Once a compensation matrix is calculated to correct for fluorescence spillover, more controls must be run to ensure an analyte’s signal is real. These controls include fluorescence minus one (FMO) and isotype controls.
FMO controls contain every antibody in your panel except for one. FMOs are useful as a gating control to identify the gating boundary for the one antibody that is missing. FMO controls are mainly used to identify background staining due to fluorescence spillover.
Isotype controls are useful for identifying nonspecific staining. The isotype control should have the same heavy chain isotype, the same light chain, the same color, and be derived from the same species as the antigen-specific antibody. For example, Tonbo APC Rat IgG1 Isotype Control (cat. no. 20-4301) should be paired with APC Rat Anti-Mouse IFN gamma (cat. no. 20-7311). Isotype controls should be used at the same concentration as the antibody for which it is a control. Ideally, the isotype control antibody should be purchased from the same company as the test antibody, especially for tandem dye conjugates as manufacturing processes may vary from vendor to vendor.
In practice, the answer to the question above is YES to all, depending on the situation and stain. You would be wasting time and money if using isotype and/or FMO controls for CD4+ versus CD8+ cells in a bivariate plot when populations are clearly resolved. However, when designing a panel with 4+ colors, both controls should be used – use FMO controls to define gates and isotype controls to identify staining issues. Once you are comfortable with a panel’s performance on a cell type of interest, you may not need FMO and isotype controls for every single antibody in the panel – just include the controls that are necessary for the experiment.
About the Author
Joseph S. Dolina, Ph.D.In 2006, Joseph obtained B.S. degrees in Biology and Chemistry from The College of New Jersey. This was shortly followed by a M.S. in Biomedical Sciences in 2007 from the University of Medicine and Dentistry of New Jersey. From 2007-2013 Joseph completed his doctoral work at the University of Virginia obtaining his M.S. and Ph.D. in Microbiology. Dr. Dolina then joined the La Jolla Institute as a postdoctoral fellow in 2014 in the Schoenberger laboratory.