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. […]

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