Flow cytometry uses fluorescent dyes to label individual cells passing through a stream, allowing for analysis of cell types and markers. The process involves staining cells with antibodies and passing them through a flow cytometer with lasers and photomultiplier tubes. Software is used to analyze the data.
Flow cytometry is the study of individual cells as they pass through a liquid stream. The components of the cell, internally or on the surface, must first be labeled with one or more fluorescent dyes. A laser in an instrument called a flow cytometer excites these fluorescent molecules to emit light at various wavelengths. The amount of fluorescence can give an indication of the percentage of various cell types 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 a one- or two-step process. Cellular proteins are often stained using antibodies directed against the protein of interest. A one-step process involves staining the cells with antibodies that are already labeled with a fluorescent dye. If labeled antibodies are not available, cells can be stained first with an unlabelled primary antibody and then with a fluorescently labeled secondary antibody.
Once the cells are labeled, they are ready for flow cytometry analysis. A tube containing the cell suspension is loaded onto the flow cytometer. Cells flow through the instrument and pass a laser in single file. As the laser hits each cell, the light is scattered forward or sideways. Forward scatter measurements give an indication of cell size, while lateral scatter is a measure of cell granularity.
The laser energy also excites molecules of fluorochrome, the fluorescent dye used to color 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. The 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 can be used in a flow cytometer. Other dyes used in flow cytometry analysis include phycoerythrin, Texas red, or allophycocyanin. Different combinations of lasers and dyes allow the researcher to gather information on many different cellular markers in the same experiment.
Software designed for use with the flow cytometer assists the researcher in visualizing the data. Cell populations are analyzed using dot plots, densities or histograms. Regions can be drawn around the cells of interest and the intensity of the fluorescence can give an indication of the percentages of various cells in the sample.
Protect your devices with Threat Protection by NordVPN
