What is flow cytometry?

Flow cytometry is a fluorescence-based assay that enables measurement of multiple characteristics, simultaneously, such as population counts and protein abundance, from individual cells suspended in a solution. It is a powerful tool that enables rapid, quantitative, and accurate measurement of cellular characteristics and provides unparalleled insight into the heterogeneity of cellular populations.

This is achieved using instrumentation that directs a single stream of cells past a light source and measures the resulting scatter and emission of light energy in various wavelengths. Researchers use fluorescent molecules with different excitation and emission characteristics so that they can be combined, detected, and differentiated to provide multiple readouts from a single cell. These fluorescent molecules may be attached to antibodies targeting specific proteins or protein modifications, or they may be dyes that directly bind to cellular components.

When to use flow cytometry

Flow cytometry is a highly versatile application that can provide simple, single-target readouts or complex subpopulation phenotyping and cell signaling analysis. Because flow cytometry relies on cells being in suspension in order to enable them to flow past the illumination source, it is readily compatible with biological samples where the cells naturally exist as a single-cell suspension (eg, white blood cells). However, adherent cells and even tissue samples may be dissociated and analyzed by flow cytometry as single-cell suspensions.

Flow cytometry is ideally suited for experiments where quantitative information is desired at a single cell level, to gain understanding at the cellular level and/or at the population level. The quantitative information being analyzed may provide insight into the number of cells of a given type, the amount of a specific protein present in that cell type, or the functional activity of that protein, to name several examples. Because multiple readouts are collected simultaneously, flow cytometry also enables correlation between those readouts at a cellular level.

Contrast this to applications such as western blotting that irreversibly combine populations of cells into a single readout. Flow cytometry allows researchers to ask complex questions, such as how the activity of 2 key signaling pathways in 3 immune cell types changes in response to a treatment, and answer the question within a few hours by labeling and running a single sample on a flow cytometer. The same question would take days of work to answer by western blot.

Not all biological research questions are capable of being addressed with flow cytometry. Spatial distribution of cells in tissue cannot be assessed using flow cytometry, since the sample needs to be dissociated into a single-cell suspension prior to analysis. For that analysis, immunohistochemistry or tissue-based immunofluorescence are preferred. Fine-scale subcellular analysis is more suited to analysis by fluorescent and/or confocal microscopy; though, imaging flow cytometers are making rapid progress on this front. Analysis of genetic mutations or large-scale mRNA analysis is better left to sequencing instruments. However, when it comes to rapid cell level quantification, few assays can top flow cytometry.

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