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Current Protocols in Immunology Feb 2018Flow cytometry is a technology that provides rapid multi-parametric analysis of single cells in solution. Flow cytometers utilize lasers as light sources to produce both... (Review)
Review
Flow cytometry is a technology that provides rapid multi-parametric analysis of single cells in solution. Flow cytometers utilize lasers as light sources to produce both scattered and fluorescent light signals that are read by detectors such as photodiodes or photomultiplier tubes. These light signals are converted into electronic signals that are analyzed by a computer and written to a standardized format (.fcs) data file. Cell populations can be analyzed and/or purified based on their fluorescent or light scattering characteristics. A variety of fluorescent reagents are utilized in flow cytometry. These include fluorescently conjugated antibodies, nucleic acid binding dyes, viability dyes, ion indicator dyes, and fluorescent expression proteins. Flow cytometry is a powerful tool that has applications in immunology, molecular biology, bacteriology, virology, cancer biology, and infectious disease monitoring. It has seen dramatic advances over the last 30 years, allowing unprecedented detail in studies of the immune system and other areas of cell biology. © 2018 by John Wiley & Sons, Inc.
Topics: Data Analysis; Flow Cytometry; Humans; Indicators and Reagents
PubMed: 29512141
DOI: 10.1002/cpim.40 -
Nature Reviews. Immunology Feb 2012The heterogeneity in the healthy human immune system, and the immunological changes that portend various diseases, have been only partially described. Their... (Review)
Review
The heterogeneity in the healthy human immune system, and the immunological changes that portend various diseases, have been only partially described. Their comprehensive elucidation has been termed the 'Human Immunology Project'. The accurate measurement of variations in the human immune system requires precise and standardized assays to distinguish true biological changes from technical artefacts. Thus, to be successful, the Human Immunology Project will require standardized assays for immunophenotyping humans in health and disease. A major tool in this effort is flow cytometry, which remains highly variable with regard to sample handling, reagents, instrument setup and data analysis. In this Review, we outline the current state of standardization of flow cytometry assays and summarize the steps that are required to enable the Human Immunology Project.
Topics: Animals; Flow Cytometry; Humans; Immunophenotyping; Leukocytes, Mononuclear; Mice; Reference Standards
PubMed: 22343568
DOI: 10.1038/nri3158 -
Current Protocols in Cytometry Jan 2013Interest in measuring the complete fluorescence spectra of individual cells in flow can be traced to the earliest days of flow cytometry. Recent advances in detectors,... (Review)
Review
Interest in measuring the complete fluorescence spectra of individual cells in flow can be traced to the earliest days of flow cytometry. Recent advances in detectors, optics, and computation have made it possible to make full spectral measurements in the sub-millisecond time frame in which flow cytometry measurements typically occur. This opens up new possibilities for applying spectroscopy to the analysis of individual cells. This unit reviews historical and contemporary approaches to spectral flow cytometry, as well as instrument design, calibration, and data analysis for spectral flow cytometry applications.
Topics: Animals; Calibration; Flow Cytometry; History, 20th Century; History, 21st Century; Humans; Models, Biological; Single-Cell Analysis; Spectrum Analysis, Raman; Statistics as Topic
PubMed: 23292705
DOI: 10.1002/0471142956.cy0127s63 -
Cells Jul 2023Unmasking the subtleties of the immune system requires both a comprehensive knowledge base and the ability to interrogate that system with intimate sensitivity. That... (Review)
Review
Unmasking the subtleties of the immune system requires both a comprehensive knowledge base and the ability to interrogate that system with intimate sensitivity. That task, to a considerable extent, has been handled by an iterative expansion in flow cytometry methods, both in technological capability and also in accompanying advances in informatics. As the field of fluorescence-based cytomics matured, it reached a technological barrier at around 30 parameter analyses, which stalled the field until spectral flow cytometry created a fundamental transformation that will likely lead to the potential of 100 simultaneous parameter analyses within a few years. The simultaneous advance in informatics has now become a watershed moment for the field as it competes with mature systematic approaches such as genomics and proteomics, allowing cytomics to take a seat at the multi-omics table. In addition, recent technological advances try to combine the speed of flow systems with other detection methods, in addition to fluorescence alone, which will make flow-based instruments even more indispensable in any biological laboratory. This paper outlines current approaches in cell analysis and detection methods, discusses traditional and microfluidic sorting approaches as well as next-generation instruments, and provides an early look at future opportunities that are likely to arise.
Topics: Flow Cytometry; Proteomics; Genomics; Technology; Microfluidics
PubMed: 37508539
DOI: 10.3390/cells12141875 -
Cytometry. Part B, Clinical Cytometry May 2017
Topics: Flow Cytometry; Humans; Leukocyte Count; Macrophages; Monocytes
PubMed: 28449204
DOI: 10.1002/cyto.b.21530 -
Cytometry. Part B, Clinical Cytometry Jan 2020Platelet flow cytometry is widely used in cardiovascular medicine as the platelet surface is rich in clinical biomarkers. Surface profiling is critical in disease... (Review)
Review
Platelet flow cytometry is widely used in cardiovascular medicine as the platelet surface is rich in clinical biomarkers. Surface profiling is critical in disease management, but current assays can abet clinical errors as they are suboptimal and prone to bias. Accordingly, the technical and analytical advances that can be used to create high quality assays with minimal error and maximal sensitivity were reviewed. Specifically, the best practices for instrument setup, quality control, panel design, titration, gating, and compensation were described. Adherence to these practices will enhance the validity and reliability of platelet flow cytometry in clinical/research settings. © 2019 International Clinical Cytometry Society.
Topics: Animals; Biomarkers; Blood Platelets; Flow Cytometry; Humans; Quality Control; Reproducibility of Results
PubMed: 30779477
DOI: 10.1002/cyto.b.21774 -
Cytometry. Part a : the Journal of the... Sep 2006A frequent goal of flow cytometric analysis is to classify cells as positive or negative for a given marker, or to determine the precise ratio of positive to negative...
A frequent goal of flow cytometric analysis is to classify cells as positive or negative for a given marker, or to determine the precise ratio of positive to negative cells. This requires good and reproducible instrument setup, and careful use of controls for analyzing and interpreting the data. The type of controls to include in various kinds of flow cytometry experiments is a matter of some debate and discussion. In this tutorial, we classify controls in various categories, describe the options within each category, and discuss the merits of each option.
Topics: Biomarkers; Flow Cytometry; Reproducibility of Results; Research Design
PubMed: 16888771
DOI: 10.1002/cyto.a.20333 -
Cytometry. Part a : the Journal of the... Nov 2022High-dimensional single-cell data has become an important tool in unraveling the complexity of the immune system and its involvement in homeostasis and a large array of...
High-dimensional single-cell data has become an important tool in unraveling the complexity of the immune system and its involvement in homeostasis and a large array of pathologies. As technological tools are developed, researchers are adopting them to answer increasingly complex biological questions. Up until recently, mass cytometry (MC) has been the main technology employed in cytometric assays requiring more than 29 markers. Recently, however, with the introduction of full spectrum flow cytometry (FSFC), it has become possible to break the fluorescence barrier and go beyond 29 fluorescent parameters. In this study, in collaboration with the Stanford Human Immune Monitoring Center (HIMC), we compared five patient samples using an established immune panel developed by the HIMC using their MC platform. Using split samples and the same antibody panel, we were able to demonstrate highly comparable results between the two technologies using multiple data analysis approaches. We report here a direct comparison of two technology platforms (MC and FSFC) using a 32-marker flow cytometric immune monitoring panel that can identify all the previously described and anticipated immune subpopulations defined by this panel.
Topics: Humans; Flow Cytometry; Immunophenotyping; Biomarkers; Data Analysis
PubMed: 35593221
DOI: 10.1002/cyto.a.24565 -
Cytometry. Part a : the Journal of the... Oct 2008A fundamental tenet of scientific research is that published results are open to independent validation and refutation. Minimum data standards aid data providers, users,...
A fundamental tenet of scientific research is that published results are open to independent validation and refutation. Minimum data standards aid data providers, users, and publishers by providing a specification of what is required to unambiguously interpret experimental findings. Here, we present the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, stating the minimum information required to report flow cytometry (FCM) experiments. We brought together a cross-disciplinary international collaborative group of bioinformaticians, computational statisticians, software developers, instrument manufacturers, and clinical and basic research scientists to develop the standard. The standard was subsequently vetted by the International Society for Advancement of Cytometry (ISAC) Data Standards Task Force, Standards Committee, membership, and Council. The MIFlowCyt standard includes recommendations about descriptions of the specimens and reagents included in the FCM experiment, the configuration of the instrument used to perform the assays, and the data processing approaches used to interpret the primary output data. MIFlowCyt has been adopted as a standard by ISAC, representing the FCM scientific community including scientists as well as software and hardware manufacturers. Adoptionof MIFlowCyt by the scientific and publishing communities will facilitate third-party understanding and reuse of FCM data.
Topics: Cell Separation; Database Management Systems; Flow Cytometry; Guidelines as Topic; Indicators and Reagents; Specimen Handling
PubMed: 18752282
DOI: 10.1002/cyto.a.20623 -
Scientific Reports Jan 2022Biomedical research relies on identification and isolation of specific cell types using molecular biomarkers and sorting methods such as fluorescence or magnetic...
Biomedical research relies on identification and isolation of specific cell types using molecular biomarkers and sorting methods such as fluorescence or magnetic activated cell sorting. Labelling processes potentially alter the cells' properties and should be avoided, especially when purifying cells for clinical applications. A promising alternative is the label-free identification of cells based on physical properties. Sorting real-time deformability cytometry (soRT-DC) is a microfluidic technique for label-free analysis and sorting of single cells. In soRT-FDC, bright-field images of cells are analyzed by a deep neural net (DNN) to obtain a sorting decision, but sorting was so far only demonstrated for blood cells which show clear morphological differences and are naturally in suspension. Most cells, however, grow in tissues, requiring dissociation before cell sorting which is associated with challenges including changes in morphology, or presence of aggregates. Here, we introduce methods to improve robustness of analysis and sorting of single cells from nervous tissue and provide DNNs which can distinguish visually similar cells. We employ the DNN for image-based sorting to enrich photoreceptor cells from dissociated retina for transplantation into the mouse eye.
Topics: Animals; Cell Aggregation; Flow Cytometry; Mice; Microfluidic Analytical Techniques; Neural Networks, Computer; Photoreceptor Cells, Vertebrate; Software
PubMed: 35046492
DOI: 10.1038/s41598-022-05007-2