The University of Iowa Carver College of Medicine

Flow Cytometry Facility

Compensation Controls for
Multi-fluorochrome Experiments

Flow Cytometry technology can yield highly accurate data, but only if the proper controls are done prior to sample analysis. This is especially important when multi-color analysis is undertaken. Spectral overlap between fluorochromes in multi-color experiments requires the use of compensation. (See http://www.drmr.com/compensation/index.html for an in depth explanation of compensation.) Without proper compensation, the data obtained would be meaningless. The proper compensation controls may vary from experiment to experiment, but some basics are the same. Thus, an unstained or isotype control and a positive control for each color are essential.

Example: For mouse splenocytes stained with FITC, PE, and PE-Cy7 conjugated antibodies, compensation controls should include:

  • tube 1) unstained or isotype stained for each color
  • tube 2) anti-CD4 FITC
  • tube 3) anti-CD8 PE
  • tube 4) anti-CD8 PE-Cy7

Experiments that cannot spare cells for compensation controls can use a bead product from Becton Dickinson called BD CompBeads. The beads can be stained with antibody specific for the Kappa light chain for mouse, rat, or rat/hamster immunoglobulins. The beads are stained as if they were cells using the same antibodies and fluorochromes that are used in the experiment. In this way, a set of compensation tubes can be produced using beads instead of cells that have both a negative and bright positive population for each color. If the beads are used, the experiment would only require the addition of an unstained or a negative tube of cells to set the initial baselines.

The Facility's mission is to serve the investigators in their quest to obtain accurate data. The lack of proper compensation may yield misleading, confusing, and inaccurate data. In order to live up to our mission of assuring quality control and reproducibility, the Facility will not run the samples if the necessary compensation controls are not provided by the investigator.