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Journal articles on the topic 'Flow cytometers'

Author: Grafiati
Published: 6 September 2023

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1

Chao, Zixi, Yong Han, Zeheng Jiao, Zheng You, and Jingjing Zhao. "Prism Design for Spectral Flow Cytometry." Micromachines 14, no. 2 (January 26, 2023): 315. http://dx.doi.org/10.3390/mi14020315.

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Flow cytometers are instruments used for the rapid quantitative analysis of cell suspension. Traditional flow cytometry uses multi-channel filters to detect fluorescence, whereas full-spectrum fluorescence based on dispersion detection is a more effective and accurate method. The application of various dispersion schemes in flow cytometry spectroscopy has been studied. From the perspective of modern detectors and demand for the miniaturization of flow cytometry, prism dispersion exhibits higher and more uniform light energy utilization, meaning that it is a more suitable dispersion method for small flow cytometers, such as microfluidic flow cytometers. Prism dispersion designs include the size, number, and placement of prisms. By deducing the formula of the final position of light passing through the prism and combining it with the formula of transmittance, the design criteria of the top angle and the incident angle of the prism in pursuit of the optimum transmittance and dispersion index can be obtained. Considering the case of multiple prisms, under the premise of pursuing a smaller size, the optimal design criteria for dispersion system composed of multiple prisms can be obtained. The design of prism dispersion fluorescence detection was demonstrated with a microfluidic flow cytometer, and the effectiveness of the design results was verified by microsphere experiments and practical biological experiments. This design criterion developed in this study is generally applicable to spectral flow cytometers.
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2

Volotovski, I. D., and S. V. Pinchuk. "Flow cytometry. Basics of technology and its application in biology." Proceedings of the National Academy of Sciences of Belarus, Biological Series 67, no. 2 (May 4, 2022): 229–42. http://dx.doi.org/10.29235/1029-8940-2022-67-2-229-242.

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The given review is an attempt of concentrated consideration of flow cytometry problem which is widely used as a fundamental research approach in various fields of biology like cell biology, biophysics, biochemistry and molecular biology and also in applied and diagnostic medicine. The method principle, construction of flow cytometers and their possibilities (study of structure and function state of cell populations and cell sorting), usage of lasers in flow cytometers, wide assortment of fluorophores and monoclonal antibodies. The concrete examples of flow cytometer methods in different experiments are given. The trends in development of flow cytometry are considered.
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Cottingham, Katie. "Incredible shrinking flow cytometers." Analytical Chemistry 77, no. 3 (February 2005): 73 A—76 A. http://dx.doi.org/10.1021/ac053330g.

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4

Anderson, April, and Jolene Bradford. "Rare Event detection using Acoustic Cytometry (144.4)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 144.4. http://dx.doi.org/10.4049/jimmunol.184.supp.144.4.

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Abstract Detection of rare events includes populations comprising less than 5% of the total cells, which involves the detection of stem cells, minimal residual disease and fetomaternal hemorrhage. Analysis of rare cell populations requires the collection of high numbers of events in order to attain a reliable measure of accuracy, leading to long acquisition times. Acoustic cytometry is a new technology which allows dilute samples to be processed quickly. We compared collection times of the Applied Biosystems® Attune™ Acoustic Focusing Cytometer to conventional cytometers. Varying numbers of CD34+ KG-1a cells were spiked into whole blood collected from a normal donor, stained with CD45 & CD34 conjugates, and then collected on traditional and acoustic cytometers. Two hundred thousand live cells were acquired and acquisition time recorded, with results compared across all instruments tested. Rare CD34+ populations were detected on all instruments, giving equivalent results. Acquisition time was significantly reduced using an acoustic cytometer compared to a standard flow cytometer. Acquisition times on the acoustic cytometer can be reduced by up to 5-10 fold compared to traditional flow cytometers, while maintaining comparable results in rare event detection. Utilizing the acoustic focusing cytometer rare event acquisition can be accomplished in one fifth of the time without sacrificing data integrity.
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Shrirao, Anil B., Zachary Fritz, Eric M. Novik, Gabriel M. Yarmush, Rene S. Schloss, Jeffrey D. Zahn, and Martin L. Yarmush. "Microfluidic flow cytometry: The role of microfabrication methodologies, performance and functional specification." TECHNOLOGY 06, no. 01 (March 2018): 1–23. http://dx.doi.org/10.1142/s2339547818300019.

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Flow cytometry is an invaluable tool utilized in modern biomedical research and clinical applications requiring high throughput, high resolution particle analysis for cytometric characterization and/or sorting of cells and particles as well as for analyzing results from immunocytometric assays. In recent years, research has focused on developing microfluidic flow cytometers with the motivation of creating smaller, less expensive, simpler, and more autonomous alternatives to conventional flow cytometers. These devices could ideally be highly portable, easy to operate without extensive user training, and utilized for research purposes and/or point-of-care diagnostics especially in limited resource facilities or locations requiring on-site analyses. However, designing a device that fulfills the criteria of high throughput analysis, automation and portability, while not sacrificing performance is not a trivial matter. This review intends to present the current state of the field and provide considerations for further improvement by focusing on the key design components of microfluidic flow cytometers. The recent innovations in particle focusing and detection strategies are detailed and compared. This review outlines performance matrix parameters of flow cytometers that are interdependent with each other, suggesting trade offs in selection based on the requirements of the applications. The ongoing contribution of microfluidics demonstrates that it is a viable technology to advance the current state of flow cytometry and develop automated, easy to operate and cost-effective flow cytometers.
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6

Shapiro, Howard M., and Nancy G. Perlmutter. "Personal cytometers: Slow flow or no flow?" Cytometry Part A 69A, no. 7 (2006): 620–30. http://dx.doi.org/10.1002/cyto.a.20284.

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7

NOGUCHI, Yoshio, and Yoshio TENJIN. "Laser flow cytometers newly developed." Review of Laser Engineering 15, no. 8 (1987): 647–56. http://dx.doi.org/10.2184/lsj.15.647.

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8

Nitta, Carolina Franco, Mackenzie Pierce, Jeanne Elia, Jen Ruiz, Art-Danniel Hipol, Nicolas Fong, Henry Qazi, et al. "A rapid and high-throughput T cell immunophenotyping assay for cellular therapy bioprocess using the Cellaca PLX image cytometer." Journal of Immunology 210, no. 1_Supplement (May 1, 2023): 250.06. http://dx.doi.org/10.4049/jimmunol.210.supp.250.06.

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Abstract Cellular therapies such as chimeric antigen receptor (CAR) T or NK cells undergo phenotypic characterization during discovery and development of novel therapies, biomanufacturing, and upstream patient cell processing for clinical use. Samples are routinely analyzed via flow cytometry for T cell, NK cell, B cell, and monocyte surface marker expression. Image cytometry systems perform cell-based assays that can be used as a convenient alternative to flow cytometry. Recently, a new high-throughput image cytometer, the Cellaca PLX system (Nexcelom, Lawrence, MA) was developed for immunophenotyping, transfection/transduction efficiency, and cell health assays. This new instrument can assess several critical quality attributes (CQAs) such as cell identity, viability, and other relevant biological functions recommended by the International Organization for Standardization using the ISO Cell Characterization that focuses on cellular therapeutic products. Here we demonstrate a rapid and high-throughput immunophenotyping and viability detection in peripheral blood mononuclear cells (PBMCs) using the Cellaca PLX. PBMCs underwent red blood cell (RBC) lysis and CD3 enrichment, were stained with Hoechst/CD3/CD4/CD8 or Hoechst/CD3/CD8/RubyDead surface marker kits and measured on the Cellaca PLX and three different flow cytometers for comparison. Results showed highly comparable cell populations between the flow cytometers and the Cellaca PLX suggesting that the image cytometer may provide a rapid and convenient alternative method for immunophenotyping. The proposed method can streamline workflows from sample staining to data analysis, quickly moving precious patient samples into downstream processes.
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9

Kotz, Kenneth T., Anne C. Petrofsky, Ramin Haghgooie, Robert Granier, Mehmet Toner, and Ronald G. Tompkins. "Inertial focusing cytometer with integrated optics for particle characterization." TECHNOLOGY 01, no. 01 (September 2013): 27–36. http://dx.doi.org/10.1142/s233954781350009x.

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Microfluidic inertial focusing has been shown as a simple and effective method to localize cells and particles within a flow cell for interrogation by an external optical system. To enable portable point of care optical cytometry, however, requires a reduction in the complexity of the large optical systems that are used in standard flow cytometers. Here, we present a new design that incorporates optical waveguides and focusing elements with an inertial focusing flow cell to make a compact robust cytometer capable of enumerating and discriminating beads, cells, and platelets.
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10

Bang, Hyunwoo, Hoyoung Yun, Won Gu Lee, Junha Park, Joonmo Lee, Seok Chung, Keunchang Cho, Chanil Chung, Dong-Chul Han, and Jun Keun Chang. "Expansion channel for microchip flow cytometers." Lab on a Chip 6, no. 10 (2006): 1381. http://dx.doi.org/10.1039/b604578b.

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11

Steen, Harald B., and Trond Stokke. "Dye exclusion artifact in flow cytometers." Cytometry 47, no. 3 (February 19, 2002): 200–205. http://dx.doi.org/10.1002/cyto.10063.

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12

Bernander, Rolf, Trond Stokke, and Erik Boye. "Flow cytometry of bacterial cells: Comparison between different flow cytometers and different DNA stains." Cytometry 31, no. 1 (January 1, 1998): 29–36. http://dx.doi.org/10.1002/(sici)1097-0320(19980101)31:1<29::aid-cyto4>3.0.co;2-e.

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13

Guenther, Garret, and William Telford. "Comparison of flow cytometers using the side population technique to identify stem cells (TECH2P.900)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 206.10. http://dx.doi.org/10.4049/jimmunol.194.supp.206.10.

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Abstract Traditional methods using the side population analysis to identify stem cells involved the use of a flow cytometer with an ultra violet laser excited dye. This presents some challenges for researchers in relation to costs, and even though other dyes such as DyeCycle Violet (DCV) are now available that use the more economical violet laser, the cost of a flow cytometer to provide this type of analysis is still an obstacle. More recently, new benchtop flow cytometers have become available that can provide the quality of a high-end flow cytometer at a much more affordable cost. In this analysis, we sought out to identify stem cells and progenitors using the side population technique in murine bone marrow using the ACEA NovoCyte flow cytometer which maximizes optical design through its use of shared PMTs. Samples were stained and evaluated with (DCV), Lineage markers, Sca, and c-kit. To identify stem cells using the side population technique, we first gated on Lineage- cells and subsequently on Sca+, c-kit+. To confirm the correct identification of these cells, this population was back gated comparing DCV red fluorescence vs DCV blue fluorescence and verified the cells are localized to the expected tail-region. This study is evidence that quality data can be generated on a budget-friendly flow cytometer and allows researchers with limited resources to use flow cytometry.
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Bradford, Jolene A., Justin Meskas, and Patricia Sardina. "Evaluation of Cytometer Sensitivity and Stability using Automated Analysis." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 131.41. http://dx.doi.org/10.4049/jimmunol.202.supp.131.41.

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Abstract Flow cytometers are standard tools of the immunologist, providing simultaneous measurement of many targets. The performance of a flow cytometer may vary between instruments, or over time within the same instrument. Evaluation of cytometer performance is essential to ensure results are consistent over time within the same instrument for longitudinal studies and day-to-day data consistency, and across multiple instruments when more than one cytometer is being utilized. Basic capabilities of each fluorescent detector can be estimated using the Q & B calculations, where Q is detection efficiency and B is background. Precision of measurements and minimum detectable dye can be derived from Q & B measurements, to evaluate sensitivity and stability. In this study hard-dyed multi-level, multi-dye beads were collected in three different identically configured cytometers over 11 days. Each day data was collected in 14 different fluorescent detectors at six flow rates. An automated approach to data analysis was utilized to provide unbiased evaluation. All data files were first evaluated with flowCut to check for spurious events. Using automated gating, beads were then identified using Forward Scatter vs. Side Scatter, and flowQB was used to calculate Q & B. This data was used to evaluate intra-instrument and inter-instrument sensitivity and stability. Results show the minimum amount of detectable dye and precision of its measurement are robust across time and different cytometers, though decreased at the highest flow rates of ≥ 500 μL/min. Using an automated unbiased approach for determining Q & B demonstrates cytometer sensitivity and consistency over time, between instruments, and across flow rates.
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15

Zhang, Ting, Mengge Gao, Xiao Chen, Chiyuan Gao, Shilun Feng, Deyong Chen, Junbo Wang, Xiaosu Zhao, and Jian Chen. "Demands and technical developments of clinical flow cytometry with emphasis in quantitative, spectral, and imaging capabilities." Nanotechnology and Precision Engineering 5, no. 4 (December 1, 2022): 045002. http://dx.doi.org/10.1063/10.0015301.

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As the gold-standard method for single-cell analysis, flow cytometry enables high-throughput and multiple-parameter characterization of individual biological cells. This review highlights the demands for clinical flow cytometry in laboratory hematology (e.g., diagnoses of minimal residual disease and various types of leukemia), summarizes state-of-the-art clinical flow cytometers (e.g., FACSLyricTM by Becton Dickinson, DxFLEX by Beckman Coulter), then considers innovative technical improvements in flow cytometry (including quantitative, spectral, and imaging approaches) to address the limitations of clinical flow cytometry in hematology diagnosis. Finally, driven by these clinical demands, future developments in clinical flow cytometry are suggested.
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16

Yang, Ruey-Jen, Lung-Ming Fu, and Hui-Hsiung Hou. "Review and perspectives on microfluidic flow cytometers." Sensors and Actuators B: Chemical 266 (August 2018): 26–45. http://dx.doi.org/10.1016/j.snb.2018.03.091.

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17

Steen, Harald B. "A sample injection device for flow cytometers." Cytometry 49, no. 2 (September 30, 2002): 70–72. http://dx.doi.org/10.1002/cyto.10147.

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18

Steen, Harald B. "Noise, Sensitivity, and Resolution of Flow Cytometers." Cytometry 13, no. 8 (1992): 822–30. http://dx.doi.org/10.1002/cyto.990130804.

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19

Kaleem, Zahid. "Flow Cytometric Analysis of Lymphomas: Current Status and Usefulness." Archives of Pathology & Laboratory Medicine 130, no. 12 (December 1, 2006): 1850–58. http://dx.doi.org/10.5858/2006-130-1850-fcaolc.

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Abstract Context.—Immunophenotyping has become a routine practice in the diagnosis and classification of most cases of non-Hodgkin lymphoma, and flow cytometry is often the method of choice in many laboratories. The role that flow cytometry plays, however, extends beyond just diagnosis and classification. Objective.—To review and evaluate the current roles of flow cytometry in non-Hodgkin lymphoma, to compare it with immunohistochemistry, and to discuss its potential future applications in the molecular diagnostic era. Data Sources.—The information contained herein is derived from peer-reviewed articles on the subject published in the English-language medical literature during the years 1980 to 2005 that were identified using PubMed (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi, 1980–2005) search, various books and other sources on flow cytometry, and the author's personal experience of more than 10 years with flow cytometric analysis of lymphomas and leukemia using Becton-Dickinson (San Jose, Calif) and Beckman-Coulter (Miami, Fla) flow cytometers. Study Selection.—Studies were selected based on adequate material and methods, statistically significant results, and adequate clinical follow-up. Data Extraction.—The data from various sources were compared when the methods used were the same or similar and appropriate controls were included. Most of the studies employed 2-color, 3-color, or 4-color flow cytometers with antibodies from Becton-Dickinson, Beckman-Coulter, or DakoCytomation (Carpinteria, Calif). Results were evaluated from studies utilizing the same or similar techniques and flow cytometers. Only objective data analyses from relevant and useful publications were included for reporting and discussion. Data Synthesis.—Flow cytometry serves a variety of roles in the field of lymphoma/leukemia including rapid diagnosis, proper classification, staging, minimal residual disease detection, central nervous system lymphoma detection, evaluation of prognostic markers, detection of target molecules for therapies, ploidy analysis of lymphoma cell DNA, and evaluation of multidrug-resistance markers. It offers many advantages in comparison to immunohistochemistry for the same roles and provides uses that are either not possible or not preferable by immunohistochemistry such as multiparameter evaluation of single cells and detection of clonality in T cells. Conclusions.—By virtue of its ability to evaluate not only surface but also cytoplasmic and nuclear antigens, flow cytometry continues to enjoy widespread use in various capacities in lymphoma evaluation and treatment. Additional roles for flow cytometry are likely to be invented in the future and should provide distinctive uses in the molecular era.
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20

Hyrkas, Jeremy, Daniel Halperin, and Bill Howe. "Time-Varying Clusters in Large-Scale Flow Cytometry." Proceedings of the AAAI Conference on Artificial Intelligence 29, no. 2 (January 25, 2015): 4022–23. http://dx.doi.org/10.1609/aaai.v29i2.19067.

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Flow cytometers measure the optical properties of particles to classify microbes. Recent innovations have allowed oceanographers to collect flow cytometry data continuously during research cruises, leading to an explosion of data and new challenges for the classification task. The massive scale, time-varying underlying populations, and noisy measurements motivate the development of new classification methods. We describe the problem, the data, and some preliminary results demonstrating the difficulty with conventional methods.
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Lucchetti, Donatella, Alessandra Battaglia, Claudio Ricciardi-Tenore, Filomena Colella, Luigi Perelli, Ruggero De Maria, Giovanni Scambia, Alessandro Sgambato, and Andrea Fattorossi. "Measuring Extracellular Vesicles by Conventional Flow Cytometry: Dream or Reality?" International Journal of Molecular Sciences 21, no. 17 (August 29, 2020): 6257. http://dx.doi.org/10.3390/ijms21176257.

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Intense research is being conducted using flow cytometers available in clinically oriented laboratories to assess extracellular vesicles (EVs) surface cargo in a variety of diseases. Using EVs of various sizes purified from the HT29 human colorectal adenocarcinoma cell line, we report on the difficulty to assess small and medium sized EVs by conventional flow cytometer that combines light side scatter off a 405 nm laser with the fluorescent signal from the EVs general labels Calcein-green and Calcein-violet, and surface markers. Small sized EVs (~70 nm) immunophenotyping failed, consistent with the scarcity of monoclonal antibody binding sites, and were therefore excluded from further investigation. Medium sized EVs (~250 nm) immunophenotyping was possible but their detection was plagued by an excess of coincident particles (swarm detection) and by a high abort rate; both factors affected the measured EVs concentration. By running samples containing equal amounts of Calcein-green and Calcein-violet stained medium sized EVs, we found that swarm detection produced false double positive events, a phenomenon that was significantly reduced, but not totally eliminated, by sample dilution. Moreover, running highly diluted samples required long periods of cytometer time. Present findings raise questions about the routine applicability of conventional flow cytometers for EV analysis.
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Pugsley, Haley Renee, and Alexandra G. Sutton. "Analysis of STAT Protein Phosphorylation in T Cells, NK Cells, and B Cells Using High Sensitivity Flow Cytometry." Journal of Immunology 210, no. 1_Supplement (May 1, 2023): 168.10. http://dx.doi.org/10.4049/jimmunol.210.supp.168.10.

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Abstract In the immune system, cytokine-induced protein phosphorylation is a key cell signaling event that modulates diverse functions, including proliferation, differentiation, apoptosis, and migration. The degree of protein phosphorylation is determined by both the cytokine concentration and the number of cytokine-specific receptors on the cell surface, which are differentially expressed across immune cell populations. Signal transducer and activator of transcription (STAT) proteins are transcription factors that translocate to the nucleus upon activation by tyrosine phosphorylation and alter gene expression. Cytokines such as interleukins (IL) initiate the signaling cascade that activates STAT proteins. In addition, phosphorylated STAT3 and STAT6 have been associated with prognosis in cancer and other malignancies. Flow cytometry is a powerful method to study the orchestrated response of immune cell subsets to stimulation by combining immunophenotyping markers with fluorescent antibodies specific to phosphorylated signaling molecules. However, because many phosphorylation events occur on weakly expressed proteins and with low stoichiometry, flow cytometers must be highly sensitive to track the subtle changes in signaling magnitude. Flow cytometers with sensitive detection, such as the Amnis ®systems, can be used to study phosphorylation events that are key to understanding the processes required for vaccine and immunotherapy development. In this study, we used the Amnis ®CellStream ®Flow Cytometer to monitor the phosphorylation of STAT3 and STAT6 in CD4+ T cells, NK cells, and B cells after stimulation with IL-4 and IL-6.
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de Rutte, Joseph, Robert Dimatteo, Sheldon Zhu, Maani M. Archang, and Dino Di Carlo. "Sorting single-cell microcarriers using commercial flow cytometers." SLAS Technology 27, no. 2 (April 2022): 150–59. http://dx.doi.org/10.1016/j.slast.2021.10.004.

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Wood, James C. S., and Robert A. Hoffman. "Evaluating fluorescence sensitivity on flow cytometers: An overview." Cytometry 33, no. 2 (October 1, 1998): 256–59. http://dx.doi.org/10.1002/(sici)1097-0320(19981001)33:2<256::aid-cyto22>3.0.co;2-s.

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25

deMello, Andrew, Anand Rane, Gregor Holzner, and Stavros Stavrakis. "Ultra-High-Throughput Multi-Parametric Imaging Flow Cytometry." EPJ Web of Conferences 215 (2019): 10001. http://dx.doi.org/10.1051/epjconf/201921510001.

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I will present a microfluidic imaging flow cytometer incorporating stroboscopic illumination, for blur-free cellular analysis at throughputs exceeding 100,000 cells per second. By combining passive (inertial or viscoelastic) focusing of cells in parallel microchannels with stroboscopic illumination, such chip-based cytometers are able to extract multi-colour fluorescence and bright-field images of single cells moving at high linear velocities. This in turn allows accurate sizing of individual cells, intracellular localization and analysis of heterogeneous cell suspensions. The method is showcased through the rapid enumeration of apoptotic cells, high-throughput discrimination cell cycle phases and localization of p-bodies.
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Lin, Bo, Leo Chan, Ning Lai, Alnoor Pirani, Tim Smith, Emily Lyettefi, and Jean Qiu. "Obtain consistent and accurate PBMC cell counting results with Cellometer automatic cell counter (65.14)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 65.14. http://dx.doi.org/10.4049/jimmunol.186.supp.65.14.

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Abstract Traditional methods for fluorescence-based PBMC analysis commonly employ manual hemacytometer and standard flow cytometry for concentration and viability measurement. Manual cell counting requires trained technicians, which is time-consuming and prone to human error. In addition, PBMC samples are usually contaminated with RBCs and platelets, which may increase manual counting difficulty. Standard flow cytometry remains expensive, large in size, and require considerable amount of maintenance, which may not be ideal for research laboratories that need a cost-effective method. In addition, traditional flow cytometers do not have imaging capabilities, which often generate some uncertainties in the fluorescence results obtained. Recently, an imaging cytometry platform has been developed by Nexcelom Bioscience. This system allows automated cell image acquisition and processing with a novel counting algorithm for accurate and consistent measurement of cell population and viability with 20 μl of PBMCs sample. In this work, we demonstrate a rapid and cost-effective method for concentration and viability measurement of PBMCs using the Cellometer imaging cytometry method. This method has the ability to resolve the issues caused by manual hemacytometer and flow cytometer. By using Cellometer imaging cytometry, the assay time for generating concentration and viability result is greatly reduced, which is significant for research development in the biomedical and clinical studies.
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Smith, R. W. "Non-Imaging Microscopies: Flow Cytometry as a Correlative Analytical Tool in the Quantification of Cell Structure, Autofluorescence, Fluorescent Probes and Cell Populations." Microscopy and Microanalysis 5, S2 (August 1999): 490–91. http://dx.doi.org/10.1017/s1431927600015774.

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Non-imaging microscopy has developed somewhat independently of both traditional light microscopy and laser confocal microscopy. Flow cytometry is the chief commercial and research technology among these microscopies, and, like other nonimaging detection systems, developed around the theme of automation in clinical laboratory medicine. It is an important correlative or parallel microscopy to several image forming microscopical methods. Cell sorting is an important option as well.The basic structure of the flow cytometer certainly parallels light, laser and electron microscopes. The flow cytometer has a light source, a set of adjustable optics to focus the beam on the specimen, objective optics to collect the light and direct it to appropriate sensors, and the sensors themselves. A real image is not formed because the sensors are not in an even plane with the projection, such as provided by the retina in light microscopy or an image plane or film plate in electron microscopy, and the objective optics may not focus in the image plane.While early flow cytometers were developed primarily for the automatic counting of cells and particles, modern instruments offer particular advantages for the analysis of fluorescence, fluorescent chemicals and probes and cellular auto fluorescence.
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Chaturvedi, Akhil, and Sai Siva Gorthi. "Automated Blood Sample Preparation Unit (ABSPU) for Portable Microfluidic Flow Cytometry." SLAS TECHNOLOGY: Translating Life Sciences Innovation 22, no. 1 (September 26, 2016): 73–80. http://dx.doi.org/10.1177/2211068216663604.

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Portable microfluidic diagnostic devices, including flow cytometers, are being developed for point-of-care settings, especially in conjunction with inexpensive imaging devices such as mobile phone cameras. However, two pervasive drawbacks of these have been the lack of automated sample preparation processes and cells settling out of sample suspensions, leading to inaccurate results. We report an automated blood sample preparation unit (ABSPU) to prevent blood samples from settling in a reservoir during loading of samples in flow cytometers. This apparatus automates the preanalytical steps of dilution and staining of blood cells prior to microfluidic loading. It employs an assembly with a miniature vibration motor to drive turbulence in a sample reservoir. To validate performance of this system, we present experimental evidence demonstrating prevention of blood cell settling, cell integrity, and staining of cells prior to flow cytometric analysis. This setup is further integrated with a microfluidic imaging flow cytometer to investigate cell count variability. With no need for prior sample preparation, a drop of whole blood can be directly introduced to the setup without premixing with buffers manually. Our results show that integration of this assembly with microfluidic analysis provides a competent automation tool for low-cost point-of-care blood-based diagnostics.
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Miyake, Ryo, Hiroshi Ohki, and Isao Yamazaki. "Investigation of Sheath Flow Chambers for Flow Cytometers. 1st Report, Stabilization of Sheath Flow." Transactions of the Japan Society of Mechanical Engineers Series B 61, no. 591 (1995): 4039–45. http://dx.doi.org/10.1299/kikaib.61.4039.

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30

Rogers, Clare, Melanie Gubbels Bupp, Unyoung Kim, Gregory DeKrey, Sharon Stranford, Rheem Medh, and Gloria Tomich. "Successful integration of practical flow cytometric experience into undergraduate education. (51.4)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 51.4. http://dx.doi.org/10.4049/jimmunol.186.supp.51.4.

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Abstract Flow cytometry is an essential tool in almost every discipline of cell biology, and is increasingly utilized in a variety of other associated yet diverse fields, including molecular biology, bioengineering, microbiology and marine biology. For many years, access to the use of flow cytometers as teaching tools, even in large universities, was limited to graduate programs, due to the cost of instrument purchase and maintenance, and to the complexity of operation. Recently, several affordable, bench-top flow cytometers have appeared on the market. The affordability and user-friendly nature of these instruments have made them attractive to the faculty of various departments at medium- and small-sized universities and colleges for use both as teaching tools and in faculty research projects. We have compiled information here on the funding sources for both purchase and maintenance of these instruments at our various institutions, and share information on applications, protocols and lab exercises that have worked well with our students. Example protocols described include applications such as immunophenotyping, apoptosis (annexin/propidium iodide), monocytic phagocytosis, intracellular and mitochondrial reactive oxygen species detection, and detection of fluorescent protein expression in bacteria.
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31

Edwards, Bruce S., and Larry A. Sklar. "Flow Cytometry." Journal of Biomolecular Screening 20, no. 6 (March 24, 2015): 689–707. http://dx.doi.org/10.1177/1087057115578273.

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Modern flow cytometers can make optical measurements of 10 or more parameters per cell at tens of thousands of cells per second and more than five orders of magnitude dynamic range. Although flow cytometry is used in most drug discovery stages, “sip-and-spit” sampling technology has restricted it to low-sample-throughput applications. The advent of HyperCyt sampling technology has recently made possible primary screening applications in which tens of thousands of compounds are analyzed per day. Target-multiplexing methodologies in combination with extended multiparameter analyses enable profiling of lead candidates early in the discovery process, when the greatest numbers of candidates are available for evaluation. The ability to sample small volumes with negligible waste reduces reagent costs, compound usage, and consumption of cells. Improved compound library formatting strategies can further extend primary screening opportunities when samples are scarce. Dozens of targets have been screened in 384- and 1536-well assay formats, predominantly in academic screening lab settings. In concert with commercial platform evolution and trending drug discovery strategies, HyperCyt-based systems are now finding their way into mainstream screening labs. Recent advances in flow-based imaging, mass spectrometry, and parallel sample processing promise dramatically expanded single-cell profiling capabilities to bolster systems-level approaches to drug discovery.
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BROWN, MICHAEL C., ROBERT A. HOFFMAN, and STEFAN J. KIRCHANSKI. "Controls for Flow Cytometers in Hematology and Cellular Immunology." Annals of the New York Academy of Sciences 468, no. 1 Clinical Cyto (June 1986): 93–103. http://dx.doi.org/10.1111/j.1749-6632.1986.tb42032.x.

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PETEGEM, M., R. CARTUYVELS, P. SCHOUWER, V. DUPPEN, W. GOOSSENS, and L. HOVE. "Comparative evaluation of three flow cytometers for reticulocyte enumeration." Clinical & Laboratory Haematology 15, no. 2 (June 28, 2008): 103–11. http://dx.doi.org/10.1111/j.1365-2257.1993.tb00133.x.

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Wang, Jun Sheng, You Nan Song, Jin Yang Sun, Hui Chu, Jin Hu Jiang, Xin Xiang Pan, Ye Qing Sun, and Dong Qing Li. "A Microfluidic Cytometer for Quantitative Evaluation of Radiation Dose by γ-H2AX." Applied Mechanics and Materials 522-524 (February 2014): 1119–22. http://dx.doi.org/10.4028/www.scientific.net/amm.522-524.1119.

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Evaluation of radiation dose is very important for the detection of radiation damage. γ-H2AX is a popular biological dosimeter to evaluate the radiation effect. Typically, bulky and expensive commercial flow cytometers are used to detect γ-H2AX. This paper presents a miniaturized and highly sensitive cytometer using a microfluidic chip for evaluating the radiation dose by detecting the mean immunofluorescence intensity of γ-H2AX. A compact optical focusing system and a shift-phase differential amplifier are designed to improve the detection sensitivity. Sample lymphocyte cells are stained by FITC fluorescent dye after being irradiated by UVC. Comparison experiments between the developed miniature cytometer and a commercial flow cytometer were conducted under different radiation doses. The developed microfluidic cytometer can also demonstrate a good linear correlation between the measured fluorescence intensity and the irradiation dose with a detection limit similar to that of the commercial flow cytometer. The developed cytometer can evaluate quantitatively the radiation dose by the mean fluorescence intensity of γ-H2AX with a significantly smaller amount of blood samples than a commercial flow cytometer.
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Sukovich, David J., Samuel C. Kim, Noorsher Ahmed, and Adam R. Abate. "Bulk double emulsification for flow cytometric analysis of microfluidic droplets." Analyst 142, no. 24 (2017): 4618–22. http://dx.doi.org/10.1039/c7an01695f.

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36

Pugsley, Haley Renee, Bryan R. Davidson, and Philip Morrissey. "Immunophenotyping extracellular vesicles using the CellStream flow cytometer." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 131.5. http://dx.doi.org/10.4049/jimmunol.202.supp.131.5.

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Abstract Extracellular vesicles are membrane derived structures that include exosomes, microvesicles and apoptotic bodies. The importance of extracellular vesicles as key mediators of intercellular communication is not well understood. Exosomes have been shown to transfer molecules between cells, potentially transmitting signals. Exosomes are released under normal physiological conditions; however, they are also believed to serve as mediators in the pathogenesis of neurological, vascular, haematological and autoimmune diseases as well as cancer. Quantifying and characterizing extracellular vesicles in a reproducible and reliable manner is challenging due to their small size (exosomes range from 30 to 100 nm in diameter). Extracellular vesicle analysis can be done using high-magnification microscopy; however, this technique has a very low throughput. Attempts to analyze extracellular vesicles using traditional PMT based flow cytometers has been hampered by the limit of detection of such small particles and their low refractive index. To overcome these limitations, we have employed the Amnis CellStream that has the advantage of high throughput flow cytometry with higher sensitivity to small particles due to the CCD based, time-delay-integration image capturing system. Data will be presented using the CellStream flow cytometer to immunophenotype extracellular vesicles derived from red blood cells and platelets.
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WANG, XIANWEN, FENG CHEN, ZHI CHENG, YAOHUA DU, and TAIHU WU. "AUTOMATED GATING OF PORTABLE CYTOMETER DATA BASED ON SKEW t MIXTURE MODELS." Journal of Mechanics in Medicine and Biology 15, no. 03 (June 2015): 1550033. http://dx.doi.org/10.1142/s0219519415500335.

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A major component of flow cytometry (FCM) data analysis involves gating, which is the process of identifying homogeneous groups of cells. With the rapid development of the portable flow cytometer, manual gating techniques have been unable to meet the demand for accurate and rapid analysis of samples. To provide a practical application for portable devices, we propose a flexible, statistical model-based clustering approach for identifying cell populations in FCM data. This approach, which mimics the manual gating process, employs a finite mixture model with a density function of skew t distribution and estimates parameters via an expectation maximization algorithm. Data analysis from an experiment on a patient’s peripheral blood samples have proven that the proposed methodology yields better results in terms of robustness against outliers than current state-of-the-art automated gating methods, has more flexibility in clustering symmetric data and leads to lower misclassification rates (misclassification rates of skew t method is 0.06442) when handling highly asymmetric data. The method we proposed will improve data analysis of portable flow cytometers, especially when the users have no professional training.
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de Bruijn, Douwe S., Koen F. A. Jorissen, Wouter Olthuis, and Albert van den Berg. "Determining Particle Size and Position in a Coplanar Electrode Setup Using Measured Opacity for Microfluidic Cytometry." Biosensors 11, no. 10 (September 23, 2021): 353. http://dx.doi.org/10.3390/bios11100353.

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Microfluidic impedance flow cytometers enable high-throughput, non-invasive, and label-free detection of single-cells. Cytometers with coplanar electrodes are easy and cheap to fabricate, but are sensitive to positional differences of passing particles, owing to the inhomogeneous electric field. We present a novel particle height compensation method, which employs the dependence of measured electrical opacity on particle height. The measured electrical opacity correlates with the particle height as a result of the constant electrical double layer series capacitance of the electrodes. As an alternative to existing compensation methods, we use only two coplanar electrodes and multi-frequency analysis to determine the particle size of a mixture of 5, 6, and 7 µm polystyrene beads with an accuracy (CV) of 5.8%, 4.0%, and 2.9%, respectively. Additionally, we can predict the bead height with an accuracy of 1.5 µm (8% of channel height) using the measured opacity and we demonstrate its application in flow cytometry with yeast. The use of only two electrodes is of special interest for simplified, easy-to-use chips with a minimum amount of instrumentation and of limited size.
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Lee, Gwo-Bin, Che-Hsin Lin, and Guan-Liang Chang. "Micro flow cytometers with buried SU-8/SOG optical waveguides." Sensors and Actuators A: Physical 103, no. 1-2 (January 2003): 165–70. http://dx.doi.org/10.1016/s0924-4247(02)00305-9.

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Tsai, Chien-Hsiung, Hui-Hsiung Hou, and Lung-Ming Fu. "An optimal three-dimensional focusing technique for micro-flow cytometers." Microfluidics and Nanofluidics 5, no. 6 (April 22, 2008): 827–36. http://dx.doi.org/10.1007/s10404-008-0284-6.

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MIYAKE, Ryo, Hiroshi OHKI, Isao YAMAZAKI, and Takeo TAKAGI. "Investigation of Sheath Flow Chambers for Flow Cytometers (Micro Machined Flow Chamber with Low Pressure Loss)." JSME International Journal Series B 40, no. 1 (1997): 106–13. http://dx.doi.org/10.1299/jsmeb.40.106.

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Ding, Mei, Roger Clark, Catherine Bardelle, Anna Backmark, Tyrrell Norris, Wendy Williams, Mark Wigglesworth, and Rob Howes. "Application of High-Throughput Flow Cytometry in Early Drug Discovery: An AstraZeneca Perspective." SLAS DISCOVERY: Advancing the Science of Drug Discovery 23, no. 7 (May 22, 2018): 719–31. http://dx.doi.org/10.1177/2472555218775074.

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Flow cytometry is a powerful tool providing multiparametric analysis of single cells or particles. The introduction of faster plate-based sampling technologies on flow cytometers has transformed the technology into one that has become attractive for higher throughput drug discovery screening. This article describes AstraZeneca’s perspectives on the deployment and application of high-throughput flow cytometry (HTFC) platforms for small-molecule high-throughput screening (HTS), structure–activity relationship (SAR) and phenotypic screening, and antibody screening. We describe the overarching HTFC workflow, including the associated automation and data analysis, along with a high-level overview of our HTFC assay portfolio. We go on to discuss the practical challenges encountered and solutions adopted in the course of our deployment of HTFC, as well as future enhancements and expansion of the technology to new areas of drug discovery.
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43

Shapiro, Howard M., and Howard M. Shapiro. "Gently Down The Stream: Flow Cytometry As Microscopy." Microscopy and Microanalysis 7, S2 (August 2001): 610–11. http://dx.doi.org/10.1017/s1431927600029123.

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Flow cytometry is an analytical technique in which optical measurements are made of cells or other biological particles as the cells or particles flow, ideally in single file, in a fluid stream past one or more optical measurement stations. Modern optical flow cytometers typically measure light scattered at small (1-5°) and large (15°-135°) angles to an illuminating laser beam, and fluorescence emitted in three or more discrete spectral bands; the most complex instruments employ three or four spatially separated illuminating beams at different wavelengths and can measure twelve fluorescence signals from each cell or particle analyzed.Flow cytometry was developed in the 1960's to speed up and automate microspectrophotometry and microfluorometry, which had previously been microscope-based, in order to facilitate development of observer-independent clinical tests which could replace the Papanicolaou smear and the differential leukocyte count. At that time, high-resolution scanning was an extremely slow process; high-intensity sources were restricted to arc lamps, and memory in which images could be stored was expensive.
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Welsh, Joshua, Julia Kepley, Ariel Rosner, Peter Horak, Jay Berzofsky, and Jennifer Jones. "Prospective Use of High-Refractive Index Materials for Single Molecule Detection in Flow Cytometry." Sensors 18, no. 8 (August 1, 2018): 2461. http://dx.doi.org/10.3390/s18082461.

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Phenotyping extracellular vesicles (EVs), where surface receptor expression is often as low as one molecule per EV, remains problematic due to the inability of commercial flow cytometers to provide single-fluorescent molecule sensitivity. While EVs are widely considered to be of great potential as diagnostic, prognostic and theranostic biomarkers, their use is currently hindered by the lack of tools available to accurately and reproducibly enumerate and phenotype them. Herein, we propose a new class of labels that leverage the biophysical properties of materials with unique complex refractive indices and demonstrate that this class of labels has the possibility of allowing single-epitope detection using conventional flow cytometry.
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Lin, Che-Hsin, and Gwo-Bin Lee. "Micromachined flow cytometers with embedded etched optic fibers for optical detection." Journal of Micromechanics and Microengineering 13, no. 3 (April 11, 2003): 447–53. http://dx.doi.org/10.1088/0960-1317/13/3/315.

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Rahman, Md Mizanur, Susane Giti, and Debashish Saha. "Flow Cytomerty: Clinical Applications in Haemato-Oncology." Journal of Armed Forces Medical College, Bangladesh 11, no. 1 (December 15, 2016): 74–80. http://dx.doi.org/10.3329/jafmc.v11i1.30677.

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In the past decade, the use of flow cytometry in the clinical haematology laboratory has grown substantially due to the development of smaller, user-friendly, less-expensive instruments and a continuous increase in the number of clinical applications. Multiple characteristics of single cells can be analyzed rapidly by flow cytometry. Both qualitative and quantitative information are obtained by flow cytometry whereas previously only in research institutions and esteemed academic centres flow cytometers were found. With advances in technology now it is possible for secondary level hospitals to use this methodology. This paper reviews the selected applications of flow cytometry in the clinical haematology laboratory in Bangladesh. This review serves to awaken the interest of stakeholders involved in the diagnosis and management of haematological malignancies (HM) in the efficacy of flow cytometry in the immunophenotypic characterization of leukaemias and lymphomas. Relevant literature including those provided by different international consensus groups on the phenotypic characterization of HM was reviewed. Additionally, recent reports on the immunophenotypic analysis of HM published in haematology, oncology, pathology, immunology and cell biology journals were also analyzed. Flow cytometric immunophenotyping of HM is highly demanding. It is highly useful in profiling the leukaemias and lymphomas and allows proper ramification along the latest WHO classification guidelines, thereby paving the way for targeted therapy and clinical trial-driven management, significantly outweighs the cost, which can be fully recovered if properly managed. In a low-resource setting like Bangladesh, limited immunohistochemistry serves to bridge the gap in technological advancement.Journal of Armed Forces Medical College Bangladesh Vol.11(1) 2015: 74-80
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47

MIYAKE, Ryo, Hiroshi OHKI, Isao YAMAZAKI, and Takeo TAKAGI. "Investigation of Sheath Flow Chambers for Flow Cytometers. 2nd Report. Micro machined Flow Chamber with Low Pressure Loss." Transactions of the Japan Society of Mechanical Engineers Series B 62, no. 597 (1996): 1693–99. http://dx.doi.org/10.1299/kikaib.62.1693.

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48

Janossy, George, Ilesh V. Jani, Nicholas J. Bradley, Arsene Bikoue, Tim Pitfield, and Debbie K. Glencross. "Affordable CD4+-T-Cell Counting by Flow Cytometry: CD45 Gating for Volumetric Analysis." Clinical and Vaccine Immunology 9, no. 5 (September 2002): 1085–94. http://dx.doi.org/10.1128/cdli.9.5.1085-1094.2002.

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ABSTRACT The flow cytometers that are currently supported by industry provide accurate CD4+-T-cell counts for monitoring human immunodeficiency virus disease but remain unaffordable for routine service work under resource-poor conditions. We therefore combined volumetric flow cytometry (measuring absolute lymphocyte counts in unit volumes of blood) and simpler protocols with generic monoclonal antibodies (MAbs) to increase cost efficiency. Volumetric absolute counts were generated using CD45/CD4 and CD45/CD8 MAb combinations in two parallel tubes. The percentage values for the various subsets were also determined within the leukocyte and lymphocyte populations utilizing a fully automated protocol. The levels of agreement between the newly developed method and the present industry standards, including both volumetric and bead-based systems using a full MAb panel for subset analysis, were tested by Bland-Altman analyses. The limits of agreement for CD4 counts generated by the volumetric methods using either CD45/CD4 (in a single tube) or the full Trio MAb panel (in three tubes) on the CytoronAbsolute flow cytometer were between −29 and +46 cells/mm3 with very little bias for CD4 counts (in favor of the Trio method: +8 CD4+ lymphocytes/mm3; 0.38% of lymphocytes). The limits of agreement for absolute CD4 counts yielded by the volumetric CD45/CD4 method and the bead-based method were between −118 and +98 cells/mm3, again with a negligible bias (−10 CD4+ lymphocytes/mm3). In the volumetric method using CD45/CD8, the strongly CD8+ cells were gated and the levels of agreement with the full Trio showed a minor bias (in favor of the Trio; +40 CD8+ cells/mm3; 5.2% of lymphocytes) without a significant influence on CD4/CD8 ratios. One trained flow cytometrist was able to process 300 to 400 stained tubes per day. This workload extrapolates to a throughput of >30,000 samples per year if both CD45/CD4 and CD45/CD8 stainings are performed for each patient or a throughput of >60,000 samples if only CD45/CD4 counts are tested in a single tube. Thus, on the basis of the high efficiency and excellent agreement with the present industry standards, volumetric flow cytometers with automated gating protocols and autobiosamplers, complemented by generic CD45, CD4, and CD8 MAbs used in two-color immunofluorescence, represent the most suitable arrangements for large regional laboratories in resource-poor settings.
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Mulama, David, Roman J. Riveria, Kimberly McKinney, Divya Gandra, Kaitlyn Smith, Nicholas Tastet, and Giselle L. Saulnier Sholler. "Abstract 3053: Comparative validation of neuroblastoma cell lines using flow cytometry and CyToF." Cancer Research 83, no. 7_Supplement (April 4, 2023): 3053. http://dx.doi.org/10.1158/1538-7445.am2023-3053.

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Abstract Introduction The validation of patient derived cell lines and other biologics has gained importance due to more frequent laboratory collaborations. Polychromatic flow cytometry has been the gold standard for detection and analysis of biochemical, structural complexity and characterization of cellular particles in biological systems. Despite the tremendous advancement in flow cytometry from the earlier simple, slow version of 1960s to more recent complex, fast spectral analyzers with comprehensive structural and functional immune profiling capability, this development has not matched the ever-increasing need for high throughput output to phenotype cells. Mass cytometry (CyToF) uniquely combines time-of-flight mass spectrometry with metal-labeling technology which can theoretically but simultaneously analyze 50+ parameters on a single cell. Despite the functional similarities in utility, mass cytometers and flow cytometers have different operating platforms that may impact their functional readouts . The goal of this study was to investigate and compare phenotypic readouts between flow cytometry and Cytof platforms using human neuroblastoma cell lines. Methods To test this question, we utilized custom designed staining panels for both flow cytometry (fluorochromes-PerCp Cy 5.5, PE and Coralite 488 respectively) and Cytof (lanthanides: 155Gd, 151Eu, and 116Cd respectively) targeting CD56, Nestin and Synaptophysin, markers that are consistent with neuroblastoma using both five established neuroblastoma cell lines (BE2C, CHLA90, SMS-KCNR, SHSY5Y) and NGP) as well as four novel cells lines established in our laboratory derived from patient specimens (SL01277, SL01404, SL01255 and SL01287). We compared the percent expression of respective markers across cell lines between the two platforms. Results Using both techniques we demonstrated that there was no difference in detection of viability in cells when validated under flow cytometry or CyToF (p&lt0.05). We also show extracellular staining of CD56 between the two platforms are comparable(p&lt0.05) across tested cell lines. Finally, we demonstrate that intracellular structures can be detected using both platforms with no significant difference (p&lt0.05). Discussion and Conclusion The congruence and reproducibility in readouts between flow cytometry and CyToF analysis indicates that either assay can be used to study biological systems. Given the high dimensionality and versatility, then CyToF offers a robust platform that can be leveraged for immunophenotypic and functional studies of cellular material. Furthermore, CyToF due to its wide breadth of multiparametric measurements available will be an instrument of choice where the study of multiple parameters is desired. Citation Format: David Mulama, Roman J. Riveria, Kimberly McKinney, Divya Gandra, Kaitlyn Smith, Nicholas Tastet, Giselle L. Saulnier Sholler. Comparative validation of neuroblastoma cell lines using flow cytometry and CyToF [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3053.
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Botha, Jaco, Haley R. Pugsley, and Aase Handberg. "Conventional, High-Resolution and Imaging Flow Cytometry: Benchmarking Performance in Characterisation of Extracellular Vesicles." Biomedicines 9, no. 2 (January 27, 2021): 124. http://dx.doi.org/10.3390/biomedicines9020124.

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Flow cytometry remains a commonly used methodology due to its ability to characterise multiple parameters on single particles in a high-throughput manner. In order to address limitations with lacking sensitivity of conventional flow cytometry to characterise extracellular vesicles (EVs), novel, highly sensitive platforms, such as high-resolution and imaging flow cytometers, have been developed. We provided comparative benchmarks of a conventional FACS Aria III, a high-resolution Apogee A60 Micro-PLUS and the ImageStream X Mk II imaging flow cytometry platform. Nanospheres were used to systematically characterise the abilities of each platform to detect and quantify populations with different sizes, refractive indices and fluorescence properties, and the repeatability in concentration determinations was reported for each population. We evaluated the ability of the three platforms to detect different EV phenotypes in blood plasma and the intra-day, inter-day and global variabilities in determining EV concentrations. By applying this or similar methodology to characterise methods, researchers would be able to make informed decisions on choice of platforms and thereby be able to match suitable flow cytometry platforms with projects based on the needs of each individual project. This would greatly contribute to improving the robustness and reproducibility of EV studies.
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