Introduction to Flow Cytometry

Flow cytometry is a method, somewhat analogous to fluorescent microscopy, in which measurements are performed on particles (cells) in liquid suspension, which flow one at a time through a focused laser beam at rates up to several thousand particles per second. Light scattered and fluorescence emitted by the particles (cells) is collected, different wavelengths of light filtered, digitized and sent to a computer for analysis. Most work done at the Wistar flow cytometry facility involves measuring the binding of fluorochrome-labeled probes to cells or cell components and the comparison of the resultant fluorescence to an unstained background.  Examples of the types of probes and assays are given below.

Cell sorting goes beyond the initial analysis, where cells are separated and recovered based upon those properties measured. Cells recovered via sorting are viable and are collected under sterile conditions. Most often subpopulations sorted are greater than 99% pure.

FACS is an acronym often used in referring to flow cytometry. It is a brand name for Fluorescence-Activated Cell Sorting, an inaccurate term used to describe standard flow cytometry analysis.

Measurements (parameters) utilized in flow cytometry

Light scatter

Forward and side scatter (FSC, SSC) are used for preliminary identification and gating of cell populations. Scatter parameters are used to exclude debris, dead cells, and unwanted aggregates. In a peripheral blood or bone marrow sample, lymphocyte, monocyte and granulocyte populations can be further defined and separately gated and analyzed on the basis of forward and side scatter.

Fluorescence

Fluorescent parameter measurement allows for investigation of cell structures and functions based upon direct staining, reactions with fluorochrome labeled probes (e.g., antibodies), or expression of fluorescent proteins.  Fluorescence signals correspond to different laser excitation and fluorescence emission wavelengths.

When different fluorochromes are used simultaneously, signal spillover can occur between fluorescence channels.  This needs to be corrected through compensation. Certain combinations of fluorochromes are not able to be used simultaneously. Please consult with someone experienced in flow cytometry prior to planning multi-fluorochrome experiments to develop workable fluorochrome panels for each instrument, or see tools for multicolor flow cytometry.

Common flow cytometry applications

Immunofluorescence involves the staining of cells with antibodies conjugated to fluorescent dyes such as FITC, PE, APC, AlexaFluors, and tandem conjugates (PE-Cy5, APC-Cy7, etc.). Cell surface antigens are usual targets of this assay, but antibodies can be directed at intracellular antigens or cytokines as well.

DNA staining is used primarily for cell cycle profiling, or as one method for measuring apoptosis. Propidium iodide (PI) is among commonly used DNA stains, and cannot enter live cells and can therefore be used for viability assays. For cell cycle or apoptosis assays using PI, cells must first be fixed in order for staining to take place. The relative quantity of PI-DNA staining corresponds to the proportion of cells in G0/G1, S, and G2/M phases, with lesser amounts of staining indicating apoptotic/necrotic cells. Cell cycle staining can be performed simultaneously with certain fluorochromes in assays to further characterize apoptosis or gene expression.

Gene expression and transfection can be measured indirectly by using a fluorescence-expressing reporter gene in a construct.  Fluorescent Protein-type constructs (EGFP, dsRed) can be used to quantify populations of those cells expressing the gene/construct.  Fluorescent are available which may be excited with common lasers, but emit fluorescence at different wavelengths.  This allows for measurement of co-transfection, as well as simultaneous detection of gene and antibody expression. Appropriate negative (background) controls for experiments involving fluorescent protein constructs should be included – the control is the same cell type, using the gene insert minus the fluorescent protein construct.

Metabolic studies and other:  Annexin-V may be labeled with various fluorochromes in order to identify cells in early stages of apoptosis. Proliferation:  CFSE and similar tracking dyes bind to cell membranes and are equally distributed when cells divide. The number of divisions cells undergo in a period of time can then be counted. Proliferation dyes can be used in conjunction with immunofluorescence. Reagents for functional assays such as measurement of radical oxygen species, changes in pH, and calcium flux are available for use in flow cytometry.

Cell sorting 
refers to an assay in which cells are separated and recovered from suspension based upon properties measured in flow cytometry analysis. Up to 4 subpopulations may be separated by instruments at Wistar. Most assays utilized for analysis may serve as the basis for sorting experiments, as long as gates and regions defining the subpopulation(s) to be sorted do not logically overlap. Throughput rate maxima range from 9,000 cells/second (~32 x 106 cells/hour) to 30,000 cells/second (>100 x 106 cells/hour). Throughput rate is dependent upon cell size; smaller cells, such as lymphocytes, may be sorted with a smaller nozzle (70μ) at the higher rates, whereas larger cells such as most cultured cell lines are sorted with the larger nozzle (100 μ) at lower rates.  The rate of collection of the separated population(s) sorted depends primarily upon the condition of the cells and the percentage of reactivity. Cells that are recovered via flow sorting are viable and collected under sterile conditions. We can most often recover subpopulations that are in excess of 99% pure. 

Common cell sorting experiments usually involve immunofluorescence assays: staining of cells with antibodies conjugated to fluorescent dyes in order to detect antigens. In addition, a great deal of sorting is done using fluorescent protein constructs in order to isolate pure populations of cells expressing a given gene/construct. Other criteria may also be utilized for cell sorting; please check with the facility.