Introduction to flow cytometry
Flow cytometry is a technique in which fluorescently labeled cells flow through a cytometer a single cell at a time. The fluorescent compounds are excited with a laser and detectors measure the light emitted from these compounds. Different fluorescent compounds emit light at different wavelengths, which allows for the discrimination of several different proteins on a cell. When multiple fluorescent compounds are used to analyze a cell population, the results typically are depicted in two-dimensional diagrams. For example, assume a heterogeneous mixture of cells is stained with fluorescently labeled antibodies specific for two proteins, then analyzed on a cytometer with results shown below. A cell that expresses only one protein detected with an antibody (labeled with green fluorescence) will shift along the X-axis but not the Y-axis. A cell that expresses only one protein detected with an antibody (labeled with red fluorescence) will shift along the Y-axis but not the X-axis. A cell expressing both antigens will shift along both the X- and Y-axis. These cells are referred to as "double positive" cells. Cells that do not express either antigen will not shift along either the X- or Y-axis, which can be seen as a population in the lower left corner. These cells are referred to as "double negative" cells.