Proper compensation is critical for accurate interpretation of your flow cytometry data. Therefore, we have compiled this “comprehensive” list of compensation tips to help improve your analysis. Sections:

What is Compensation & Why Do I Need it?

Compensation is necessary whenever a fluorophore may be detected in multiple channels. The purpose of compensation is to account for any spill-over signal in all neighboring channels where it is also detected. Ultimately, compensation is used to ensure that the fluorescence detected in each channel derives from the specific fluorochrome being measured.

Best Practices for Accurate Compensation

This list of general guidelines is intended to help researchers set correct fluorescence compensation on any cytometer. However, we also recommend that you refer to the cytometer manufacturer/analysis software user manuals for more detailed information and compensation guidelines specific to your instrument/software.

  1. Viability Dyes Use a viability dye to exclude dead cells and debris from your analysis.
  2. Panel Design Use FluoroFinder’s panel design tool to select fluorochrome combinations with minimal emission overlap. Reducing spillover upfront during experiment design will greatly reduce the amount of compensation needed later.
  3. Compensation Controls Use single-color compensation controls containing both positive and negative populations for each fluorochrome. The positive population should comprise at least 10% of the total cell population and the signal must be at least as bright as any of the fluorophores that will be used in the experiment. The fluorochrome used for these compensation controls must match the experimental fluorochrome exactly. Also, the same detector settings must be used to ensure equal sensitivity. If possible, these positive and negative populations should be the same cell type, so that the background auto-fluorescence levels are equal. Different cell types may be used, so long as they both express the markers of interest. Alternatively, if a marker of interest is not expressed in the control cells, you may use a different antibody (with the same fluorochrome) against a more common marker. In these cases, compensation (or capture) beads are recommended.
  4. Compensation Order Start your compensation on the fluorochrome with the highest wavelength emission. Then proceed, step-by-step, down to the fluorochromes with lower wavelength emission. As you go, be sure to check compensation in each channel where spillover occurs.
  5. Correct Compensation Correct compensation is set when the median fluorescence intensities of the positive and negative control populations are equal.
  6. Manual vs. Automated Use automated software tools where available. Modern analysis software includes automated compensation algorithms that will provide superior compensation levels. Consult your core manager or the available user manual for details about your specific analysis software. Always remember to save the single-color control settings to allow for post-acquisition compensation adjustments as necessary.
  7. Tandem Dyes Tandem dyes are composed of two covalently attached fluorescent molecules, a donor and an acceptor. Upon excitation of the donor, photons are passed to the acceptor via a process called fluorescence resonance energy transfer, causing the acceptor to fluoresce. This allows the tandem dye to appear as a single unique fluorophore, exhibiting the excitation properties of the donor and the emission properties of the acceptor. Because the photon transfer efficiency in tandem pairs can differ each time the conjugation is performed, the resulting spectral overlap values can vary from lot to lot. Therefore, each tandem dye antibody lot used in the experiment will require its own compensation control to ensure that their spectral characteristics are identical (PMID: 4415368).

For additional information on compensation techniques, see: Excyte Compensation blog BioLegend Compensation blog Bio-Rad Compensation blog BD Bioscience Compensation PDF