic anhydride (TMA) and to the contact sensitizer dinitrochlorobenzene (DNCB) used at concentrations with comparable immunogenic potential. Mice were exposed twice on the flanks (days 0, 5) to 10% TMA or 1% DNCB and challenged three times on the ears (days 10, 11, 12) with 2.5% TMA or 0.25% DNCB. Flow cytometry analysesmore » were conducted on draining lymph node cells harvested on days 13 and 18. Comparing TMA and DNCB immune responses on day 13, we found obvious differences that persisted for most of them on day 18. An increased proportion of IgE+ cells correlated to total serum IgE level and an enhancement of MHC II molecule expression were observed in the lymph node B lymphocytes from TMA-treated mice. The percentage of IL-4-producing CD4+ lymphocytes and the IL-4 receptor expression were clearly higher following TMA exposure. In contrast, higher proportions of IL-2-producing cells were detected in CD4+ and CD8+ cells from DNCB-treated mice. Both chemicals induced a significant increase in the percentage of IFN-{gamma}-producing cells among CD8+ lymphocytes but to a greater proportion following TMA treatment. In conclusion, this study encourages the use of flow cytometry to discriminate between contact and respiratory sensitizers by identifying divergent expression of immune response parameters.« less
Expression of {beta}{sub 1} integrins in human endometrial stromal and decidual cells

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

Shiokawa, Shigetatsu; Yoshimura, Yasunori; Nakamura, Yukio

The present study was undertaken to investigate the expression of {beta}{sub 1} integrins in human endometrium and decidua using flow cytometry, immunohistochemistry, and immunoprecipitation. Fluorescence-activated flow cytometry demonstrated the greater expression of the {beta}{sub 1}, {alpha}{sub 1}, {alpha}{sub 2}, and {alpha}{sub 5} subunits of the {beta}{sub 1} integrin family in cultured stromal cells from the midsecretory phase, than in those of the early proliferative phase. The addition of estradiol (E{sub 2}) and progesterone (P) to cultured stromal cells in the early proliferative phase increased the expression of {beta}{sub 1} integrins in vitro. Flow cytometry also demonstrated the expression of themore » {beta}{sub 1}, {alpha}{sub 1}, {alpha}{sub 2}, {alpha}{sub 3}, {alpha}{sub 5}, and {alpha}{sub 6} subunits of {beta}{sub 1} integrin family in cultured decidual cells, and the enriched-fraction of prolactin (PRL)-producing decidual cells isolated by Percoll gradients showed high levels of {beta}{sub 1} integrins expression. Immunohistochemistry confirmed the {beta}{sub 1} integrin cell surface phenotypes in cultured decidual cells observed by flow cytometry. In summary, the present study demonstrated that endometrial stromal and decidual cells expressed {beta}{sub 1} integrin subunits at their surfaces. The expression exhibited a variability throughout the menstrual cycles, being predominantly detected in the secretory phase, and was maintained highly in the decidua. Thus, {beta}{sub 1} integrins in human endometrium and decidua may be important in mediating the organization of extracellular matrix proteins derived from embryos during the early stage of implantation. 43 refs., 7 figs., 2 tabs.« less
Interference of flavonoids with fluorescent intracellular probes: methodological implications in the evaluation of the oxidative burst by flow cytometry.

PubMed

Peluso, Ilaria; Manafikhi, Husseen; Reggi, Raffaella; Palmery, Maura

2014-08-01

The evaluation of oxidative burst is particularly relevant in many pathological and subclinical conditions. Flow cytometry provides quick and accurate measures of the reactive oxygen species production by leukocytes in most situations. However, spurious results, related to probes' efflux may be observed in several instances. Many factors affect the evaluation of the oxidative burst with fluorescent probes that require intracellular deacetylation and could be substrate of the multidrug resistance proteins (MDR). After discussing the implications of the efflux of fluorophores in the normalization strategies in flow cytometry assays, we have pointed out the possible interference of flavonoids with fluorescet probes' staining and signal. We have also reviewed the results from human intervention studies regarding the evaluation of oxidative burst with these probes. In vitro, at concentrations close to post-ingestion circulating levels, some flavonoids and their metabolites could interfere with probes' staining and fluorescence signal through different mechanisms, such as the inhibition of esterases, the modulation of the MDR-mediate efflux of probe and the inhibition of the oxidation of probe. These effects may explain the contrasting results obtained by human intervention studies. Finally, also inflammatory state or the use of drugs substrate of MDR proteins could affect the evaluation of the oxidative burst with intracellular probes. © 2014 International Society for Advancement of Cytometry.
Determination of viability of Aeromonas hydrophila in increasing concentrations of sodium chloride at different temperatures by flow cytometry and plate count technique.

PubMed

Pianetti, Anna; Manti, Anita; Boi, Paola; Citterio, Barbara; Sabatini, Luigia; Papa, Stefano; Rocchi, Marco Bruno Luigi; Bruscolini, Francesca

2008-10-31

Aeromonads in waters and foods can represent a risk to human health. Factors such as sodium chloride concentration and temperature can affect growth and viability of several food and water-borne pathogens. The behaviour of an Aeromonas hydrophila strain in the presence of 1.7%, 3.4% and 6% NaCl concentrations at 24 degrees C and 4 degrees C was studied over a 188 day period. Viability and membrane potential were assessed by flow cytometry; growth was evaluated by plate count technique. Flow cytometry evidenced that A. hydrophila retained viability over the period although varying according to temperature and salt concentrations. Colony Forming Units were generally lower in number than viable cells especially in the presence of 6% NaCl, indicating the occurrence of stressed cells which maintain metabolic activity yet are not able to grow on agar plates. In conclusion, A. hydrophila showed a long-term halotolerance even at elevated (6%) NaCl concentrations and a lesser sensitivity to salt at low temperature; therefore, low temperature and salt, which are two important factors limiting bacterial growth, do not assure safety in the case of high initial contamination. Finally, cytometry appears a valid tool for the rapid detection of the viability of pathogenic bacteria in food and environmental matrices to control and prevent health risks.
Laser doppler blood flow imaging using a CMOS imaging sensor with on-chip signal processing.

PubMed

He, Diwei; Nguyen, Hoang C; Hayes-Gill, Barrie R; Zhu, Yiqun; Crowe, John A; Gill, Cally; Clough, Geraldine F; Morgan, Stephen P

2013-09-18

The first fully integrated 2D CMOS imaging sensor with on-chip signal processing for applications in laser Doppler blood flow (LDBF) imaging has been designed and tested. To obtain a space efficient design over 64 × 64 pixels means that standard processing electronics used off-chip cannot be implemented. Therefore the analog signal processing at each pixel is a tailored design for LDBF signals with balanced optimization for signal-to-noise ratio and silicon area. This custom made sensor offers key advantages over conventional sensors, viz. the analog signal processing at the pixel level carries out signal normalization; the AC amplification in combination with an anti-aliasing filter allows analog-to-digital conversion with a low number of bits; low resource implementation of the digital processor enables on-chip processing and the data bottleneck that exists between the detector and processing electronics has been overcome. The sensor demonstrates good agreement with simulation at each design stage. The measured optical performance of the sensor is demonstrated using modulated light signals and in vivo blood flow experiments. Images showing blood flow changes with arterial occlusion and an inflammatory response to a histamine skin-prick demonstrate that the sensor array is capable of detecting blood flow signals from tissue.
A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins

NASA Astrophysics Data System (ADS)

Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.

2011-09-01

Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells.
A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins

PubMed Central

Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.

2011-01-01

Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells. PMID:21974603
A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins.

PubMed

Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R

2011-09-01

Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells. © 2011 American Institute of Physics
Microfluidic free-flow zone electrophoresis and isotachophoresis using carbon black nano-composite PDMS sidewall membranes.

PubMed

Fu, Xiaotong; Mavrogiannis, Nicholas; Ibo, Markela; Crivellari, Francesca; Gagnon, Zachary R

2017-01-01

We present a new type of free-flow electrophoresis (FFE) device for performing on-chip microfluidic isotachophoresis and zone electrophoresis. FFE is performed using metal gallium electrodes, which are isolated from a main microfluidic flow channel using thin micron-scale polydimethylsiloxane/carbon black (PDMS/CB) composite membranes integrated directly into the sidewalls of the microfluidic channel. The thin membrane allows for field penetration and effective electrophoresis, but serves to prevent bubble generation at the electrodes from electrolysis. We experimentally demonstrate the ability to use this platform to perform on-chip free-flow electrophoretic separation and isotachophoretic concentration. Due to the small size and simple fabrication procedure, this PDMS/CB platform could be used as a part of an on-chip upstream sample preparation toolkit for portable microfluidic diagnostic applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Application of flow cytometry to wine microorganisms.

PubMed

Longin, Cédric; Petitgonnet, Clément; Guilloux-Benatier, Michèle; Rousseaux, Sandrine; Alexandre, Hervé

2017-04-01

Flow cytometry (FCM) is a powerful technique allowing detection and enumeration of microbial populations in food and during food process. Thanks to the fluorescent dyes used and specific probes, FCM provides information about cell physiological state and allows enumeration of a microorganism in a mixed culture. Thus, this technique is increasingly used to quantify pathogen, spoilage microorganisms and microorganisms of interest. Since one decade, FCM applications to the wine field increase greatly to determine population and physiological state of microorganisms performing alcoholic and malolactic fermentations. Wine spoilage microorganisms were also studied. In this review we briefly describe FCM principles. Next, a deep revision concerning enumeration of wine microorganisms by FCM is presented including the fluorescent dyes used and techniques allowing a yeast and bacteria species specific enumeration. Then, the last chapter is dedicated to fluorescent dyes which are used to date in fluorescent microscopy but applicable in FCM. This chapter also describes other interesting "future" techniques which could be applied to study the wine microorganisms. Thus, this review seeks to highlight the main advantages of the flow cytometry applied to wine microbiology. Copyright Â© 2016 Elsevier Ltd. All rights reserved.
Flow cytometry analysis of hormone receptors on human peripheral blood mononuclear cells to identify stress-induced neuroendocrine effects

NASA Technical Reports Server (NTRS)

Meehan, R. T.

1986-01-01

Understanding the role of circulating peptide hormones in the pathogenesis of space-flight induced disorders would be greatly facilitated by a method which monitors chronic levels of hormones and their effects upon in vivo cell physiology. Single and simultaneous multiparameter flow cytometry analysis was employed to identify subpopulations of mononuclear cells bearing receptors for ACTH, Endorphin, and Somatomedin-C using monoclonal antibodies and monospecific antisera with indirect immunofluorescence. Blood samples were obtained from normal donors and subjects participating in decompression chamber studies (acute stress), medical student academic examination (chronic stress), and a drug study (Dexamethasone). Preliminary results indicate most ACTH and Endorphin receptor positive cells are monocytes and B-cells, exhibit little diurnal variation but the relative percentages of receptor positive cells are influenced by exposure to various stressors and ACTH inhibition. This study demonstrates the capability of flow cytometry analysis to study cell surface hormone receptor regulation which should allow insight into neuroendocrine modulation of the immune and other cellular systems during exposure to stress or microgravity.
Detection of internal structure by scattered light intensity: Application to kidney cell sorting

NASA Technical Reports Server (NTRS)

Goolsby, C. L.; Kunze, M. E.

1985-01-01

Scattered light measurements in flow cytometry were sucessfully used to distinguish cells on the basis of differing morphology and internal structure. Differences in scattered light patterns due to changes in internal structure would be expected to occur at large scattering angles. Practically, the results of these calculations suggest that in experimental situations an array of detectors would be useful. Although in general the detection of the scattered light intensity at several intervals within the 10 to 60 region would be sufficient, there are many examples where increased sensitivity could be acheived at other angles. The ability to measure at many different angular intervals would allow the experimenter to empirically select the optimum intervals for the varying conditions of cell size, N/C ratio, granule size and internal structure from sample to sample. The feasibility of making scattered light measurements at many different intervals in flow cytometry was demonstrated. The implementation of simplified versions of these techniques in conjunction with independant measurements of cell size could potentially improve the usefulness of flow cytometry in the study of the internal structure of cells.
Tips and tricks for flow cytometry-based analysis and counting of microparticles.

PubMed

Poncelet, Philippe; Robert, Stéphane; Bailly, Nicolas; Garnache-Ottou, Francine; Bouriche, Tarik; Devalet, Bérangère; Segatchian, Jerard H; Saas, Philippe; Mullier, François

2015-10-01

Submicron-sized extra-cellular vesicles generated by budding from the external cell membranes, microparticles (MPs) are important actors in transfusion as well as in other medical specialties. After briefly positioning their role in the characterization of labile blood products, this technically oriented chapter aims to review practical points that need to be considered when trying to use flow cytometry for the analysis, characterization and absolute counting of MP subsets. Subjects of active discussions relative to instrumentation will include the choice of the trigger parameter, possible standardization approaches requiring instrument quality-control, origin and control of non-specific background and of coincidence artifacts, choice of the type of electronic signals, optimal sheath fluid and sample speed. Questions related to reagents will cover target antigens and receptors, multi-color reagents, negative controls, enumeration of MPs and limiting artifacts due to unexpected (micro-) coagulation of plasma samples. Newly detected problems are generating innovative solutions and flow cytometry will continue to remain the technology of choice for the analysis of MPs, in the domain of transfusion as well as in many diverse specialties. Copyright © 2015 Elsevier Ltd. All rights reserved.
Differences in leukocyte differentiation molecule abundances on domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis) neutrophils identified by flow cytometry.

PubMed

Highland, Margaret A; Schneider, David A; White, Stephen N; Madsen-Bouterse, Sally A; Knowles, Donald P; Davis, William C

2016-06-01

Although both domestic sheep (DS) and bighorn sheep (BHS) are affected by similar respiratory bacterial pathogens, experimental and field data indicate BHS are more susceptible to pneumonia. Cross-reactive monoclonal antibodies (mAbs) for use in flow cytometry (FC) are valuable reagents for interspecies comparative immune system analyses. This study describes cross-reactive mAbs that recognize leukocyte differentiation molecules (LDMs) and major histocompatibility complex antigens on DS and BHS leukocytes. Characterization of multichannel eosinophil autofluorescence in this study permitted cell-type specific gating of granulocytes for evaluating LDMs, specifically on neutrophils, by single-label FC. Evaluation of relative abundances of LDMs by flow cytometry revealed greater CD11a, CD11b, CD18 (β2 integrins) and CD 172a (SIRPα) on DS neutrophils and greater CD14 (lipopolysaccharide receptor) on BHS neutrophils. Greater CD25 (IL-2) was identified on BHS lymphocytes following Concavalin A stimulation. While DS and BHS have similar total peripheral blood leukocyte counts, BHS have proportionately more neutrophils. Published by Elsevier Ltd.

A flow cytometry-optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments.

PubMed

Norman, Anders; Hansen, Lars Hestbjerg; Sørensen, Søren J

2006-02-28

Whole-cell biosensors have become popular tools for detection of ecotoxic compounds in environmental samples. We have developed an assay optimized for flow cytometry with detection of genotoxic compounds in mind. The assay features extended pre-incubation and a cell density of only 10(6)-10(7) cells/mL, and proved far more sensitive than a previously published assay using the same biosensor strain. By applying the SOS-green fluorescent protein (GFP) whole-cell biosensor directly to soil microcosms we were also able to evaluate both the applicability and sensitivity of a biosensor based on SOS-induction in whole soil samples. Soil microcosms were spiked with a dilution-series of crude broth extract from the mitomycin C-producing streptomycete Streptomyces caespitosus. Biosensors extracted from these microcosms after 1 day of incubation at 30 degrees C were easily distinguished from extracts of non-contaminated soil particles when using flow cytometry, and induction of the biosensor by mitomycin C was detectable at concentrations as low as 2.5 ng/g of soil.
Biology and flow cytometry of proangiogenic hematopoietic progenitors cells.

PubMed

Rose, Jonathan A; Erzurum, Serpil; Asosingh, Kewal

2015-01-01

During development, hematopoiesis and neovascularization are closely linked to each other via a common bipotent stem cell called the hemangioblast that gives rise to both hematopoietic cells and endothelial cells. In postnatal life, this functional connection between the vasculature and hematopoiesis is maintained by a subset of hematopoietic progenitor cells endowed with the capacity to differentiate into potent proangiogenic cells. These proangiogenic hematopoietic progenitors comprise a specific subset of bone marrow (BM)-derived cells that homes to sites of neovascularization and possess potent paracrine angiogenic activity. There is emerging evidence that this subpopulation of hematopoietic progenitors plays a critical role in vascular health and disease. Their angiogenic activity is distinct from putative "endothelial progenitor cells" that become structural cells of the endothelium by differentiation into endothelial cells. Proangiogenic hematopoietic progenitor cell research requires multidisciplinary expertise in flow cytometry, hematology, and vascular biology. This review provides a comprehensive overview of proangiogenic hematopoietic progenitor cell biology and flow cytometric methods to detect these cells in the peripheral blood circulation and BM. © 2014 International Society for Advancement of Cytometry.
Real Time Detection of Protein Trafficking with High Throughput Flow Cytometry (HTFC) and Fluorogen Activating Protein (FAP) Base Biosensor

PubMed Central

Wu, Yang; Tapia, Phillip H.; Jarvik, Jonathan; Waggoner, Alan S.; Sklar, Larry A.

2014-01-01

We combined fluorogen activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform allows drug discovery for trafficking receptors, such as G-protein coupled receptors, receptor tyrosine kinases and ion channels, that were previously not suitable for high throughput screening by flow cytometry.. The system has been validated using the β2-adrenergic receptor (β2AR) system and extended to other GPCRs. When a chemical library containing ~1,200 off-patent drugs was screened against cells expressing FAP tagged β2AR, all known β2AR active ligands in the library were successfully identified, together with a few compounds that were later confirmed to regulate receptor internalization in a non-traditional manner. The unexpected discovery of new ligands by this approach indicates the potential of using this protocol for GPCR de-orphanization. In addition, screens of multiplexed targets promise improved efficiency with minor protocol modification. PMID:24510772
[Evaluation of transfection effectiveness using fluorescein-labelled oligonucleotides and entraster-R siRNA transfection into Plasmodium falciparum].

PubMed

Zhou, Hong-Chang; Gao, Yu-Hui; Shao, Sheng-Wen; Zhang, Hui; Zhang, Ting

2013-12-01

The cultured Plasmodium falciparum parasites were synchronized twice by 5% sorbitol treatment twice (8-hour window), and then incubated at 37 degrees C for 16 h. Parasites were transfected with fluorescein-labelled oligonucleotides (group A) or fluorescein-labelled oligonucleotides+Entranster-R siRNA transfection reagent (group B). After 5 h a part of parasites was evaluated by fluorescence microscopy and flow cytometry. The rest of parasites were washed with RPMI 1640 medium, and then incubated with 500 microl new medium containing 2% fresh erythrocytes for another 12 h, and detected by flow cytometry. The fluorescein-labelled oligonucleotides were localized in erythrocytes in group B, but nearly no fluorescence was observed for group A. Flow cytometry analysis indicated that the transfection efficiency of group B [(47.40 +/- 3.39)%] was higher than that of group A [(0.60 +/- 0.27)%]. In the second cell cycle, the transfection efficiency in group B was (26.85 +/- 2.90)%, while that of group A was nearly zero. The results indicated that Entranster-R siRNA transfection reagent may increase the oligonucleotides transfection efficiency.
Ultrafast Microfluidic Cellular Imaging by Optical Time-Stretch.

PubMed

Lau, Andy K S; Wong, Terence T W; Shum, Ho Cheung; Wong, Kenneth K Y; Tsia, Kevin K

2016-01-01

There is an unmet need in biomedicine for measuring a multitude of parameters of individual cells (i.e., high content) in a large population efficiently (i.e., high throughput). This is particularly driven by the emerging interest in bringing Big-Data analysis into this arena, encompassing pathology, drug discovery, rare cancer cell detection, emulsion microdroplet assays, to name a few. This momentum is particularly evident in recent advancements in flow cytometry. They include scaling of the number of measurable colors from the labeled cells and incorporation of imaging capability to access the morphological information of the cells. However, an unspoken predicament appears in the current technologies: higher content comes at the expense of lower throughput, and vice versa. For example, accessing additional spatial information of individual cells, imaging flow cytometers only achieve an imaging throughput ~1000 cells/s, orders of magnitude slower than the non-imaging flow cytometers. In this chapter, we introduce an entirely new imaging platform, namely optical time-stretch microscopy, for ultrahigh speed and high contrast label-free single-cell (in a ultrafast microfluidic flow up to 10 m/s) imaging and analysis with an ultra-fast imaging line-scan rate as high as tens of MHz. Based on this technique, not only morphological information of the individual cells can be obtained in an ultrafast manner, quantitative evaluation of cellular information (e.g., cell volume, mass, refractive index, stiffness, membrane tension) at nanometer scale based on the optical phase is also possible. The technology can also be integrated with conventional fluorescence measurements widely adopted in the non-imaging flow cytometers. Therefore, these two combinatorial and complementary measurement capabilities in long run is an attractive platform for addressing the pressing need for expanding the "parameter space" in high-throughput single-cell analysis. This chapter provides the general guidelines of constructing the optical system for time stretch imaging, fabrication and design of the microfluidic chip for ultrafast fluidic flow, as well as the image acquisition and processing.
CytometryML with DICOM and FCS

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2018-02-01

Abstract: Flow Cytometry Standard, FCS, and Digital Imaging and Communications in Medicine standard, DICOM, are based on extensive, superb domain knowledge, However, they are isolated systems, do not take advantage of data structures, require special programs to read and write the data, lack the capability to interoperate or work with other standards and FCS lacks many of the datatypes necessary for clinical laboratory data. The large overlap between imaging and flow cytometry provides strong evidence that both modalities should be covered by the same standard. Method: The XML Schema Definition Language, XSD 1.1 was used to translate FCS and/or DICOM objects. A MIFlowCyt file was tested with published values. Results: Previously, a significant part of an XML standard based upon a combination of FCS and DICOM has been implemented and validated with MIFlowCyt data. Strongly typed translations of FCS keywords have been constructed in XML. These keywords contain links to their DICOM and FCS equivalents.
Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

PubMed

Juzwa, W; Duber, A; Myszka, K; Białas, W; Czaczyk, K

2016-09-01

In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.
A critical evaluation of a flow cytometer used for detecting enterococci in recreational waters.

PubMed

King, Dawn N; Brenner, Kristen P; Rodgers, Mark R

2007-06-01

The current U. S. Environmental Protection Agency-approved method for enterococci (Method 1600) in recreational water is a membrane filter (MF) method that takes 24 hours to obtain results. If the recreational water is not in compliance with the standard, the risk of exposure to enteric pathogens may occur before the water is identified as hazardous. Because flow cytometry combined with specific fluorescent antibodies has the potential to be used as a rapid detection method for microorganisms, this technology was evaluated as a rapid, same-day method to detect enterococci in bathing beach waters. The flow cytometer chosen for this study was a laser microbial detection system designed to detect labeled antibodies. A comparison of MF counts with flow cytometry counts of enterococci in phosphate buffer and sterile-filtered recreational water showed good agreement between the two methods. However, when flow cytometry was used, the counts were several orders of magnitude higher than the MF counts with no correlation to Enterococcus spike concentrations. The unspiked sample controls frequently had higher counts than the samples spiked with enterococci. Particles within the spiked water samples were probably counted as target cells by the flow cytometer because of autofluorescence or non-specific adsorption of antibody and carryover to subsequent samples. For these reasons, this technology may not be suitable for enterococci detection in recreational waters. Improvements in research and instrument design that will eliminate high background and carryover may make this a viable technology in the
Investigation of the mechanism of action of 3-(4-bromophenyl)-5-acyloxymethyl-2,5-dihydrofuran-2-one against Candida albicans by flow cytometry.

PubMed

Vale-Silva, Luís A; Buchta, Vladimír; Vokurková, Doris; Pour, Milan

2006-05-01

The mechanism of action of the antifungal agent 3-(4-bromophenyl)-5-acyloxymethyl-2,5-dihydrofuran-2-one against Candida albicans was investigated by flow cytometry, using propidium iodide, DiBAC4(3), and FUN-1 as the fluorescent dyes. A related but less active agent, together with amphotericin B and fluconazole, was tested in parallel for comparison of the results. The incrustoporine derivative was found to have a potent fungicidal activity on C. albicans, resulting in damage of cell membrane.
Quaternized wood as sorbent for hexavalent chromium.

PubMed

Low, K S; Lee, C K; Lee, C Y

2001-01-01

The potential of quaternized wood (QW) chips in removing hexavalent chromium from synthetic solution and chrome waste under both batch and continuous-flow conditions was investigated. Sorption was found to be dependent on pH, metal concentration, and temperature. QW chips provide higher sorption capacity and wider pH range compared with untreated wood chips. The equilibrium data could be fitted into the Langmuir isotherm model, and maximum sorption capacities were calculated to be 27.03 and 25.77 mg/g in synthetic chromate solution and chrome waste, respectively. The presence of sulfate in high concentration appeared to suppress the uptake of chromium by QW chips. Column studies showed that bed depth influenced the breakthrough time greatly whereas flow rate of influent had little effect on its sorption on the column.
Peripheral T-cell lymphomas of follicular helper T-cell type frequently display an aberrant CD3(-/dim)CD4(+) population by flow cytometry: an important clue to the diagnosis of a Hodgkin lymphoma mimic.

PubMed

Alikhan, Mir; Song, Joo Y; Sohani, Aliyah R; Moroch, Julien; Plonquet, Anne; Duffield, Amy S; Borowitz, Michael J; Jiang, Liuyan; Bueso-Ramos, Carlos; Inamdar, Kedar; Menon, Madhu P; Gurbuxani, Sandeep; Chan, Ernest; Smith, Sonali M; Nicolae, Alina; Jaffe, Elaine S; Gaulard, Philippe; Venkataraman, Girish

2016-10-01

Nodal follicular helper T-cell-derived lymphoproliferations (specifically the less common peripheral T-cell lymphomas of follicular type) exhibit a spectrum of histologic features that may mimic reactive hyperplasia or Hodgkin lymphoma. Even though angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma of follicular type share a common biologic origin from follicular helper T-cells and their morphology has been well characterized, flow cytometry of peripheral T-cell lymphomas of follicular type has not been widely discussed as a tool for identifying this reactive hyperplasia/Hodgkin lymphoma mimic. We identified 10 peripheral T-cell lymphomas of follicular type with available flow cytometry data from five different institutions, including two cases with peripheral blood evaluation. For comparison, we examined flow cytometry data for 8 classical Hodgkin lymphomas (including 1 lymphocyte-rich classical Hodgkin lymphoma), 15 nodular lymphocyte predominant Hodgkin lymphomas, 15 angioimmunoblastic T-cell lymphomas, and 26 reactive nodes. Lymph node histology and flow cytometry data were reviewed, specifically for the presence of a CD3(-/dim)CD4(+) aberrant T-cell population (described in angioimmunoblastic T-cell lymphomas), besides other T-cell aberrancies. Nine of 10 (90%) peripheral T-cell lymphomas of follicular type showed a CD3(-/dim)CD4(+) T-cell population constituting 29.3% (range 7.9-62%) of all lymphocytes. Five of 10 (50%) had nodular lymphocyte predominant Hodgkin lymphoma or lymphocyte-rich classical Hodgkin lymphoma-like morphology with scattered Hodgkin-like cells that expressed CD20, CD30, CD15, and MUM1. Three cases had a nodular growth pattern and three others exhibited a perifollicular growth pattern without Hodgkin-like cells. Epstein-Barr virus was positive in 1 of 10 cases (10%). PCR analysis showed clonal T-cell receptor gamma gene rearrangement in all 10 peripheral T-cell lymphomas of follicular type. By flow cytometry, 11 of 15 (73.3%) angioimmunoblastic T-cell lymphomas showed the CD3(-/dim)CD4(+) population (mean: 19.5%, range: 3-71.8%). Using a threshold of 3% for CD3(-/dim)CD4(+) T cells, all 15 nodular lymphocyte predominant Hodgkin lymphoma controls and 8 classical Hodgkin lymphomas were negative (Mann-Whitney P=0.01, F-PTCL vs Hodgkin lymphomas), as were 25 of 26 reactive lymph nodes. The high frequency of CD3(-/dim)CD4(+) aberrant T cells is similar in angioimmunoblastic T-cell lymphomas and peripheral T-cell lymphomas of follicular type, and is a useful feature in distinguishing peripheral T-cell lymphomas of follicular type from morphologic mimics such as reactive hyperplasia or Hodgkin lymphoma.
FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry

PubMed Central

Arrigucci, Riccardo; Bushkin, Yuri; Radford, Felix; Lakehal, Karim; Vir, Pooja; Pine, Richard; Martin, December; Sugarman, Jeffrey; Zhao, Yanlin; Yap, George S; Lardizabal, Alfred A; Tyagi, Sanjay; Gennaro, Maria Laura

2017-01-01

We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes ~30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T cells with specific antigens is described. PMID:28518171
Experimental Study of Subcooled Flow Boiling Heat Transfer on a Smooth Surface in Short-Term Microgravity

NASA Astrophysics Data System (ADS)

Zhang, Yonghai; Liu, Bin; Zhao, Jianfu; Deng, Yueping; Wei, Jinjia

2018-06-01

The flow boiling heat transfer characteristics of subcooled air-dissolved FC-72 on a smooth surface (chip S) were studied in microgravity by utilizing the drop tower facility in Beijing. The heater, with dimensions of 40 × 10 × 0.5 mm3 (length × width × thickness), was combined with two silicon chips with the dimensions of 20 × 10 × 0.5 mm3. High-speed visualization was used to supplement observation in the heat transfer and vapor-liquid two-phase flow characteristics. In the low and moderate heat fluxes region, the flow boiling of chip S at inlet velocity V = 0.5 m/s shows almost the same regulations as that in pool boiling. All the wall temperatures at different positions along the heater in microgravity are slightly lower than that in normal gravity, which indicates slight heat transfer enhancement. However, in the high heat flux region, the pool boiling of chip S shows much evident deterioration of heat transfer compared with that of flow boiling in microgravity. Moreover, the bubbles of flow boiling in microgravity become larger than that in normal gravity due to the lack of buoyancy Although the difference of the void fraction in x-y plain becomes larger with increasing heat flux under different gravity levels, it shows nearly no effect on heat transfer performance except for critical heat flux (CHF). Once the void fraction in y-z plain at the end of the heater equals 1, the vapor blanket will be formed quickly and transmit from downstream to upstream along the heater, and CHF occurs. Thus, the height of channel is an important parameter to determine CHF in microgravity at a fixed velocity. The flow boiling of chip S at inlet velocity V = 0.5 m/s shows higher CHF than that of pool boiling because of the inertia force, and the CHF under microgravity is about 78-92% of that in normal gravity.
Clinical cytometry and progress in HLA antibody detection.

PubMed

Bray, Robert A; Tarsitani, Christine; Gebel, Howard M; Lee, Jar-How

2011-01-01

For most solid organ and selected stem cell transplants, antibodies against mismatched HLA antigens can lead to early and late graft failure. In recognition of the clinical significance of these antibodies, HLA antibody identification is one of the most critical functions of histocompatibility laboratories. Early methods employed cumbersome and insensitive complement-dependent cytotoxicity assays with a visual read-out. A little over 20 years ago flow cytometry entered the realm of antibody detection with the introduction of the flow cytometric crossmatch. Cytometry's increased sensitivity and objectivity quickly earned it popularity as a preferred crossmatch method especially for sensitized recipients. Although a sensitive method, the flow crossmatch was criticized as being "too sensitive" as false positive reactions were a know drawback. In part, the shortcomings of the flow crossmatch were due to the lack of corresponding sensitive and specific HLA antibody screening assays. However, in the mid 1990s, solid phase assays, capable of utilizing standard flow cytometers, were developed. These assays used microparticles coated with purified HLA molecules. Hence, the era of solid-phase, microparticle technology for HLA antibody detection was born permitting the sensitive and specific detection of HLA antibody. It was now possible to provide better correlation between HLA antibody detection and the flow cytometric crossmatch. This flow-based technology was soon followed by adaptation to the Luminex platform permitting a mutltiplexed approach for the identification and characterization of HLA antibodies. It is hoped that these technologies will ultimately lead to the identification of parameters that best correlate with and/or predict transplant outcomes. Copyright © 2011 Elsevier Inc. All rights reserved.
Piecewise uniform conduction-like flow channels and method therefor

DOEpatents

Cummings, Eric B [Livermore, CA; Fiechtner, Gregory J [Livermore, CA

2006-02-28

A low-dispersion methodology for designing microfabricated conduction channels for on-chip electrokinetic-based systems is presented. The technique relies on trigonometric relations that apply for ideal electrokinetic flows, allowing faceted channels to be designed on chips using common drafting software and a hand calculator. Flows are rotated and stretched along the abrupt interface between adjacent regions with differing permeability. Regions bounded by interfaces form flow "prisms" that can be combined with other designed prisms to obtain a wide range of turning angles and expansion ratios while minimizing dispersion. Designs are demonstrated using two-dimensional numerical solutions of the Laplace equation.
CytometryML binary data standards

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2005-03-01

CytometryML is a proposed new Analytical Cytology (Cytomics) data standard, which is based on a common set of XML schemas for encoding flow cytometry and digital microscopy text based data types (metadata). CytometryML schemas reference both DICOM (Digital Imaging and Communications in Medicine) codes and FCS keywords. Flow Cytometry Standard (FCS) list-mode has been mapped to the DICOM Waveform Information Object. The separation of the large binary data objects (list mode and image data) from the XML description of the metadata permits the metadata to be directly displayed, analyzed, and reported with standard commercial software packages; the direct use of XML languages; and direct interfacing with clinical information systems. The separation of the binary data into its own files simplifies parsing because all extraneous header data has been eliminated. The storage of images as two-dimensional arrays without any extraneous data, such as in the Adobe Photoshop RAW format, facilitates the development by scientists of their own analysis and visualization software. Adobe Photoshop provided the display infrastructure and the translation facility to interconvert between the image data from commercial formats and RAW format. Similarly, the storage and parsing of list mode binary data type with a group of parameters that are specified at compilation time is straight forward. However when the user is permitted at run-time to select a subset of the parameters and/or specify results of mathematical manipulations, the development of special software was required. The use of CytometryML will permit investigators to be able to create their own interoperable data analysis software and to employ commercially available software to disseminate their data.
System-on-fluidics immunoassay device integrating wireless radio-frequency-identification sensor chips.

PubMed

Yazawa, Yoshiaki; Oonishi, Tadashi; Watanabe, Kazuki; Shiratori, Akiko; Funaoka, Sohei; Fukushima, Masao

2014-09-01

A simple and sensitive point-of-care-test (POCT) device for chemiluminescence (CL) immunoassay was devised and tested. The device consists of a plastic flow-channel reactor and two wireless-communication sensor chips, namely, a photo-sensor chip and a temperature-sensor chip. In the flow-channel reactor, a target antigen is captured by an antibody immobilized on the inner wall of the flow-channel and detected with enzyme labeled antibody by using CL substrate. The CL signal corresponding to the amount of antigen is measured by a newly developed radio-frequency-identification (RFID) sensor, which enables batteryless operation and wireless data communication with an external reader. As for the POCT device, its usage environment, especially temperature, varies for each measurement. Hence, temperature compensation is a key issue in regard to eliminating dark-signal fluctuation, which is a major factor in deterioration of the precision of the POCT device. A two-stage temperature-compensation scheme was adopted. As for the first stage, the signals of two photodiodes, one with an open window and one with a sealed window, integrated on the photo-sensor chip are differentiated to delete the dark signal. As for the second stage, the differentiated signal fluctuation caused by a temperature variation is compensated by using the other sensor chip (equipped with a temperature sensor). The dark-level fluctuation caused by temperature was reduced from 0.24 to 0.02 pA/°C. The POCT device was evaluated as a CL immunoassay of thyroid-stimulating hormone (TSH). The flow rate of the CL reagent in the flow channel was optimized. As a result, the detection limit of the POCT device was 0.08 ng/ml (i.e., 0.4 μIU/ml). Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
The cytometric future: it ain't necessarily flow!

PubMed

Shapiro, Howard M

2011-01-01

Initial approaches to cytometry for classifying and characterizing cells were based on microscopy; it was necessary to collect relatively high-resolution images of cells because only a few specific reagents usable for cell identification were available. Although flow cytometry, now the dominant cytometric technology, typically utilizes lenses similar to microscope lenses for light collection, improved, more quantitative reagents allow the necessary information to be acquired in the form of whole-cell measurements of the intensities of light transmission, scattering, and/or fluorescence.Much of the cost and complexity of both automated microscopes and flow cytometers arises from the necessity for them to measure one cell at a time. Recent developments in digital camera technology now offer an alternative in which one or more low-magnification, low-resolution images are made of a wide field containing many cells, using inexpensive light-emitting diodes (LEDs) for illumination. Minimalist widefield imaging cytometers can provide a smaller, less complex, and substantially less expensive alternative to flow cytometry, critical in systems intended for in resource-poor areas. Minimalism is, likewise, a good philosophy in developing instrumentation and methodology for both clinical and large-scale research use; it simplifies quality assurance and compliance with regulatory requirements, as well as reduces capital outlays, material costs, and personnel training requirements. Also, importantly, it yields "greener" technology.
In vivo flow cytometry and time-resolved near-IR angiography and lymphography

NASA Astrophysics Data System (ADS)

Galanzha, Ekaterina I.; Tuchin, Valery V.; Brock, Robert W.; Zharov, Vladimir P.

2007-05-01

Integration of photoacoustic and photothermal techniques with high-speed, high-resolution transmission and fluorescence microscopy shows great potential for in vivo flow cytometry and indocyanine green (ICG) near-infrared (IR) angiography of blood and lymph microvessels. In particular, the capabilities of in vivo flow cytometry using rat mesentery and nude mouse ear models are demonstrated for real-time quantitative detection of circulating and migrating individual blood and cancer cells in skin, mesentery, lymph nodes, liver, kidney; studying vascular dynamics with a focus on lymphatics; monitoring cell traffic between blood and lymph systems; high-speed imaging of cell deformability in flow; and label-free real-time monitoring of single cell extravasation from blood vessel lumen into tissue. As presented, the advantages of ICG IR-angiography include estimation of time resolved dye dynamics (appearance and clearance) in blood and lymph microvessels using fluorescent and photoacoustic modules of the integrated technique. These new approaches are important for monitoring and quantifying metastatic and apoptotic cells; comparative measurements of plasma and cell velocities; analysis of immune responses; monitoring of circulating macromolecules, chylomicrons, bacteria, viruses and nanoparticles; molecular imaging. In the future, we believe that the integrated technique presented will have great potential for translation to early disease diagnoses (e.g. cancer) or assessment of innovative therapeutic interventions in humans.
Webcam-based flow cytometer using wide-field imaging for low cell number detection at high throughput.

PubMed

Balsam, Joshua; Bruck, Hugh Alan; Rasooly, Avraham

2014-09-07

Here we describe a novel low-cost flow cytometer based on a webcam capable of low cell number detection in a large volume which may overcome the limitations of current flow cytometry. Several key elements have been combined to yield both high throughput and high sensitivity. The first element is a commercially available webcam capable of 187 frames per second video capture at a resolution of 320 × 240 pixels. The second element in this design is a 1 W 450 nm laser module for area-excitation, which combined with the webcam allows for rapid interrogation of a flow field. The final element is a 2D flow-cell which overcomes the flow limitation of hydrodynamic focusing and allows for higher sample throughput in a wider flow field. This cell allows for the linear velocity of target cells to be lower than in a conventional "1D" hydrodynamic focusing flow-cells typically used in cytometry at similar volumetric flow rates. It also allows cells to be imaged at the full frame rate of the webcam. Using this webcam-based flow cytometer with wide-field imaging, it was confirmed that the detection of fluorescently tagged 5 μm polystyrene beads in "1D" hydrodynamic focusing flow-cells was not practical for low cell number detection due to streaking from the motion of the beads, which did not occur with the 2D flow-cell design. The sensitivity and throughput of this webcam-based flow cytometer was then investigated using THP-1 human monocytes stained with SYTO-9 florescent dye in the 2D flow-cell. The flow cytometer was found to be capable of detecting fluorescently tagged cells at concentrations as low as 1 cell per mL at flow rates of 500 μL min(-1) in buffer and in blood. The effectiveness of detection was concentration dependent: at 100 cells per mL 84% of the cells were detected compared to microscopy, 10 cells per mL 79% detected and 1 cell per mL 59% of the cells were detected. With the blood samples spiked to 100 cells per mL, the average concentration for all samples was 91.4 cells per mL, with a 95% confidence interval of 86-97 cells per mL. These low cell concentrations and the large volume capabilities of the system may overcome the limitations of current cytometry, and are applicable to rare cell (such as circulating tumor cell) detection The simplicity and low cost of this device suggests that it may have a potential use in developing point-of-care clinical flow cytometry for resource-poor settings associated with global health.

Multi-angle lensless digital holography for depth resolved imaging on a chip.

PubMed

Su, Ting-Wei; Isikman, Serhan O; Bishara, Waheb; Tseng, Derek; Erlinger, Anthony; Ozcan, Aydogan

2010-04-26

A multi-angle lensfree holographic imaging platform that can accurately characterize both the axial and lateral positions of cells located within multi-layered micro-channels is introduced. In this platform, lensfree digital holograms of the micro-objects on the chip are recorded at different illumination angles using partially coherent illumination. These digital holograms start to shift laterally on the sensor plane as the illumination angle of the source is tilted. Since the exact amount of this lateral shift of each object hologram can be calculated with an accuracy that beats the diffraction limit of light, the height of each cell from the substrate can be determined over a large field of view without the use of any lenses. We demonstrate the proof of concept of this multi-angle lensless imaging platform by using light emitting diodes to characterize various sized microparticles located on a chip with sub-micron axial and lateral localization over approximately 60 mm(2) field of view. Furthermore, we successfully apply this lensless imaging approach to simultaneously characterize blood samples located at multi-layered micro-channels in terms of the counts, individual thicknesses and the volumes of the cells at each layer. Because this platform does not require any lenses, lasers or other bulky optical/mechanical components, it provides a compact and high-throughput alternative to conventional approaches for cytometry and diagnostics applications involving lab on a chip systems.
Highly multiparametric analysis by mass cytometry.

PubMed

Ornatsky, Olga; Bandura, Dmitry; Baranov, Vladimir; Nitz, Mark; Winnik, Mitchell A; Tanner, Scott

2010-09-30

This review paper describes a new technology, mass cytometry, that addresses applications typically run by flow cytometer analyzers, but extends the capability to highly multiparametric analysis. The detection technology is based on atomic mass spectrometry. It offers quantitation, specificity and dynamic range of mass spectrometry in a format that is familiar to flow cytometry practitioners. The mass cytometer does not require compensation, allowing the application of statistical techniques; this has been impossible given the constraints of fluorescence noise with traditional cytometry instruments. Instead of "colors" the mass cytometer "reads" the stable isotope tags attached to antibodies using metal-chelating labeling reagents. Because there are many available stable isotopes, and the mass spectrometer provides exquisite resolution between detection channels, many parameters can be measured as easily as one. For example, in a single tube the technique allows for the ready detection and characterization of the major cell subsets in blood or bone marrow. Here we describe mass cytometric immunophenotyping of human leukemia cell lines and leukemia patient samples, differential cell analysis of normal peripheral and umbilical cord blood; intracellular protein identification and metal-encoded bead arrays. Copyright © 2010 Elsevier B.V. All rights reserved.
Rapid differentiation of cocci/mixed bacteria from rods in voided urine culture of women with uncomplicated urinary tract infections.

PubMed

Yang, Chun-Chun; Yang, Stephen Shei-Dei; Hung, Hui-Ching; Chiang, I-Ni; Peng, Chiung-Hui; Chang, Shang-Jen

2017-09-01

To evaluate the ability of laser flow cytometry to predict cocci/mixed growth in the pre-analytical phase of urine specimens. We retrospectively reviewed urine samples from women with uncomplicated urinary tract infections from urologic clinics for study. Urine analyses were performed with laser flow cytometry (UF1000i, Sysmex, Kobe, Japan) and then diagrams were generated (forward scatter vs. fluorescent light scatter). Each specimen (bacteria count >357 BACT/μL) was classified as either cocci bacteria or rods/mixed growth according to the diagrams. Standard urine cultures were performed, and the agreement between cultures and the UF1000i interpretations was analyzed with kappa statistics. Finally, 491 specimens met the criteria for analysis. Among the 376 specimens with single bacteria growth, there were 26 gram-positive cocci (13 Streptococci spp., 7 Staphylococci spp., 6 Enterococci spp.), 1 gram-positive rods (Corynebacterium spp.), and 349 gram-negative rods (273 Escherichia coli, 33 Klebsiella spp., 29 Proteus spp., 6 Citrobacter spp., 4 Enterobacter spp., 3 Pseudomonas spp., and 1 Providencia spp.). There were 115 specimens with two bacteria species or more that were regarded as mixed growth. Agreement of rods or cocci/mixed growth between the laser flow cytometry and urine cultures yielded a kappa value of 0.58. The positive and negative predictive rate of the UF1000i for cocci/mixed growth in voided urine culture was 81.8% and 84.7%, respectively. Through laser flow cytometry, we can predict growth of cocci/mixed growth in the pre-analytical phase of urine culture, thus avoiding unnecessary urine culture and waiting time. © 2016 Wiley Periodicals, Inc.
Influence of Macrophages on the Rooster Spermatozoa Quality.

PubMed

Kuzelova, L; Vasicek, J; Chrenek, P

2015-08-01

The goal of this study was to evaluate the occurrence of macrophages in rooster semen and to investigate their impact on the spermatozoa quality. Ross 308 breeder males (n = 30) with no evidence of genital tract infections were used to determine the concentration of macrophages using fluorescently conjugated acetylated low-density lipoprotein (AcLDL). Subsequently, the roosters were divided into two groups on the basis of semen macrophage concentration, and semen quality was compared in two heterospermic samples. We applied computer-assisted semen analysis (CASA) system to determine motility parameters. Fluorescence microscopy and flow cytometry were used to evaluate occurrence of apoptotic and dead spermatozoa. Spermatozoa fertility potential was examined after intravaginal artificial insemination of hens. Eighteen roosters (control group) contained 0.2-3% of macrophages within spermatozoa population and ten roosters (macrophage group) had 10-15% of macrophages. Males from macrophage group had lower (p < 0.05) motility parameters (total and progressive movement, velocity curved line) and increased concentration of dead spermatozoa detected by flow cytometry and fluorescence microscopy (p < 0.001 and p ˂ 0.05, respectively). Differences (p < 0.05) between fluorescent microscopy and flow cytometry in results on spermatozoa apoptosis and viability were observed. No significant difference was found between groups in fertility of spermatozoa. In conclusion, the higher presence of macrophages in rooster semen may have a negative effect on some parameters of rooster spermatozoa evaluated in vitro. Furthermore, our study suggests that flow cytometry allows more precise examination of spermatozoa viability and apoptosis in a very short time compared with the fluorescent microscopy. © 2015 Blackwell Verlag GmbH.
The arbuscular mycorrhizal fungus Glomus intraradices is haploid and has a small genome size in the lower limit of eukaryotes.

PubMed

Hijri, Mohamed; Sanders, Ian R

2004-02-01

The genome size, complexity, and ploidy of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was determined using flow cytometry, reassociation kinetics, and genomic reconstruction. Nuclei of G. intraradices from in vitro culture, were analyzed by flow cytometry. The estimated average length of DNA per nucleus was 14.07+/-3.52 Mb. Reassociation kinetics on G. intraradices DNA indicated a haploid genome size of approximately 16.54 Mb, comprising 88.36% single copy DNA, 1.59% repetitive DNA, and 10.05% fold-back DNA. To determine ploidy, the DNA content per nucleus measured by flow cytometry was compared with the genome estimate of reassociation kinetics. G. intraradices was found to have a DNA index (DNA per nucleus per haploid genome size) of approximately 0.9, indicating that it is haploid. Genomic DNA of G. intraradices was also analyzed by genomic reconstruction using four genes (Malate synthase, RecA, Rad32, and Hsp88). Because we used flow cytometry and reassociation kinetics to reveal the genome size of G. intraradices and show that it is haploid, then a similar value for genome size should be found when using genomic reconstruction as long as the genes studied are single copy. The average genome size estimate was 15.74+/-1.69 Mb indicating that these four genes are single copy per haploid genome and per nucleus of G. intraradices. Our results show that the genome size of G. intraradices is much smaller than estimates of other AMF and that the unusually high within-spore genetic variation that is seen in this fungus cannot be due to high ploidy.
Leukocyte Populations in Human Preterm and Term Breast Milk Identified by Multicolour Flow Cytometry

PubMed Central

Trend, Stephanie; de Jong, Emma; Lloyd, Megan L.; Kok, Chooi Heen; Richmond, Peter; Doherty, Dorota A.; Simmer, Karen; Kakulas, Foteini; Strunk, Tobias; Currie, Andrew

2015-01-01

Background Extremely preterm infants are highly susceptible to bacterial infections but breast milk provides some protection. It is unknown if leukocyte numbers and subsets in milk differ between term and preterm breast milk. This study serially characterised leukocyte populations in breast milk of mothers of preterm and term infants using multicolour flow cytometry methods for extended differential leukocyte counts in blood. Methods Sixty mothers of extremely preterm (<28 weeks gestational age), very preterm (28–31 wk), and moderately preterm (32–36 wk), as well as term (37–41 wk) infants were recruited. Colostrum (d2–5), transitional (d8–12) and mature milk (d26–30) samples were collected, cells isolated, and leukocyte subsets analysed using flow cytometry. Results The major CD45+ leukocyte populations circulating in blood were also detectable in breast milk but at different frequencies. Progression of lactation was associated with decreasing CD45+ leukocyte concentration, as well as increases in the relative frequencies of neutrophils and immature granulocytes, and decreases in the relative frequencies of eosinophils, myeloid and B cell precursors, and CD16- monocytes. No differences were observed between preterm and term breast milk in leukocyte concentration, though minor differences between preterm groups in some leukocyte frequencies were observed. Conclusions Flow cytometry is a useful tool to identify and quantify leukocyte subsets in breast milk. The stage of lactation is associated with major changes in milk leukocyte composition in this population. Fresh preterm breast milk is not deficient in leukocytes, but shorter gestation may be associated with minor differences in leukocyte subset frequencies in preterm compared to term breast milk. PMID:26288195
Where Do All the Phytoplankton Go? Challenges in Keeping Track of Viable Cells in Phytoplankton Communities Using Flow Cytometry and Cell Staining

NASA Astrophysics Data System (ADS)

Simmons, L. J.; Fobbe, D. J.; Berges, J. A.

2016-02-01

Understanding the dynamics of phytoplankton communities has traditionally focused on differences in growth and related processes among taxa. It is now appreciated that differences in mortality could be equally important in contributing to these dynamics. Studying mortality in communities is difficult, especially on relevant time scales, which could be as short as hours to days. Flow cytometry can potentially provide solutions, because it can allow discrimination of different taxa, and when combined with staining, distinguish live and dead cells. We applied flow cytometry and staining to phytoplankton communities in a model system: a small, well-studied, urban pond in southeastern Wisconsin. Using flow cytometry, it was possible to resolve up to six dominant taxa (most <37 µm) and track them through an annual cycle. However, the axes traditionally used, forward scatter (FSC, related to cell size) and red fluorescence (FL3, related to chlorophyll a content) offered poor discrimination. Addition of orange fluorescence (FL2, traditionally related to phycobilipigments) and side scatter (SSC, related to cell surface characteristics) improved separation of taxa, but reproducibility (i.e. the specific position of the taxa on axes) was also more sensitive to environmental variation in the case of the fluorescence parameters. Dead cells could be distinguished by green fluorescence (FL1, using SYTOX Green©), but the stain also affected other fluorescence channels, requiring compensation. Correlations of numbers of dead cells with environmental factors (e.g. temperature, nutrient concentrations, irradiance) were generally poor, suggesting the greater importance of biotic versus abiotic variables in community mortality dynamics. Ongoing work is focusing on the effects of viral pathogens, grazing and allelopathic interactions using experimental manipulations and individual-based modeling.
Characterisation of the immune response to type I collagen in scleroderma

PubMed Central

Warrington, Kenneth J; Nair, Usha; Carbone, Laura D; Kang, Andrew H; Postlethwaite, Arnold E

2006-01-01

This study was conducted to examine the frequency, phenotype, and functional profile of T lymphocytes that proliferate in response to type I collagen (CI) in patients with scleroderma (SSc). Peripheral blood mononuclear cells (PBMCs) from SSc patients, healthy controls, and rheumatoid arthritis disease controls were labeled with carboxy-fluorescein diacetate, succinimidyl ester (CFSE), cultured with or without antigen (bovine CI) for 14 days, and analysed by flow cytometry. Surface markers of proliferating cells were identified by multi-color flow cytometry. T-cell lines were derived after sorting for proliferating T cells (CFSElow). Cytokine expression in CI-responsive T cells was detected by intracellular staining/flow cytometry and by multiplex cytokine bead assay (Bio-Plex). A T-cell proliferative response to CI was detected in 8 of 25 (32%) SSc patients, but was infrequent in healthy or disease controls (3.6%; p = 0.009). The proliferating T cells expressed a CD4+, activated (CD25+), memory (CD45RO+) phenotype. Proliferation to CI did not correlate with disease duration or extent of skin involvement. T-cell lines were generated using in vitro CI stimulation to study the functional profile of these cells. Following activation of CI-reactive T cells, we detected intracellular interferon (IFN)-γ but not interleukin (IL)-4 by flow cytometry. Supernatants from the T-cell lines generated in vitro contained IL-2, IFN-γ, GM-CSF (granulocyte macrophage-colony-stimulating factor), and tumour necrosis factor-α, but little or no IL-4 and IL-10, suggesting that CI-responsive T cells express a predominantly Th1 cytokine pattern. In conclusion, circulating memory CD4 T cells that proliferate to CI are present in a subset of patients with SSc, but are infrequent in healthy or disease controls. PMID:16879746
Evaluation of cryopreserved stallion semen from Tori and Estonian breeds using CASA and flow cytometry.

PubMed

Kavak, A; Johannisson, A; Lundeheim, N; Rodriguez-Martinez, H; Aidnik, M; Einarsson, S

2003-04-15

Methods to evaluate the quality of frozen-thawed stallion semen are still needed, particularly those considering the sperm function. The present study evaluated sperm motility, membrane and acrosome integrity and the capacitation status of frozen-thawed spermatozoa from seven Tori and six Estonian breed stallions by way of computer assisted sperm analysis (CASA), a triple fluorophore stain combination and Merocyanine 540, respectively, the latter ones using flow cytometry. Two ejaculates from each stallion were cryopreserved using the Hannover method in 0.5 ml plastic straws. Two straws per ejaculate per stallion were thawed at 37 degrees C for 30s. Motility was analysed with CASA immediately after thawing, while for flow cytometry spermatozoa were cleansed by 70:40% Percoll discontinuous density gradient separation before analysed for sperm viability, acrosome integrity (stained with SNARF, PI and FITC-PSA) and capacitation status (stained with Merocyanine 540/Yo-Pro-1). Results (as least square means) were as follows: the motility of frozen-thawed semen was 43.4% for Tori stallions and 42.3% for Estonian stallions (P>0.05). After Percoll separation 79.3% of the spermatozoa from Tori stallions had intact acrosomes and 1.7% of them showed early signs of capacitation. The same parameters for Estonian stallions were 84.5 and 2.3%, respectively. There were no statistically significant differences between breeds or ejaculates within breed for any evaluated parameter. We conclude that triple staining and flow cytometry are valuable techniques to evaluate frozen-thawed stallion spermatozoa, and that no differences in quality of frozen semen were registered between Tori and Estonian breed stallions, allowing implementation of this technology in the Estonian horse population.
Red and near-infrared fluorophores inspired by chlorophylls: consideration of practical brightness in multicolor flow cytometry and biomedical sciences

NASA Astrophysics Data System (ADS)

Taniguchi, Masahiko; Hu, Gongfang; Liu, Rui; Du, Hai; Lindsey, Jonathan S.

2018-02-01

Demands in flow cytometry for increased multiplexing (for detection of multiple antigens) and brightness (for detection of rare entities) require new fluorophores (i.e., "colors") with spectrally distinct fluorescence outside the relatively congested visible spectral region. Flow cytometry fluorophores typically must function in aqueous solution upon bioconjugation and ideally should exhibit a host of photophysical features: (i) strong absorption, (ii) sizable Stokes shift, (iii) modest if not strong fluorescence, and (iv) narrow fluorescence band. Tandem dyes have long been pursued to achieve a large effective Stokes shift, increased brightness, and better control over the excitation and emission wavelengths. Here, the attractive photophysical features of chlorophylls and bacteriochlorophylls - Nature's chosen photoactive pigments for photosynthesis - are described with regards to use in flow cytometry. A chlorophyll (or bacteriochlorophyll) constitutes an intrinsic tandem dye given the red (or near-infrared) fluorescence upon excitation in the higher energy ultraviolet (UV) or visible absorption bands (due to rapid internal conversion to the lowest energy state). Synthetic (bacterio)chlorins are available with strong absorption (near-UV molar absorption coefficient ɛ(λexc) 105 M-1cm-1), modest fluorescence quantum yield (Φf = 0.05-0.30), and narrow fluorescence band (10-25 nm) tunable from 600-900 nm depending on synthetic design. The "relative practical brightness" is given by intrinsic brightness [ɛ(λexc) x Φf] times ηf, the fraction of the fluorescence band that is captured by an emission filter in a multicolor experiment. The spectroscopic features of (bacterio)chlorins are evaluated quantitatively to illustrate practical brightness for this novel class of fluorophores in a prospective 8-color panel.
Human Eosinophils Express Functional CCR7

PubMed Central

Ueki, Shigeharu; Estanislau, Jessica; Weller, Peter F.

2013-01-01

Human eosinophils display directed chemotactic activity toward an array of soluble chemokines. Eosinophils have been observed to migrate to draining lymph nodes in experimental models of allergic inflammation, yet it is unknown whether eosinophils express CCR7, a key chemokine receptor in coordinating leukocyte trafficking to lymph nodes. The purpose of this study is to demonstrate expression of CCR7 by human eosinophils and functional responses to CCL19 and CCL21, the known ligands of CCR7. Human eosinophils were purified by negative selection from healthy donors. CCR7 expression of freshly purified, unstimulated eosinophils and of IL-5–primed eosinophils was determined by flow cytometry and Western blot. Chemotaxis to CCL19 and CCL21 was measured in transwell assays. Shape changes to CCL19 and CCL21 were analyzed by flow cytometry and microscopy. Calcium fluxes of fluo-4 AM–loaded eosinophils were recorded by flow cytometry after chemokine stimulation. ERK phosphorylation of CCL19- and CCL21-stimulated eosinophils was measured by Western blot and Luminex assay. Human eosinophils expressed CCR7 as demonstrated by flow cytometry and Western blots. Eosinophils exhibited detectable cell surface expression of CCR7. IL-5–primed eosinophils exhibited chemotaxis toward CCL19 and CCL21 in a dose-dependent fashion. Upon stimulation with CCL19 or CCL21, IL-5–primed eosinophils demonstrated dose-dependent shape changes with polarization of F-actin and exhibited calcium influxes. Finally, primed eosinophils stimulated with CCL19 or CCL21 exhibited increased phosphorylation of ERK in response to both CCR7 ligands. We demonstrate that human eosinophils express CCR7 and have multipotent responses to the known ligands of CCR7. PMID:23449735
A GEIL flow cytometry consensus proposal for quantification of plasma cells: application to differential diagnosis between MGUS and myeloma.

PubMed

Frébet, Elise; Abraham, Julie; Geneviève, Franck; Lepelley, Pascale; Daliphard, Sylvie; Bardet, Valérie; Amsellem, Sophie; Guy, Julien; Mullier, Francois; Durrieu, Francoise; Venon, Marie-Dominique; Leleu, Xavier; Jaccard, Arnaud; Faucher, Jean-Luc; Béné, Marie C; Feuillard, Jean

2011-05-01

Flow cytometry is the sole available technique for quantification of tumor plasma-cells in plasma-cell disorders, but so far, no consensus technique has been proposed. Here, we report on a standardized, simple, robust five color flow cytometry protocol developed to characterize and quantify bone marrow tumor plasma-cells, validated in a multicenter manner. CD36 was used to exclude red blood cell debris and erythroblasts, CD38 and CD138 to detect plasma-cells, immunoglobulin light chains, CD45, CD56, CD19, and CD117 + CD34 to simultaneously characterize abnormal plasma-cells and quantify bone marrow precursors. This approach was applied in nine centers to 229 cases, including 25 controls. Tumor plasma-cells were detected in 96.8% of cases, all exhibiting an immunoglobulin peak over 1g/L. Calculation of a plasma-cells/precursors (PC/P) ratio allowed quantification of the plasma-cell burden independently from bone marrow hemodilution. The PC/P ratio yielded the best results in terms of sensitivity (81%) and specificity (84%) for differential diagnosis between MGUS and myeloma, when compared with other criteria. Combination of both the PC/P ratio and percentage of abnormal plasma-cells allowed the best differential diagnosis, but these criteria were discordant in 25% cases. Indirect calculation of CD19 negative PC/R ratio gave the best results in terms of sensitivity (87%). This standardized multiparameter flow cytometric approach allows for the detection and quantification of bone marrow tumor plasma-cell infiltration in nearly all cases of MGUS and myeloma, independently of debris and hemodilution. This approach may also prove useful for the detection of minimal residual disease. Copyright © 2010 International Clinical Cytometry Society.
Comparison between the effects of ultrasound and gamma-rays on the inactivation of Saccharomyces cerevisiae: analyses of cell membrane permeability and DNA or RNA synthesis by flow cytometry.

PubMed

Oyane, Ikuko; Takeda, Tomo; Oda, Yasunori; Sakata, Takashi; Furuta, Masakazu; Okitsu, Kenji; Maeda, Yasuaki; Nishimura, Rokuro

2009-04-01

The effects of 200 kHz ultrasonic irradiation on DNA or RNA formation and membrane permeability of yeast cells were investigated by flow cytometry and compared with those of (60)Co gamma-ray radiation. Colony counting analyses were also performed for comparison. It was observed that the colony-forming activity of yeast cells was not affected by small doses of ultrasonic irradiation, but was closely related to the amounts of sonolytically formed hydrogen peroxide at concentrations of more than 80 microM. On the other hand, gamma-rays directly retarded colony-forming ability in addition to the effects of radiolytically formed hydrogen peroxide. The results obtained by flow cytometry also indicated that the amounts of DNA or RNA formed decreased with an increase in ultrasonic irradiation time without any threshold. These results indicated that flow cytometry can show early growth activities, but that colony counting analyses are insufficient to evaluate continuous and quantitative changes in these activities. In addition, by analyzing the amounts of DNA or RNA formed in the presence of the same amount of hydrogen peroxide, it was found that DNA or RNA formation behavior in the presence of hydrogen peroxide with no irradiation was similar to that following ultrasonic irradiation. These results suggested that similar chemical effects due to the formation of hydrogen peroxide were produced during ultrasonic irradiation. In addition, physical effects of ultrasound, such as shock wave, hardly contributed to cell inactivation and cell membrane damage, because relatively high frequency ultrasound was used here. In the case of gamma-ray radiation, direct physical effects on the cells were clearly observed.
Polyploidy in the Olive Complex (Olea europaea): Evidence from Flow Cytometry and Nuclear Microsatellite Analyses

PubMed Central

Besnard, G.; Garcia-Verdugo, C.; Rubio De Casas, R.; Treier, U. A.; Galland, N.; Vargas, P.

2008-01-01

Background Phylogenetic and phylogeographic investigations have been previously performed to study the evolution of the olive tree complex (Olea europaea). A particularly high genomic diversity has been found in north-west Africa. However, to date no exhaustive study has been addressed to infer putative polyploidization events and their evolutionary significance in the diversification of the olive tree and its relatives. Methods Representatives of the six olive subspecies were investigated using (a) flow cytometry to estimate genome content, and (b) six highly variable nuclear microsatellites to assess the presence of multiple alleles at co-dominant loci. In addition, nine individuals from a controlled cross between two individuals of O. europaea subsp. maroccana were characterized with microsatellites to check for chromosome inheritance. Key Results Based on flow cytometry and genetic analyses, strong evidence for polyploidy was obtained in subspp. cerasiformis (tetraploid) and maroccana (hexaploid), whereas the other subspecies appeared to be diploids. Agreement between flow cytometry and genetic analyses gives an alternative approach to chromosome counting to determine ploidy level of trees. Lastly, abnormalities in chromosomes inheritance leading to aneuploid formation were revealed using microsatellite analyses in the offspring from the controlled cross in subsp. maroccana. Conclusions This study constitutes the first report for multiple polyploidy in olive tree relatives. Formation of tetraploids and hexaploids may have played a major role in the diversification of the olive complex in north-west Africa. The fact that polyploidy is found in narrow endemic subspecies from Madeira (subsp. cerasiformis) and the Agadir Mountains (subsp. maroccana) suggests that polyploidization has been favoured to overcome inbreeding depression. Lastly, based on previous phylogenetic analyses, we hypothesize that subsp. cerasiformis resulted from hybridization between ancestors of subspp. guanchica and europaea. PMID:18024415
High-pressure microfluidic control in lab-on-a-chip devices using mobile polymer monoliths.

PubMed

Hasselbrink, Ernest F; Shepodd, Timothy J; Rehm, Jason E

2002-10-01

We have developed a nonstick polymer formulation for creating moving parts inside of microfluidic channels and have applied the technique to create piston-based devices that overcome several microfluidic flow control challenges. The parts were created bycompletely filling the channels of a glass microfluidic chip with the monomer/ solvent/initiator components of a nonstick photopolymer and then selectively exposing the chip to UV light in order to define mobile pistons (or other quasi-two-dimensional shapes) inside the channels. Stops defined in the substrate prevent the part from flushing out of the device but also provide sealing surfaces so that valves and other flow control devices are possible. Sealing against pressures greater than 30 MPa (4,500 psi) and actuation times less than 33 ms are observed. An on-chip check valve, a diverter valve, and a 10-nL pipet are demonstrated. This valving technology, coupled with high-pressure electrokinetic pumps, should make it possible to create a completely integrated HPLC system on a chip.
Low intensity ultrasound induces apoptosis via MPT channel on mitochondrial membrane: Target for regulating cancer therapy or not?

NASA Astrophysics Data System (ADS)

Feng, Yi; Wan, Mingxi

2017-03-01

To discuss how the mitochondrion is involved in low intensity ultrasound induced apoptosis, HepG2 cells were irradiated by low intensity focused ultrasound (ISPTA = 3W/cm2, 1 min) and then cultured from 3-12 h post irradiation in the study. The morphological alteration was examined by light and fluorescent microscopy respectively. Cell viability and apoptosis were examined by trypan blue staining and flow cytometry with double staining of FITC-labelled Annexin-V/PI. Key proteins responded to irradiation were screened out by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and shotgun proteomic methods with Agilent 1100 HPLC-Chip-MS technology. Representative apoptotic morphological characteristics and increased percentage of apoptotic cells were achieved. Six important proteins (4 up-regulated and 2 down-regulated) were selected and analyzed. It revealed low intensity focused ultrasound could induce apoptosis in HepG2 cells and the US-induced apoptosis was mitochondria-dependent and caspases-dependent. Moreover, mitochondrial membrane permeability transition (MPT) is related to ultrasound induced apoptosis, but VDAC may be not the main MPT channel. Understanding it could help to assist the cancer therapy by regulating the MPT as the target.
Pokemon silencing leads to Bim-mediated anoikis of human hepatoma cell QGY7703.

PubMed

Liu, Kun; Liu, Feng; Zhang, Nannan; Liu, Shiying; Jiang, Yuyang

2012-01-01

Pokemon is an important proto-oncogene that plays a critical role in cellular oncogenic transformation and tumorigenesis. Anoikis, which is regulated by Bim-mediated apoptosis, is critical to cancer cell invasion and metastasis. We investigated the role of Pokemon in anoikis, and our results show that Pokemon renders liver cells resistant to anoikis via suppression of Bim transcription. We knocked-down Pokemon in human hepatoma cells QGY7703 with small interfering RNAs (siRNA). Knockdown of Pokemon alone did not significantly affect the growth and survival of QGY7703 cells but notably enhanced their sensitivity to apoptotic stress due to the presence of chemical agents or cell detachment, thereby inducing anoikis, as evidenced by flow cytometry and caspase-3 activity assays. In contrast, ectopic expression of Pokemon in HL7702 cells led to resistance to anoikis. Dual-luciferase reporter and ChIP assays illustrated that Pokemon suppressed Bim transcription via direct binding to its promoter. Our results suggest that Pokemon prevents anoikis through the suppression of Bim expression, which facilitates tumor cell invasion and metastasis. This Pokemon-Bim pathway may be an effective target for therapeutic intervention for cancer.
Pokemon Silencing Leads to Bim-Mediated Anoikis of Human Hepatoma Cell QGY7703

PubMed Central

Liu, Kun; Liu, Feng; Zhang, Nannan; Liu, Shiying; Jiang, Yuyang

2012-01-01

Pokemon is an important proto-oncogene that plays a critical role in cellular oncogenic transformation and tumorigenesis. Anoikis, which is regulated by Bim-mediated apoptosis, is critical to cancer cell invasion and metastasis. We investigated the role of Pokemon in anoikis, and our results show that Pokemon renders liver cells resistant to anoikis via suppression of Bim transcription. We knocked-down Pokemon in human hepatoma cells QGY7703 with small interfering RNAs (siRNA). Knockdown of Pokemon alone did not significantly affect the growth and survival of QGY7703 cells but notably enhanced their sensitivity to apoptotic stress due to the presence of chemical agents or cell detachment, thereby inducing anoikis, as evidenced by flow cytometry and caspase-3 activity assays. In contrast, ectopic expression of Pokemon in HL7702 cells led to resistance to anoikis. Dual-luciferase reporter and ChIP assays illustrated that Pokemon suppressed Bim transcription via direct binding to its promoter. Our results suggest that Pokemon prevents anoikis through the suppression of Bim expression, which facilitates tumor cell invasion and metastasis. This Pokemon-Bim pathway may be an effective target for therapeutic intervention for cancer. PMID:22754333
Integration of electro-optical mechanical systems and medicine: where are we and where can we go?

NASA Astrophysics Data System (ADS)

Gourley, Mark F.; Gourley, Paul L.

1997-03-01

The marriage of microfabricated materials with microbiological systems will allow advances in medicine to proceed at an unprecedented pace. Biomedical research is placing new demands on speed and limits of detection to assay body tissues and fluids. Emerging microfabricated chip technologies from the engineering community offer researchers novel types of analysis of human samples. In guiding these developments, the ability to swiftly and accurately gain useful information for identification and establish a diagnosis, is of utmost importance. Current examples of such technology include DNA amplification and analysis, and fluorescent cell analysis by flow cytometry. Potential applications include the development of rapid techniques for examining large number of cells in tissue or in blood. These could serve as screening tools for the detection and quantification of abnormal cell types; for example malignant or HIV infected cells. Micro/nanofabrication methods will make these devices compact, providing access of this technology to point of care providers; in a clinic, ambulance, or on a battlefield. Currently, these tools are in the construction phase. Upon delivery to researchers, validation of these instruments leads to clinical demand that requires approval from the Food and Drug Administration. This paper outlines criteria that successful devices must satisfy.
Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy.

PubMed

Reipert, S; Reipert, B M; Allen, T D

1994-09-01

The aim of the work is to visualise nuclear pore complexes (NPCs) in mammalian cells by high resolution scanning electron microscopy. A detergent-free isolation protocol was employed to obtain clean nuclei from the haemopoietic cell line K 562. Nuclear isolation was performed by mechanical homogenisation under hypotonic conditions followed by purification of the nuclear fraction. The isolated nuclei were attached to silicon chips, fixed, critical point dried, and sputter coated with a thin film (3-4 nm) of tantalum. Analysis of the nuclear surface by scanning electron microscopy (SEM) revealed a strong sensitivity of the outer nuclear membrane (ONM) to disruption during the isolation procedure. A significant reduction of the characteristic pattern of damage to the ONM was achieved by means of an isopicnic centrifugation on an isoosmolar balanced Percoll gradient. Analysis of the population of isolated nuclei by flow cytometry showed no signs of cell cycle specific losses of nuclei during isolation. The SEM investigations of the morphology of the nuclear envelope (NE) and of substructural details of NPCs and polyribosomes were performed using an in-lens field emission scanning electron microscope.

Evaluation of imaging biomarkers for identification of single cancer cells in blood

NASA Astrophysics Data System (ADS)

Odaka, Masao; Kim, Hyonchol; Girault, Mathias; Hattori, Akihiro; Terazono, Hideyuki; Matsuura, Kenji; Yasuda, Kenji

2015-06-01

A method of discriminating single cancer cells from whole blood cells based on their morphological visual characteristics (i.e., “imaging biomarker”) was examined. Cells in healthy rat blood, a cancer cell line (MAT-LyLu), and cells in cancer-cell-implanted rat blood were chosen as models, and their bright-field (BF, whole-cell morphology) and fluorescence (FL, nucleus morphology) images were taken by an on-chip multi-imaging flow cytometry system and compared. Eight imaging biomarker indices, i.e., cellular area in a BF image, nucleus area in an FL image, area ratio of a whole cell and its nucleus, distance of the mass center between a whole cell and nucleus, cellular and nucleus perimeter, and perimeter ratios were calculated and analyzed using the BF and FL images taken. Results show that cancer cells can be clearly distinguished from healthy blood cells using correlation diagrams for cellular and nucleus areas as two different categories. Moreover, a portion of cancer cells showed a low nucleus perimeter ratio less than 0.9 because of the irregular nucleus morphologies of cancer cells. These results indicate that the measurements of imaging biomarkers are practically applicable to identifying cancer cells in blood.
Simulation of cooling efficiency via miniaturised channels in multilayer LTCC for power electronics

NASA Astrophysics Data System (ADS)

Pietrikova, Alena; Girasek, Tomas; Lukacs, Peter; Welker, Tilo; Müller, Jens

2017-03-01

The aim of this paper is detailed investigation of thermal resistance, flow analysis and distribution of coolant as well as thermal distribution inside multilayer LTCC substrates with embedded channels for power electronic devices by simulation software. For this reason four various structures of internal channels in the multilayer LTCC substrates were designed and simulated. The impact of the volume flow, structures of channels, and power loss of chip was simulated, calculated and analyzed by using the simulation software Mentor Graphics FloEFDTM. The structure, size and location of channels have the significant impact on thermal resistance, pressure of coolant as well as the effectivity of cooling power components (chips) that can be placed on the top of LTCC substrate. The main contribution of this paper is thermal analyze, optimization and impact of 4 various cooling channels embedded in LTCC multilayer structure. Paper investigate, the effect of volume flow in cooling channels for achieving the least thermal resistance of LTCC substrate that is loaded by power thermal chips. Paper shows on the impact of the first chips thermal load on the second chip as well as. This possible new technology could ensure in the case of practical realization effective cooling and increasing reliability of high power modules.
CytoSPADE: high-performance analysis and visualization of high-dimensional cytometry data

PubMed Central

Linderman, Michael D.; Simonds, Erin F.; Qiu, Peng; Bruggner, Robert V.; Sheode, Ketaki; Meng, Teresa H.; Plevritis, Sylvia K.; Nolan, Garry P.

2012-01-01

Motivation: Recent advances in flow cytometry enable simultaneous single-cell measurement of 30+ surface and intracellular proteins. CytoSPADE is a high-performance implementation of an interface for the Spanning-tree Progression Analysis of Density-normalized Events algorithm for tree-based analysis and visualization of this high-dimensional cytometry data. Availability: Source code and binaries are freely available at http://cytospade.org and via Bioconductor version 2.10 onwards for Linux, OSX and Windows. CytoSPADE is implemented in R, C++ and Java. Contact: michael.linderman@mssm.edu Supplementary Information: Additional documentation available at http://cytospade.org. PMID:22782546
In vivo flow cytometry of circulating clots using negative photothermal and photoacoustic contrasts.

PubMed

Galanzha, Ekaterina I; Sarimollaoglu, Mustafa; Nedosekin, Dmitry A; Keyrouz, Salah G; Mehta, Jawahar L; Zharov, Vladimir P

2011-10-01

Conventional photothermal (PT) and photoacousic (PA) imaging, spectroscopy, and cytometry are preferentially based on positive PT/PA effects, when signals are above background. Here, we introduce PT/PA technique based on detection of negative signals below background. Among various new applications, we propose label-free in vivo flow cytometry of circulating clots. No method has been developed for the early detection of clots of different compositions as a source of thromboembolism including ischemia at strokes and myocardial infarction. When a low-absorbing, platelet-rich clot passes a laser-irradiated vessel volume, a transient decrease in local absorption results in an ultrasharp negative PA hole in blood background. Using this phenomenon alone or in combination with positive contrasts, we demonstrated identification of white, red, and mixed clots on a mouse model of myocardial infarction and human blood. The concentration and size of clots were measured with threshold down to few clots in the entire circulation with size as low as 20 μm. This multiparameter diagnostic platform using portable personal high-speed flow cytometer with negative dynamic contrast mode has potential to real-time defining risk factors for cardiovascular diseases, and for prognosis and prevention of stroke or use clot count as a marker of therapy efficacy. Possibility for label-free detection of platelets, leukocytes, tumor cells or targeting themby negative PA probes (e.g., nonabsorbing beads or bubbles) is also highlighted. Copyright © 2011 International Society for Advancement of Cytometry.
Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry.

PubMed

Smurthwaite, Cameron A; Hilton, Brett J; O'Hanlon, Ryan; Stolp, Zachary D; Hancock, Bryan M; Abbadessa, Darin; Stotland, Aleksandr; Sklar, Larry A; Wolkowicz, Roland

2014-01-01

The discovery of the green fluorescent protein from Aequorea victoria has revolutionized the field of cell and molecular biology. Since its discovery a growing panel of fluorescent proteins, fluorophores and fluorescent-coupled staining methodologies, have expanded the analytical capabilities of flow cytometry. Here, we exploit the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins. For genetic engineering, we utilize retroviral technology, which allows for the expression of ectopic genetic information in a stable manner in mammalian cells. We have genetically barcoded both adherent and nonadherent cells with different fluorescent proteins. Multiplexing power was increased by combining both the number of distinct fluorescent proteins, and the fluorescence intensity in each channel. Moreover, retroviral expression has proven to be stable for at least a 6-month period, which is critical for applications such as biological screens. We have shown the applicability of fluorescent barcoded multiplexing to cell-based assays that rely themselves on genetic barcoding, or on classical staining protocols. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. Once cell lines are developed, no further manipulation or staining is required, decreasing time, nonspecific background associated with staining protocols, and cost. The increasing number of discovered and/or engineered fluorescent proteins with unique absorbance/emission spectra, combined with the growing number of detection devices and lasers, increases multiplexing versatility, making fluorescent genetic barcoding a powerful tool for flow cytometry-based analysis. © 2013 International Society for Advancement of Cytometry.
Chromatin status of apoptosis genes correlates with sensitivity to chemo-, immune- and radiation therapy in colorectal cancer cell lines.

PubMed

Benard, Anne; Janssen, Connie M; van den Elsen, Peter J; van Eggermond, Marja C J A; Hoon, Dave S B; van de Velde, Cornelis J H; Kuppen, Peter J K

2014-12-01

The apoptosis pathway of programmed cell death is frequently deregulated in cancer. An intact apoptosis pathway is required for proper response to anti-cancer treatment. We investigated the chromatin status of key apoptosis genes in the apoptosis pathway in colorectal cancer cell lines in relation to apoptosis induced by chemo-, immune- or radiation therapy. Using chromatin immunoprecipitation (ChIP), we measured the presence of transcription-activating histone modifications H3Ac and H3K4me3 and silencing modifications H3K9me3 and H3K27me3 at the gene promoter regions of key apoptosis genes Bax, Bcl2, Caspase-9, Fas (CD95) and p53. Cell lines DLD1, SW620, Colo320, Caco2, Lovo and HT29 were treated with cisplatin, anti-Fas or radiation. The apoptotic response was measured by flow cytometry using propidium iodide and annexin V-FITC. The chromatin status of the apoptosis genes reflected the activation status of the intrinsic (Bax, Bcl2, Caspase-9 and p53) and extrinsic (Fas) pathways. An active intrinsic apoptotic pathway corresponded to sensitivity to cisplatin and radiation treatment of cell lines DLD1, SW620 and Colo320. An active Fas promoter corresponded to an active extrinsic apoptotic pathway in cell line DLD1. mRNA expression data correlated with the chromatin status of the apoptosis genes as measured by ChIP. In conclusion, the results presented in this study indicate that the balance between activating and silencing histone modifications, reflecting the chromatin status of apoptosis genes, can be used to predict the response of tumor cells to different anti-cancer therapies and could provide a novel target to sensitize tumors to obtain adequate treatment responses.
Mechanism of gemcitabine-induced suppression of human cholangiocellular carcinoma cell growth.

PubMed

Toyota, Yuka; Iwama, Hisakazu; Kato, Kiyohito; Tani, Joji; Katsura, Akiko; Miyata, Miwa; Fujiwara, Shintaro; Fujita, Koji; Sakamoto, Teppei; Fujimori, Takayuki; Okura, Ryoichi; Kobayashi, Kiyoyuki; Tadokoro, Tomoko; Mimura, Shima; Nomura, Takako; Miyoshi, Hisaaki; Morishita, Asahiro; Kamada, Hideki; Yoneyama, Hirohito; Okano, Keiichi; Suzuki, Yasuyuki; Masaki, Tsutomu

2015-10-01

Although gemcitabine (2',2'-difluorocytidine monohydrochloride) is a common anticancer agent of cholangiocellular carcinoma (CCC), its growth inhibitory effects and gemcitabine resistance in CCC cells are poorly understood. Our aims were to uncover the mechanism underlying the antitumor effect of gemcitabine and to analyze the mechanism regulating in vitro CCC cell gemcitabine resistance. In addition, we sought to identify miRNAs associated with the antitumor effects of gemcitabine in CCCs. Using a cell proliferation assay and flow cytometry, we examined the ability of gemcitabine to inhibit cell proliferation in three types of human CCC cell lines (HuCCT-1, Huh28, TKKK). We also employed western blotting to investigate the effects of gemcitabine on cell cycle-related molecules in CCC cells. In addition, we used array chips to assess gemcitabine-mediated changes in angiogenic molecules and activated tyrosine kinase receptors in CCC cells. We used miRNA array chips to comprehensively analyze gemcitabine-induced miRNAs and examined clusters of differentially expressed miRNAs in cells with and without gemcitabine treatment. Gemcitabine inhibited cell proliferation in a dose- and time-dependent manner in HuCCT-1 cells, whereas cell proliferation was unchanged in Huh28 and TKKK cells. Gemcitabine inhibited cell cycle progression in HuCCT-1 cells from G0/G1 to S phase, resulting in G1 cell cycle arrest due to the reduction of cyclin D1 expression. In addition, gemcitabine upregulated the angiogenic molecules IL-6, IL-8, ENA-78 and MCP-1. In TKKK cells, by contrast, gemcitabine did not arrest the cell cycle or modify angiogenic molecules. Furthermore, in gemcitabine-sensitive HuCCT-1 cells, gemcitabine markedly altered miRNA expression. The miRNAs and angiogenic molecules altered by gemcitabine contribute to the inhibition of tumor growth in vitro.
Numerical Simulation and Performance Optimization of a Magnetophoretic Bio-separation chip

NASA Astrophysics Data System (ADS)

Golozar, Matin; Darabi, Jeff; Molki, Majid

Separation of micro/nanoparticles is important in biomedicine and biotechnology. This research presents the modeling and optimization of a magnetophoretic bio-separation chip for the isolation of biomaterials, such as circulating tumor cells (CTCs) from the peripheral blood. The chip consists of a continuous flow through microfluidic channels that contains locally engineered magnetic field gradients. The high gradient magnetic field produced by the magnets is spatially non-uniform and gives rise to an attractive force on magnetic particles that move through the flow channel. The computational model takes into account the magnetic and fluidic forces as well as the effect of the volume fraction of particles on the continuous phase. The model is used to investigate the effect of two-way particle-fluid coupling on both the capture efficiency and the flow pattern in the separation chip. The results show that the microfluidic device has the capability of separating CTCs from their native environment. Additionally, a parametric study is performed to investigate the effects of the channel height, substrate thickness, magnetic bead size, bioparticle size, and the number of beads per cell on the cell separation performance.
Features of free software packages in flow cytometry: a comparison between four non-commercial software sources.

PubMed

Sahraneshin Samani, Fazel; Moore, Jodene K; Khosravani, Pardis; Ebrahimi, Marzieh

2014-08-01

Flow cytometers designed to analyze large particles are enabling new applications in biology. Data analysis is a critical component of the process FCM. In this article we compare features of four free software packages including WinMDI, Cyflogic, Flowing software, and Cytobank.
Potentiometric chip-based multipumping flow system for the simultaneous determination of fluoride, chloride, pH, and redox potential in water samples.

PubMed

Chango, Gabriela; Palacio, Edwin; Cerdà, Víctor

2018-08-15

A simple potentiometric chip-based multipumping flow system (MPFS) has been developed for the simultaneous determination of fluoride, chloride, pH, and redox potential in water samples. The proposed system was developed by using a poly(methyl methacrylate) chip microfluidic-conductor using the advantages of flow techniques with potentiometric detection. For this purpose, an automatic system has been designed and built by optimizing the variables involved in the process, such as: pH, ionic strength, stirring and sample volume. This system was applied successfully to water samples getting a versatile system with an analysis frequency of 12 samples per hour. Good correlation between chloride and fluoride concentration measured with ISE and ionic chromatography technique suggests satisfactory reliability of the system. Copyright © 2018 Elsevier B.V. All rights reserved.
PDMS free-flow electrophoresis chips with integrated partitioning bars for bubble segregation.

PubMed

Köhler, Stefan; Weilbeer, Claudia; Howitz, Steffen; Becker, Holger; Beushausen, Volker; Belder, Detlev

2011-01-21

In this work, a microfluidic free-flow electrophoresis device with a novel approach for preventing gas bubbles from entering the separation area is presented. This is achieved by integrating partitioning bars to reduce the channel depth between electrode channels and separation chamber in order to obtain electrical contact and simultaneously prevent bubbles from entering the separation area. The three-layer sandwich chip features a reusable carrier plate with integrated ports for fluidic connection combined with a softlithographically cast microfluidic PDMS layer and a sealing glass slide. This design allows for a straightforward and rapid chip prototyping process. The performance of the device is demonstrated by free-flow zone electrophoretic separations of fluorescent dye mixtures as well as by the separation of labeled amines and amino acids with separation voltages up to 297 V.
Multiparametric flow cytometry in the diagnosis and characterization of low-grade pulmonary mucosa-associated lymphoid tissue lymphomas.

PubMed

Zaer, F S; Braylan, R C; Zander, D S; Iturraspe, J A; Almasri, N M

1998-06-01

Primary mucosa associated lymphoid tissue (MALT) lymphomas are rare neoplasms that seem to have a better prognosis than nodal lymphomas. Morphologic diagnosis of these lesions may be difficult because of features that overlap with those of benign lymphoid infiltrates. In this study, we assessed the contribution of multi-parametric flow cytometry in demonstrating clonality and further characterizing pulmonary MALT lymphomas. Based on a clinical or pathologic suspicion of MALT-lymphoma, 3 transbronchial biopsies, 4 fine needle aspirates, 1 core needle biopsy, and 13 wedge excisions of lung were submitted fresh (unfixed) to our laboratory for evaluation. Among the 13 cases diagnosed as MALT lymphomas, B-cell monoclonality was established by identifying expression of a single immunoglobulin light chain on CD20 or CD19-positive cells in 12 cases. One case lacked expression of both light chains on B-cells. Of 11 lymphoma cases in which CD5 and CD10 surface antigens were assessed, no cases expressed CD10, and 1 case demonstrated weak CD5 expression. Nine of 10 cases studied were diploid and 1 case was hyperdiploid. All of the lymphomas displayed low (< or = 3%) S-phase fractions consistent with low grade processes. In 10 patients with short follow-up, none died of their disease and the majority had no evidence of lymphoma dissemination. In seven of the remaining eight cases, B-cells were polyclonal consistent with reactive processes. In one morphologically reactive case, flow cytometric analysis was unsuccessful because of poor cell viability. The pulmonary MALT lymphomas in this study represent a group of B-cell tumors with distinctive morphologic, immunophenotypic, and cell kinetic characteristics. Multi-parametric flow cytometry is useful for confirming B-cell monoclonality and illustrating an antigenic profile compatible with this diagnosis. Flow cytometry can be particularly helpful when working with small biopsies and cytologic samples with limited diagnostic material and may abrogate the need for more aggressive surgical procedures.
FlowCam: Quantification and Classification of Phytoplankton by Imaging Flow Cytometry.

PubMed

Poulton, Nicole J

2016-01-01

The ability to enumerate, classify, and determine biomass of phytoplankton from environmental samples is essential for determining ecosystem function and their role in the aquatic community and microbial food web. Traditional micro-phytoplankton quantification methods using microscopic techniques require preservation and are slow, tedious and very laborious. The availability of more automated imaging microscopy platforms has revolutionized the way particles and cells are detected within their natural environment. The ability to examine cells unaltered and without preservation is key to providing more accurate cell concentration estimates and overall phytoplankton biomass. The FlowCam(®) is an imaging cytometry tool that was originally developed for use in aquatic sciences and provides a more rapid and unbiased method for enumerating and classifying phytoplankton within diverse aquatic environments.
Cytometric analysis of shape and DNA content in mammalian sperm

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

Gledhill, B.L.

1983-10-10

Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. Sperm were analyzed by flow cytometry and slit-scan flow analysis for injury following the exposure of testes to mutagens. The utility of flow cytometry in genotoxin screening and monitoring of occupational exposure was evaluated. The technique proved valuable in separation of X- and Y-chromosome bearing sperm and the potential applicability of this technique in artificial insemination and a solution, ofmore » accurately assessing the DNA content of sperm were evaluated-with reference to determination of X- and Y-chromosome bearing sperm.« less
Computing Optic Flow with ArduEye Vision Sensor

DTIC Science & Technology

2013-01-01

processing algorithm that can be applied to the flight control of other robotic platforms. 15. SUBJECT TERMS Optical flow, ArduEye, vision based ...2 Figure 2. ArduEye vision chip on Stonyman breakout board connected to Arduino Mega (8) (left) and the Stonyman vision chips (7...robotic platforms. There is a significant need for small, light , less power-hungry sensors and sensory data processing algorithms in order to control the
Combining Cell Type-Restricted Adenoviral Targeting with Immunostaining and Flow Cytometry to Identify Cells-of-Origin of Lung Cancer.

PubMed

Best, Sarah A; Kersbergen, Ariena; Asselin-Labat, Marie-Liesse; Sutherland, Kate D

2018-01-01

Lung cancers display considerable intertumoral heterogeneity, leading to the classification of distinct tumor subtypes. Our understanding of the genetic aberrations that underlie tumor subtypes has been greatly enhanced by recent genomic sequencing studies and state-of-the-art gene targeting technologies, highlighting evidence that distinct lung cancer subtypes may be derived from different "cells-of-origin". Here, we describe the intra-tracheal delivery of cell type-restricted Ad5-Cre viruses into the lungs of adult mice, combined with immunohistochemical and flow cytometry strategies for the detection of lung cancer-initiating cells in vivo.
Flow cytometry, morphometry and histopathology as biomarkers of benzo[a]pyrene exposure in brown bullheads (ameiurus nebulosus)

USGS Publications Warehouse

Grady, Andrew W.; McLaughlin, Ronald M.; Caldwell, Charles W.; Schmitt, Christopher J.; Stalling, David L.

1992-01-01

Brown bullheads were given a single intraperitoneal dose of 0, 5, 25 or 125 mg kg−1 benzo[a]pyrene (BaP), a carcinogenic polycyclic aromatic hydrocarbon, and evaluated over 18 months. Flow cytometric analyses of hepatocyte DNA content indicated an increase in DNA synthesis in BaP-exposed fish prior to day 14 post-exposure. Thereafter, all flow cytometric variables returned to initial levels. Histopathological evaluation of livers from fish sampled at 18 months revealed significant differences among treatments in the amount of hepatic macrophage ceroid pigmentation and basophilic staining intensity. No neoplasms or changes in blood cell DNA content were detected. Significant morphometric variations existed among fish, but differences between sexes overshadowed differences attributable to dose. Flow cytometry yielded no evidence of long-term DNA alterations from a single exposure to BaP; however, the differences detected by DNA analysis shortly after the toxic event suggest that flow cytometric cell cycle analysis may be useful for documenting continuing exposures.
Positional dependence of particles in microfludic impedance cytometry.

PubMed

Spencer, Daniel; Morgan, Hywel

2011-04-07

Single cell impedance cytometry is a label-free electrical analysis method that requires minimal sample preparation and has been used to count and discriminate cells on the basis of their impedance properties. This paper shows experimental and numerically simulated impedance signals for test particles (6 μm diameter polystyrene) flowing through a microfluidic channel. The variation of impedance signal with particle position is mapped using numerical simulation and these results match closely with experimental data. We demonstrate that for a nominal 40 μm × 40 μm channel, the impedance signal is independent of position over the majority of the channel area, but shows large experimentally verifiable variation at extreme positions. The parabolic flow profile in the channel ensures that most of the sample flows through the area of uniform signal. At high flow rates inertial focusing is observed; the particles flow in equal numbers through two equilibrium positions reducing the coefficient of variance (CV) in the impedance signals to negligible values.
Measurement and Characterization of Apoptosis by Flow Cytometry.

PubMed

Telford, William; Tamul, Karen; Bradford, Jolene

2016-07-01

Apoptosis is an important mechanism in cell biology, playing a critical regulatory role in virtually every organ system. It has been particularly well characterized in the immune system, with roles ranging from immature immune cell development and selection to down-regulation of the mature immune response. Apoptosis is also the primary mechanism of action of anti-cancer drugs. Flow cytometry has been the method of choice for analyzing apoptosis in suspension cells for more than 25 years. Numerous assays have been devised to measure both the earliest and latest steps in the apoptotic process, from the earliest signal-transduction events to the late morphological changes in cell shape and granularity, proteolysis, and chromatin condensation. These assays are particularly powerful when combined into multicolor assays determining several apoptotic characteristics simultaneously. The multiparametric nature of flow cytometry makes this technology particularly suited to measuring apoptosis. In this unit, we will describe the four main techniques for analyzing caspase activity in apoptotic cells, combined with annexin V and cell permeability analysis. These relatively simple multiparametric assays are powerful techniques for assessing cell death. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.
Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

PubMed

Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG 1 , kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

A novel flow cytometry-based assay for the quantification of antibody-dependent pneumococcal agglutination

PubMed Central

van der Gaast—de Jongh, Christa E.; Diavatopoulos, Dimitri A.; de Jonge, Marien I.

2017-01-01

The respiratory pathogen Streptococcus pneumoniae is a major cause of diseases such as otitis media, pneumonia, sepsis and meningitis. The first step towards infection is colonization of the nasopharynx. Recently, it was shown that agglutinating antibodies play an important role in the prevention of mucosal colonization with S. pneumoniae. Here, we present a novel method to quantify antibody-dependent pneumococcal agglutination in a high-throughput manner using flow cytometry. We found that the concentration of agglutinating antibodies against pneumococcal capsule are directly correlated with changes in the size and complexity of bacterial aggregates, as measured by flow cytometry and confirmed by light microscopy. Using the increase in size, we determined the agglutination index. The cutoff value was set by measuring a series of non-agglutinating antibodies. With this method, we show that not only anti-polysaccharide capsule antibodies are able to induce agglutination but that also anti-PspA protein antibodies have agglutinating capabilities. In conclusion, we have described and validated a novel method to quantify pneumococcal agglutination, which can be used to screen sera from murine or human vaccination studies, in a high-throughput manner. PMID:28288168
Detection and isolation of rare cells by 2-step enrichment high-speed flow cytometry/cell sorting and single cell LEAP laser ablation

NASA Astrophysics Data System (ADS)

Zordan, M. D.; Leary, James F.

2011-02-01

The clonal isolation of rare cells, especially cancer and stem cells, in a population is important to the development of improved medical treatment. We have demonstrated that the Laser-Enabled Analysis and Processing (LEAP, Cyntellect Inc., San Diego, CA) instrument can be used to efficiently produce single cell clones by photoablative dilution. Additionally, we have also shown that cells present at low frequencies can be cloned by photoablative dilution after they are pre-enriched by flow cytometry based cell sorting. Circulating tumor cells were modeled by spiking isolated peripheral blood cells with cells from the lung carcinoma cell line A549. Flow cytometry based cell sorting was used to perform an enrichment sort of A549 cells directly into a 384 well plate. Photoablative dilution was performed with the LEAPTM instrument to remove any contaminating cells, and clonally isolate 1 side population cell per well. We were able to isolate and grow single clones of side population cells using this method at greater than 90% efficiency. We have developed a 2 step method that is able to perform the clonal isolation of rare cells based on a medically relevant functional phenotype.
Noninvasive and label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Wei, Xunbin

2015-03-01

Melanoma is a malignant tumor of melanocytes. Circulating melanoma cell has high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC). PAFC is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. In our research, we developed in vitro experiments to prove the ability of PAFC system of detecting PA signals from melanoma cells. For in vivo experiments, we constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells B16F10 with subcutaneous injection. PA signals were detected in the blood vessels of mouse ears in vivo. By counting circulating melanoma cells termly, we obtained the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation. Our PAFC system is an efficient tool to monitor melanoma metastases, cancer recurrence and therapeutic efficacy.
The role of multiparametric flow cytometry in the detection of minimal residual disease in acute leukaemia.

PubMed

Lee, Denise; Grigoriadis, George; Westerman, David

2015-12-01

Flow cytometry is the most accessible method for minimal residual disease (MRD) detection due to its availability in most haematological centres. Using a precise combination of different antibodies, immunophenotypic detection of MRD in acute leukaemia can be performed by identifying abnormal combinations or expressions of antigens on malignant cells at diagnosis, during and post treatment. These abnormal phenotypes, referred to as leukaemia-associated immunophenotypes (LAIPs) are either absent or expressed at low frequency in normal bone marrow (BM) cells and are used to monitor the behaviour and quantitate the amount of residual disease following treatment. In paediatric acute lymphoblastic leukaemia (ALL), the level of MRD by multiparametric flow cytometry (MPFC) during therapy is recognised as an important predictor of outcome. Although less extensively studied, adult ALL and adult and paediatric acute myeloid leukaemia (AML) have also demonstrated similar findings. The challenge now is incorporating this information for risk-stratification so that therapy can be tailored individually and ultimately improve outcome while also limiting treatment-related toxicity. In this review we will elaborate on the current and future role of MPFC in MRD in acute leukaemia while also addressing its limitations.
A Fully Automated High-Throughput Flow Cytometry Screening System Enabling Phenotypic Drug Discovery.

PubMed

Joslin, John; Gilligan, James; Anderson, Paul; Garcia, Catherine; Sharif, Orzala; Hampton, Janice; Cohen, Steven; King, Miranda; Zhou, Bin; Jiang, Shumei; Trussell, Christopher; Dunn, Robert; Fathman, John W; Snead, Jennifer L; Boitano, Anthony E; Nguyen, Tommy; Conner, Michael; Cooke, Mike; Harris, Jennifer; Ainscow, Ed; Zhou, Yingyao; Shaw, Chris; Sipes, Dan; Mainquist, James; Lesley, Scott

2018-05-01

The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.
Fountain Flow cytometry, a new technique for the rapid detection and enumeration of microorganisms in aqueous samples.

PubMed

Johnson, Paul E; Deromedi, Anthony J; Lebaron, Philippe; Catala, Philippe; Cash, Jennifer

2006-12-01

Pathogenic microorganisms are known to cause widespread waterborne disease worldwide. There is an urgent need to develop a technique for the real-time detection of pathogens in environmental samples at low concentrations, <10 microorganisms/ml, in large sample volumes, > or =100 ml. A novel method, Fountain Flowtrade mark cytometry, for the rapid and sensitive detection of individual microorganisms in aqueous samples is presented. Each sample is first incubated with a fluorescent label and then passed as a stream in front of a laser, which excites the label. The fluorescence is detected with a CCD imager as the sample flows toward the imager along its optical axis. The feasibility of Fountain Flow cytometry (FFC) is demonstrated by the detection of Escherichia coli labeled with ChemChrome CV6 and SYBR Gold in buffer and natural river water. Detections of labeled E. coli were made in aqueous suspensions with an efficiency of 96% +/- 14% down to a concentration approximately 200 bacteria/ml. The feasibility of FFC is demonstrated by the detection of E. coli in buffer and natural river water. FFC should apply to the detection of a wide range of pathogenic microorganisms including amoebae.
Dynamics of DNA replication during premeiosis and early meiosis in wheat.

PubMed

Rey, María-Dolores; Prieto, Pilar

2014-01-01

Meiosis is a specialised cell division that involves chromosome replication, two rounds of chromosome segregation and results in the formation of the gametes. Meiotic DNA replication generally precedes chromosome pairing, recombination and synapsis in sexually developing eukaryotes. In this work, replication has been studied during premeiosis and early meiosis in wheat using flow cytometry, which has allowed the quantification of the amount of DNA in wheat anther in each phase of the cell cycle during premeiosis and each stage of early meiosis. Flow cytometry has been revealed as a suitable and user-friendly tool to detect and quantify DNA replication during early meiosis in wheat. Chromosome replication was detected in wheat during premeiosis and early meiosis until the stage of pachytene, when chromosomes are associated in pairs to further recombine and correctly segregate in the gametes. In addition, the effect of the Ph1 locus, which controls chromosome pairing and affects replication in wheat, was also studied by flow cytometry. Here we showed that the Ph1 locus plays an important role on the length of meiotic DNA replication in wheat, particularly affecting the rate of replication during early meiosis in wheat.
Dynamics of DNA Replication during Premeiosis and Early Meiosis in Wheat

PubMed Central

Rey, María-Dolores; Prieto, Pilar

2014-01-01

Meiosis is a specialised cell division that involves chromosome replication, two rounds of chromosome segregation and results in the formation of the gametes. Meiotic DNA replication generally precedes chromosome pairing, recombination and synapsis in sexually developing eukaryotes. In this work, replication has been studied during premeiosis and early meiosis in wheat using flow cytometry, which has allowed the quantification of the amount of DNA in wheat anther in each phase of the cell cycle during premeiosis and each stage of early meiosis. Flow cytometry has been revealed as a suitable and user-friendly tool to detect and quantify DNA replication during early meiosis in wheat. Chromosome replication was detected in wheat during premeiosis and early meiosis until the stage of pachytene, when chromosomes are associated in pairs to further recombine and correctly segregate in the gametes. In addition, the effect of the Ph1 locus, which controls chromosome pairing and affects replication in wheat, was also studied by flow cytometry. Here we showed that the Ph1 locus plays an important role on the length of meiotic DNA replication in wheat, particularly affecting the rate of replication during early meiosis in wheat. PMID:25275307
Measurement of Mitochondrial Mass by Flow Cytometry during Oxidative Stress.

PubMed

Doherty, Edward; Perl, Andras

2017-07-01

Properly assessing mitochondrial health is crucial to understand their role in disease. MitoTracker green (MTG) and nonylacridine orange (NAO) are fluorescent probes which have been commonly used to assess mitochondrial mass. This is based on the assumption that both MTG and NAO accumulate in mitochondria regardless of the mitochondrial transmembrane potential (ΔΨ m ). Here, we utilized flow cytometry to evaluate the performance of these probes for assessment of mitochondrial mass relative to forward (FSC) and side scatter (SSC) in human peripheral blood lymphocytes (PBL). In isolated mitochondria, two subpopulations were identified by FSC and SSC measurements which were matched to subpopulations stained by MTG and NAO. The performance of these dyes was examined under oxidative and nitrosative stress induced by rotenone and NOC-18 while N -acetylcysteine (NAC) was employed as an antioxidant. Production of reactive oxygen species (ROS) and ΔΨ m were monitored in parallel. With respect to representation of mitochondrial mass, neither MTG nor NAO was affected by ΔΨ m . However, MTG showed significant correlation with cytosolic and mitochondrial ROS production and nitrosative stress. Our data suggest that NAO may be more suitable than MTG for assessment of mitochondrial mass by flow cytometry during oxidative stress.
[Preparation of chicken red blood cells for calibration of flow cytometry].

PubMed

Yin, Jian; Zhao, Shutao; Wu, Xiaodong; Wang, Ce; Wu, Yunliang

2013-01-01

To prepare stable chicken red blood cells for the calibration of flow cytometry. The traditional isolation method of chicken red blood cells was modified by incorporating gelatin technique, Ca2+-free HBSS treatment and low-speed centrifugation. The effect of fluorescence staining of the cells was improved by the addition of TritonX-100 to enhance the membrane permeability and Rnase enzymes to disintegrate RNA tiles. The modified method was compared with the traditional method for viability of the freshly isolated cells and the DNA content coefficient of variation (CV) of the fixed cells. Chicken red blood cells obtained by the modified method showed a significantly higher viability than those obtained by the traditional method [(98.5∓3.5)% vs (93.5∓2.7)%, P<0.05]. After glutaraldehyde fixation, the isolated cells with the modified method were stable during the 90-day preservation with a significantly lower CV than the cells obtained by the traditional method [(6.0∓0.3)% to 6.2∓0.4% vs (8.6∓0.5)% to (13.1∓1.4)%, P<0.01]. The chicken red blood cells isolated using the modified method can be applicable for calibration of flow cytometry.
Honey bee hemocyte profiling by flow cytometry.

PubMed

Marringa, William J; Krueger, Michael J; Burritt, Nancy L; Burritt, James B

2014-01-01

Multiple stress factors in honey bees are causing loss of bee colonies worldwide. Several infectious agents of bees are believed to contribute to this problem. The mechanisms of honey bee immunity are not completely understood, in part due to limited information about the types and abundances of hemocytes that help bees resist disease. Our study utilized flow cytometry and microscopy to examine populations of hemolymph particulates in honey bees. We found bee hemolymph includes permeabilized cells, plasmatocytes, and acellular objects that resemble microparticles, listed in order of increasing abundance. The permeabilized cells and plasmatocytes showed unexpected differences with respect to properties of the plasma membrane and labeling with annexin V. Both permeabilized cells and plasmatocytes failed to show measurable mitochondrial membrane potential by flow cytometry using the JC-1 probe. Our results suggest hemolymph particulate populations are dynamic, revealing significant differences when comparing individual hive members, and when comparing colonies exposed to diverse conditions. Shifts in hemocyte populations in bees likely represent changing conditions or metabolic differences of colony members. A better understanding of hemocyte profiles may provide insight into physiological responses of honey bees to stress factors, some of which may be related to colony failure.
Performance of computer vision in vivo flow cytometry with low fluorescence contrast

NASA Astrophysics Data System (ADS)

Markovic, Stacey; Li, Siyuan; Niedre, Mark

2015-03-01

Detection and enumeration of circulating cells in the bloodstream of small animals are important in many areas of preclinical biomedical research, including cancer metastasis, immunology, and reproductive medicine. Optical in vivo flow cytometry (IVFC) represents a class of technologies that allow noninvasive and continuous enumeration of circulating cells without drawing blood samples. We recently developed a technique termed computer vision in vivo flow cytometry (CV-IVFC) that uses a high-sensitivity fluorescence camera and an automated computer vision algorithm to interrogate relatively large circulating blood volumes in the ear of a mouse. We detected circulating cells at concentrations as low as 20 cells/mL. In the present work, we characterized the performance of CV-IVFC with low-contrast imaging conditions with (1) weak cell fluorescent labeling using cell-simulating fluorescent microspheres with varying brightness and (2) high background tissue autofluorescence by varying autofluorescence properties of optical phantoms. Our analysis indicates that CV-IVFC can robustly track and enumerate circulating cells with at least 50% sensitivity even in conditions with two orders of magnitude degraded contrast than our previous in vivo work. These results support the significant potential utility of CV-IVFC in a wide range of in vivo biological models.
Expression and functional analysis of novel molecule - Latcripin-13 domain from Lentinula edodes C91-3 produced in prokaryotic expression system.

PubMed

Wang, Jia; Zhong, Mintao; Liu, Ben; Sha, Li; Lun, Yongzhi; Zhang, Wei; Li, Xingyun; Wang, Xiaoli; Cao, Jing; Ning, Anhong; Huang, Min

2015-01-25

The shiitake mushroom Lentinula edodes has health benefits and is used to treat various diseases due to its immunomodulatory and antineoplastic properties. In the present study, the Latcripin-13 domain, isolated from L. edodes, was expressed in Escherichia coli Rosetta-gami(DE3) in the form of inclusion bodies. The Latcripin-13 domain was purified by Ni-His affinity chromatography with high purity and refolded by urea gradient dialysis. The product showed biological activity in A549 cells, a human lung cancer cell line, by flow cytometry and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method. The MTT assay and the flow cytometry results revealed that there was a great difference between the Latcripin-13 domain-treated group and the control group (p<0.05). Similarly, cell apoptosis observed by transmission electron microscopy (TEM) supported the flow cytometry results. This work demonstrated that the Latcripin-13 domain can induce apoptosis of A549 cells, which will bring new insights into the development of new antitumor drugs in the future. Copyright © 2014 Elsevier B.V. All rights reserved.
Honey Bee Hemocyte Profiling by Flow Cytometry

PubMed Central

Marringa, William J.; Krueger, Michael J.; Burritt, Nancy L.; Burritt, James B.

2014-01-01

Multiple stress factors in honey bees are causing loss of bee colonies worldwide. Several infectious agents of bees are believed to contribute to this problem. The mechanisms of honey bee immunity are not completely understood, in part due to limited information about the types and abundances of hemocytes that help bees resist disease. Our study utilized flow cytometry and microscopy to examine populations of hemolymph particulates in honey bees. We found bee hemolymph includes permeabilized cells, plasmatocytes, and acellular objects that resemble microparticles, listed in order of increasing abundance. The permeabilized cells and plasmatocytes showed unexpected differences with respect to properties of the plasma membrane and labeling with annexin V. Both permeabilized cells and plasmatocytes failed to show measurable mitochondrial membrane potential by flow cytometry using the JC-1 probe. Our results suggest hemolymph particulate populations are dynamic, revealing significant differences when comparing individual hive members, and when comparing colonies exposed to diverse conditions. Shifts in hemocyte populations in bees likely represent changing conditions or metabolic differences of colony members. A better understanding of hemocyte profiles may provide insight into physiological responses of honey bees to stress factors, some of which may be related to colony failure. PMID:25285798
Characterization of Oct4-GFP transgenic mice as a model to study the effect of environmental estrogens on the maturation of male germ cells by using flow cytometry.

PubMed

Porro, Valentina; Pagotto, Romina; Harreguy, María Belén; Ramírez, Sofía; Crispo, Martina; Santamaría, Clarisa; Luque, Enrique H; Rodríguez, Horacio A; Bollati-Fogolín, Mariela

2015-11-01

Oct4 is involved in regulation of pluripotency during normal development and is down-regulated during formation of postnatal reservoir of germ cells. We propose thatOct4/GFP transgenic mouse, which mimics the endogenous expression pattern of Oct4, could be used as a mammalian model to study the effects of environmental estrogens on the development of male germ cells. Oct4/GFP maturation profile was assessed during postnatal days -PND- 3, 5, 7, 10, 14 and 80, using flow cytometry. Then, we exposed pregnant mothers to 17α-ethinylestradiol (EE2) from day post coitum (dpc) 5 to PND7. Percentage of Oct4/GFP-expressing cells and levels of expression of Oct4/GPF were increased in PND7 after EE2 exposure. These observations were confirmed by analysis of GFP and endogenous Oct4 protein in the seminiferous tubules and by a reduction in epididymal sperm count in adult mice. We introduced Oct4/GFP mouse together with flow cytometry as a tool to evaluate changes in male germ cells development. Copyright © 2015 Elsevier Ltd. All rights reserved.
Growth fraction in non-small cell lung cancer estimated by proliferating cell nuclear antigen and comparison with Ki-67 labeling and DNA flow cytometry data.

PubMed Central

Fontanini, G.; Pingitore, R.; Bigini, D.; Vignati, S.; Pepe, S.; Ruggiero, A.; Macchiarini, P.

1992-01-01

Results generated by the immunohistochemical staining with PC10, a new monoclonal antibody recognizing PCNA (a nuclear protein associated with cell proliferation) in formalin-fixed and paraffin-embedded tissue were compared with those of Ki-67 labeling and DNA flow cytometry in 47 consecutive non-small cell lung cancer (NSCLC). PCNA reactivity was observed in all samples and confined to the nuclei of cancer cells. Its frequency ranged from 0 to 80% (37.7 +/- 23.6) and larger sized, early-staged and DNA aneuploid tumors expressed a significant higher number of PCNA-reactive cells. The PCNA and Ki-67 labeling rates were closely correlated (r = 0.383, P = 0.009). By flow cytometry, we observed a good correlation among PCNA labeling and S-phase fraction (r = 0.422, P = .0093) and G1 phase (r = 0.303, P = .051) of the cell cycle. Results indicate that PCNA labeling with PC10 is a simple method for assessing the proliferative activity in formalin-fixed, paraffin-embedded tissue of NSCLC and correlates well with Ki-67 labeling and S-phase fraction of the cell cycle. Images Figure 2 PMID:1361306
High-Throughput Flow Cytometry Identifies Small-Molecule Inhibitors for Drug Repurposing in T-ALL.

PubMed

Perez, Dominique R; Nickl, Christian K; Waller, Anna; Delgado-Martin, Cristina; Woods, Travis; Sharma, Nitesh D; Hermiston, Michelle L; Loh, Mignon L; Hunger, Stephen P; Winter, Stuart S; Chigaev, Alexandre; Edwards, Bruce; Sklar, Larry A; Matlawska-Wasowska, Ksenia

2018-05-01

Kinase inhibitors have dramatically increased patient survival in a multitude of cancers, including hematological malignancies. However, kinase inhibitors have not yet been integrated into current clinical trials for patients with T-cell-lineage acute lymphoblastic leukemia (T-ALL). In this study, we used a high-throughput flow cytometry (HTFC) approach to test a collection of small-molecule inhibitors, including 26 FDA-approved tyrosine kinase inhibitors in a panel of T-ALL cell lines and patient-derived xenografts. Because hypoxia is known to cause resistance to chemotherapy, we developed a synthetic niche that mimics the low oxygen levels found in leukemic bone marrow to evaluate the effects of hypoxia on the tested inhibitors. Drug sensitivity screening was performed using the Agilent BioCel automated liquid handling system integrated with the HyperCyt HT flow cytometry platform, and the uptake of propidium iodide was used as an indication of cell viability. The HTFC dose-response testing identified several compounds that were efficacious in both normal and hypoxic conditions. This study shows that some clinically approved kinase inhibitors target T-ALL in the hypoxic niche of the bone marrow.
Novel full-spectral flow cytometry with multiple spectrally-adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement.

PubMed

Futamura, Koji; Sekino, Masashi; Hata, Akihiro; Ikebuchi, Ryoyo; Nakanishi, Yasutaka; Egawa, Gyohei; Kabashima, Kenji; Watanabe, Takeshi; Furuki, Motohiro; Tomura, Michio

2015-09-01

Flow cytometric analysis with multicolor fluoroprobes is an essential method for detecting biological signatures of cells. Here, we present a new full-spectral flow cytometer (spectral-FCM). Unlike conventional flow cytometer, this spectral-FCM acquires the emitted fluorescence for all probes across the full-spectrum from each cell with 32 channels sequential PMT unit after dispersion with prism, and extracts the signals of each fluoroprobe based on the spectral shape of each fluoroprobe using unique algorithm in high speed, high sensitive, accurate, automatic and real-time. The spectral-FCM detects the continuous changes in emission spectra from green to red of the photoconvertible protein, KikGR with high-spectral resolution and separates spectrally-adjacent fluoroprobes, such as FITC (Emission peak (Em) 519 nm) and EGFP (Em 507 nm). Moreover, the spectral-FCM can measure and subtract autofluorescence of each cell providing increased signal-to-noise ratios and improved resolution of dim samples, which leads to a transformative technology for investigation of single cell state and function. These advances make it possible to perform 11-color fluorescence analysis to visualize movement of multilinage immune cells by using KikGR-expressing mice. Thus, the novel spectral flow cytometry improves the combinational use of spectrally-adjacent various FPs and multicolor fluorochromes in metabolically active cell for the investigation of not only the immune system but also other research and clinical fields of use. © 2015 International Society for Advancement of Cytometry.
Treatment of domestic wastewater by subsurface flow constructed wetlands filled with gravel and tire chip media.

PubMed

Richter, A Y; Weaver, R W

2003-12-01

Subsurface flow constructed wetlands (SFCWs) are becoming increasingly common in on-site treatment of wastewater. Gravel is the most popular form of wetland fill medium, but tire chips provide more porosity, are less dense, and less expensive. This study determines the treatment efficiency of SFCWs filled with gravel or tire chip media to treat domestic wastewater. The influent and effluent of six SFCWs filled with tire chip medium and six SFCWs filled with gravel were monitored for 5 to 16 consecutive months. Parameters measured included pH, biochemical oxygen demand (BOD5), total and volatile suspended solids, NH4, P, and fecal and total coliforms. The only clear difference between medium types in wetland performance was for P. Soluble P in the effluent averaged 1.6 +/- 1.0 mg l(-1) in the tire chip-filled wetlands and 4.8 +/- 3.2 mg l(-1) in the gravel-filled wetlands. Most likely, Fe from exposed wires in shredded steel-belted tires complexed with P to create an insoluble compound. Tire chips may be a better fill medium for SFCWs than gravel because of higher porosity, lower cost, and greater reduction of P in effluent.
The Means: Cytometry and Mass Spectrometry Converge in a Single Cell Deep Profiling Platform

PubMed Central

Weis-Garcia, Frances; Bandura, Dmitry; Baranov, Vladimir; Ornatsky, Olga; Tanner, Scott

2013-01-01

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is a distinct flavor of mass spectrometry that has had little association with cell biology: it remains the state of the art for the determination of the atomic composition of materials. Unrelatedly, flow cytometry is the superior method for distinguishing the heterogeneity of cells through the determination of antigen signatures using tagged antibodies. Simply replacing fluorophore tags with stable isotopes of the heavy metals, and measuring these cell-by-cell with ICP-MS, dramatically increases the number of probes that can be simultaneously measured in cytometry and enables a transformative increase in the resolution of rare cell populations in complex biological samples. While this can be thought of as a novel incarnation of single-cell targeted proteomics, the metal-labeling reagents, ICP-MS of single cells, and accompanying informatics comprise a new field of technology termed Mass Cytometry. While the conception of mass cytometry is simple the embodiment to address the issues of multi-parameter flow cytometry has been far more challenging. There are many elements, and many more stable isotopes of those elements, that might be used as distinct reporter tags. Still, there are many approaches to conjugating metals to antibodies (or other affinity reagents) and work in this area along with developing new applications is ongoing. The mass resolution and linear (quantitative) dynamic range of ICP-MS allows those many stable isotopes to be measured simultaneously and without the spectral overlap issues that limit fluorescence assay. However, the adaptation of ICP-MS to allow high-speed simultaneous measurement with single cell distinction at high throughput required innovation of the cell introduction system, ion optics (sampling, transmission and beam-shaping), mass analysis, and signal handling and processing. An overview of “the nuts and bolts” of Mass Cytometry is presented.

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