In addition to compensation controls, there are several other controls that can, and in most cases, should be used to help resolve issue in staining. Fluorescence Minus One (FMO) controls can help identify gating boundaries, isotype controls can help identify staining issues and unstained controls show you the background or autofluorescence of the system.
FMO controls are ideal for showing gating boundaries, even with compensation issues. FMO controls contain every stain in the panel except the one you are controlling for in that sample. For example, a FITC FMO control would contain all fluorochromes except FITC. (You can include isotype controls in FMO controls if you would like, but isotype controls DO NOT accurately show staining boundaries.) The following figure shows how a gate on an unstained or isotype would be set incorrectly as compared to an FMO control. Furthermore, the second pane demonstrates how FMO controls can resolve gating boundaries even with improper compensation:
Isotype controls do not provide gating controls. Every antibody has specific and nonspecific binding properties. Isotype controls are different antibodies than antigen specific antibodies and will have different activity. Isotype controls are good staining controls to identify potential problems in staining, particularly if a primary and secondary antibody are used. However, isotypes do not reliably identify what is negative and what is positive. If users insist on using isotype
controls, makes sure they also titrate them as well, since nonspecific activity at super saturating levels will increase total measured binding and skew the “negative” population more positive.
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*Images courtesy of Mario Roederer