Flow cytometry is the study of single cells as they pass through a liquid stream. Components of the cell, either internally or on the surface, must first be labeled with one or more fluorescent dyes. A laser inside an instrument called a flow cytometer excites these fluorescent molecules so that they emit light at various wavelengths. The amount of fluorescence can give an indication as to what percentage of various cell types are present in the sample. Flow cytometry is used in laboratories for a variety of applications including cancer research, immunology, and cell cycle analysis.
To perform flow cytometry analysis, a suspension of single cells must be stained with fluorescent dyes, which can be done in either a one-step or two-step process. Cellular proteins are often stained using antibodies directed against the protein of interest. A one-step process involves staining cells with antibodies that are already labeled with fluorescent dye. If labeled antibodies are not available, cells can be stained first with an unlabeled primary antibody, and then with a fluorescent labeled secondary antibody.
Once the cells are labeled, they are ready for flow cytometry analysis. A tube containing the suspension of cells is loaded on the flow cytometer. The cells flow through the instrument and pass in front of a laser in single file. As the laser hits each cell, the light is scattered in a forward or side direction. Forward scatter measurements give an indication of cell size, while side scatter is a measure of granularity of the cells.
The energy from the laser also excites the fluorochrome molecules, the fluorescent dye used to stain the cells. A common laser used for flow cytometry is the argon-ion laser which emits light at a wavelength of 488 nanometers (nm). This light excites the molecules of the green dye fluorescein, which then emits light at a wavelength of 525 nm. Photomultiplier tubes (PMTs) in the flow cytometer have light filters that detect the light emitted by the various fluorochromes. The instrument can have several PMTs that detect light at different wavelengths.
Red-diode, violet diode, or He-Ne (helium-neon) are other lasers that may be used in a flow cytometer. Other fluorochromes used in flow cytometry analysis include phycoerythrin, Texas red, or allophycocyanin. Different combinations of lasers and fluorochromes allow the researcher to collect information about many different cellular markers in the same experiment.
Software designed for use with the flow cytometer aids the researcher in visualizing the data. Cell populations are analyzed using dot plots, density plots, or histograms. Regions can be drawn around the cells of interest and fluorescence intensity can give an indication of percentages of various cells in the sample.
