Flow Cytometry

Flow cytometry introduction

Flow cytometry is a technology that uses single or multiple lasers to provide a multi-parametric analysis of single cells. Each cell or particle is analyzed by visible light scatter or by fluorescence as cells rapidly flow past each laser. Independent of light scatter analysis, fluorescence measurements are achieved by transfection and expression of common fluorescent proteins (e.g. Green Fluorescent Protein, GFP) and staining with fluorescently conjugated antibodies or fluorescent dyes. Flow cytometry is a powerful technology that is commonly used in molecular and cellular biology research applications, including immunology, cancer biology, and disease research.   

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Flow Cytometry Antibodies and Labels

Performing flow cytometry analysis involves using various flow cytometry antibodies and labels. Flow cytometry antibodies are used to specifically bind to different cell markers, helping to characterize the cell type and assess any changes in protein expression as part of the experiment. Numerous types of labels can be used to visualize the antibodies for detection in the flow cytometer depending on the experimental design including nucleic acid dyes, cell viability dyes, polymer dyes, quantum dots, small organic molecules, and fluorescent proteins. Antibodies are typically labeled through direct conjugation (either as commercially available products or conjugation kits allowing the end user to perform the reaction) or through the use of secondary antibodies. 

Flow Cytometry Applications

Numerous fields of study use flow cytometry and often analyze multiple parameters within diverse populations of cells. Common molecular biology applications include analysis of recombinantly expressed fluorescent proteins that have induced expression in various cell types to elucidate gene function or for in vivo cell tracking. Whereas the staining of DNA allows for cell cycle analysis, the use of antibodies allow for a wide range of analyses including, signal transduction, RNA, and protein expression. However, immunophenotyping is one of most commonly used applications of flow cytometry. With immunophenotyping, fluorochrome-conjugated antibodies target multiple cell surface antigens to identify specific immune cells within population of cells.

Flow Cytometry Data Analysis

Flow cytometry data analysis often involves identifying a specific region of cells or cluster and applying additional parameters within the experiment, including investigating if additional cell identifying factors are present to gain further insight on cellular identity. Numerous computer software programs are available and commonly used to facilitate this kind of data analysis and other cell cycle analyses.

Flow Cytometry Instrumentation

A summary of the primary components used in flow cytometers includes electronics, optics, and fluidics. The fluidics serves as the liquid delivery system to direct the sample to the optic components, consisting of excitation optics (laser) and the collection optics that consist of photodiodes and photomultiplier tubes (PMTs). Importantly, the electronics components convert the information from the detectors into digital signals that are interpreted by computers. Combinations of additional systems facilitate more advanced sample analyses, including the use of cell sorters to purify and collect samples, and imaging cytometers that combine both flow cytometry with fluorescence microscopy. 

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