Spectral overlap between fluorochromes in multi-color experiments requires the use of fluorescence compensation controls. (See http://www.drmr.com/compensation/index.html for an in-depth explanation of compensation.)

The Three Rules of Compensation

• Controls need to be as bright or brighter than any sample the compensation will be applied to.
• Background fluorescence should be the same for the positive and negative control (e.g, positive cells vs negative cells, or positive beads vs negative beads).
• Compensation controls MUST match the exact experimental fluorochrome.

The proper compensation controls include a negative control (unstained cells are recommended) and one tube each of cells (or beads) stained positively with each of the fluorochromes used in the experiment. The negative control (unstained cells) establishes the background fluorescence of the experimental samples and is used to set the baseline PMT (photomultiplier tube) voltages of the instrument. Each of the compensation tubes is subsequently run to establish the spill-over values of each fluorochrome into the other fluorescent channels. It is important that each compensation tube have a population of brightly stained cells (or beads) in order for the spill-over values to be accurately determined.

Several vendors sell beads specifically for use as compensation controls. The beads are stained as if they were cells using the same antibodies and fluorochromes that are used in the experiment, producing both a negative and bright positive population for each color. For experiments that cannot spare cells for compensation, do not have enough positive events, or have only low antigen expression, compensation beads are recommended. If beads are not used, then cells expressing high levels of antigen (does not have to be an antigen of interest in the experiment) are stained with a fluorochrome-conjugated antibody that yields brightly stained cells. Because of the necessity to have brightly stained cells at a relatively high frequency (i.e, above 10% of the population) for accurate compensation, it may be necessary to use the same antibody that stains the high-density antigen while varying the fluorochrome for each tube (see the following example), except when using tandem fluorochromes (see following section about using tandem fluorochromes).

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

• tube 1) unstained splenocytes
• tube 2) anti-CD8 FITC stained splenocytes (or antibody against some other high-density antigen)
• tube 3) anti-CD8 PE stained splenocytes (or antibody against some other high-density antigen)
• tube 3) anti-CD8 PerCP stained splenocytes (or antibody against some other high-density antigen)
• tube 4) anti-CD8 APC stained splenocytes (or antibody against some other high-density antigen)

Or if using beads

• tube 1) unstained splenocytes
• tube 2) anti-* FITC stained beads 
• tube 3) anti-* PE stained beads
• tube 3) anti-* PerCP stained beads
• tube 4) anti-* APC stained beads

There are exceptions to these rules when using tandem fluorochromes. See Tandem Fluorochrome Conjugated Antibody Best Practices

            * any antibody that is compatible with the beads

List of Compensation Bead Vendors

Please Note: The facility's mission is to serve investigators in their quest to obtain accurate data. The lack of proper compensation controls may yield misleading, confusing, and inaccurate data. In order to live up to the Facility's mission of assuring quality control and reproducibility, facility staff will not assist with running samples when the necessary compensation controls are not provided by the investigator.