High quality flow cytometry assay data can be obtained with well-maintained flow cytometers and high-quality processing and staining protocols. At the center of these operations, are the inclusion of the appropriate control samples, which is critical to guaranteeing the quality of flow cytometry assay data. Isotype controls and fluorescence minus one (FMO) controls are critical in assuring that the correct fluorescence signals are being measured, and also that these controls are separate from compensation controls which address spectral overlap effects. Consider these aspects of FMOs and isotype controls as you plan your next flow cytometry experiment.
FMO controls are all the fluorescent antibodies added into a tube from a staining panel minus one, and this control is done for each antibody. By running FMO controls, users can observe spillover effects of other fluorescent labels that are detected in the channel of the absent antibody. FMO controls help users create accurate acquisition and analysis gates and may help determine if a fluorescent antibody is undesirable due to excessive spillover.
Isotype controls are antibodies with the same isotype and fluorophore as a particular antibody from a staining panel. Isotype control staining measures nonspecific staining by the antibody domains that are not specific to antigen binding. Isotype controls include all isotypes from a given panel and should be obtained from the same manufacturer as the staining panel antibodies to properly control for fluorescence conjugation.
Both FMOs and isotype controls are essential for flow cytometry assays with large staining panels, but only one of these controls is typically needed for a four-color assay with well-defined cell populations.
As you develop and implement new flow cytometry assays, be sure to include FMO and isotype controls to assure your staining panel is accurate and reliable.