10 Tips for Setting Up Compensation

When performing any experiment, planning and setting up the proper controls are critical for success. In a multicolor flow cytometry experiment, this includes running compensation controls in addition to your experimental or biological controls. Compensation is necessary in any multicolor experiment due to spectral overlap. In general, fluorophores have a wider emission spectra than the filter which is used to detect them. This causes spillover from one fluorophore into the detectors of other fluorophores. Compensation is applied to account for this spillover and allow you to properly identify your cells of interest.
10 tips to set up the ideal compensation controls
  1. Start by preparing individual single color controls for each marker in your panel. Ideally, the control should contain both a negative and positive population. If the marker is expressed on 100% of the cells, an unstained universal negative sample needs to be employed. If the marker is, for example, staining a cytokine or another marker not always expressed, the positive control should be done on cells stimulated to produce that expression.
  2. Ensure that the positive signal in your compensation control is at least as bright as your samples. If you are looking at a rare marker, you may want to consider using compensation beads instead of cells.
  3. Use the same fluorophore for compensation as you do to stain your samples. Using the same reagents for compensation is particularly important when staining with tandem dyes (such as PE/Cyanine7) due to potential lot-to-lot variation in the transfer of energy between the donor and acceptor molecules. Learn more about tandem dyes.
  4. Ensure that the autofluorescence of the negative and positive population are the same. If all of your compensation controls are stained using beads, the beads will all have the same degree of autofluorescence. You can use both beads and cells to compile your compensation profile; however, you can not choose the option to employ a universal negative control because the cells and beads will not have the same level of autofluorescence.
  5. Keep your PMT voltages as balanced as possible. The absolute compensation values are not only dependent on the spectral overlap between two fluorophores but also the PMT voltage. Remember the default CST settings may not be optimal for your sample, instead, use stained cells to adjust the PMT voltages for your experiment. To maintain balanced PMT voltages, it is important to build a balanced panel. For more information on building a balanced panel, check out our Multicolor Staining Guide.
  6. Treat your compensation controls and your samples the same way. For example, if you are fixing your samples, perform fixation on your compensation controls as well.
  7. Collect enough events to accurately calculate an MFI for the positive and negative population.
  8. Apply compensation to all parameters in your experiment. Remember some fluorophores can be minimally excited by multiple lasers which may cause spillover into channels that are not excited by the same laser.

    Using a Spectra Analyzer, you can see that the PE/Cyanine5 can be excited by the 633 nm laser, making it difficult to compensate the spillover into APC.

  9. Run freshly stained compensation controls for each experiment. Proper controls should be set up for each experiment, even if you've already generated a similar compensation matrix in the past.
  10. If you are using compensation beads, ensure that they will bind the isotype of your antibody of interest. Some compensation beads are specific to certain species or kappa light chains.
Using these guidelines, you can set up compensation for your multi-color experiment with ease. Remember, having high absolute compensation values (even values over 100%) aren't inherently problematic as long as you can resolve the positive and negative populations. For more information on choosing the proper controls for your experiment check out our Flow Cytometry Troubleshooting webpage or contact the technical services team at tech@biolegend.com.
Contributed by Kelsey Swartz, PhD.

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