A Fluorescence Minus One (FMO) control is a tube of cells stained with all fluorochromes used in the experiment except one. A multi-color immunofluorescent experiment has one FMO control for each fluorochrome. FMO controls are used to determine the cut-off point between background fluorescence and positive populations in multi-color immunofluorescent experiments. They are very useful and therefore highly recommended where a positive cell population is presented as a smear instead of being distinctly separate from the negative population. The lack of distinction between positive and negative populations is exacerbated by "spreading" of the negative populations due to the contributions of fluorescence overlap compensation from multiple fluorochromes. In cases where negative population spreading or positive population smearing is present, it is not recommended to use either unstained or isotype controls to determine positive population cut-off points.

Example: For mouse splenocytes stained with FITC, PE, PerCP, and APC conjugated antibodies, FMO controls should include:

• FITC FMO control) cells stained with PE, PerCP, and APC conjugated antibodies (no FITC)
• PE FMO control) cells stained with FITC, PerCP, and APC conjugated antibodies (no PE)
• PerCP FMO control) cells stained with FITC, PE, and APC conjugated antibodies (no PerCP)
• APC FMO control) cells stained with FITC, PE, and PerCP conjugated antibodies (no APC)