Flow cytometers must be calibrated prior to fluorescence intensity measurements because of inherent instrument variability. To correct for this variability, a standard particle (fixed chicken red blood cells, or CRBCs) must be analyzed on the instrument prior to each experiment and photomultiplier tube (PMT) voltages adjusted accordingly to place the CRBC fluorescence emission peaks into predetermined target channels. - [Read Calibration of Becton Dickinson Flow Cytometers for Relative Fluorescence Intensity Measurements]
The more commonly available single-laser cytometers can also be used to measure multicellular conjugates, but due to overlaps in emission spectra, the extent of labeling cells with fluorophores must be controlled much more carefully when the single-laser machines are used. This protocol describes the labeling of cells and analysis of conjugates with either dual-laser or single laser flow cytometers. - [Read Measurement of Intercellular Conjugates by Flow Cytometry Protocol]