Flow Cytometry: An Overview

Abstract

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.

Keywords: flow cytometry; fluorescence; light scatter; reagents.

Figure 1

Example of CFSE staining used for proliferation analysis. Human CD4+ T cells were stained with CFSE and then stimulated for 5 days with an antigen. Each peak of CFSE staining represents one generation of cell division.

Figure 2

Example of BrdU, Ki67 and PCNA used to measure proliferation. Cells from H23 lung cancer cell line were fixed and then stained with BrdU, Ki67 or PCNA and DAPI. The BrdU sample was pulsed for 2 hours with BrdU prior to staining. The samples were counterstained with DAPI to indicate cell cycle as well as proliferation. The positive cells are indicated in the rectangular region.

Figure 3

Example of gating for standard data analysis using FlowJo 10.3. Cells are first gated to remove doublets, for viability, for light scatter and then for specific lineage markers. This example is looking at CM9(SIV-gag) Dextramer staining on CD8 cells in PBMC from a vaccinated Rhesus macaque.