Miltenyi Biotec blog

8 tips to improve compensation in multicolor flow experiments

Search the term "flow cytometry compensation" and you’ll get thousands upon thousands of results. It’s little surprise. Multicolor flow cytometry is one of the most commonly used techniques for acquiring a large quantity of information on several cell subpopulations from a single sample. And a critical component of any multicolor flow experiment, besides choosing the right flow cytometry antibodies,  is compensation.

Why do we need to perform compensation? When using multiple fluorochromes in an experiment, you run the risk that the fluorescence emission of one fluorochrome is detected in a detector designed to measure signal from another fluorochrome. As you can imagine, this can seriously impact the accuracy and quality of your data. Compensation removes the signal of a specific fluorochrome from all neighboring channels where it is detected as well.

 For a complete overview of the compensation topic, you can also watch our recent webinar.

8 tips for setting up proper compensation controls in multicolor flow experiments

Tip 1: Compensation control sample must be stained with only one color.

When a cell population emits fluorescence simultaneously into two channels, this can be due to two reasons:

  1. The cell population expresses a marker that is stained with a fluorochrome-conjugated antibody and fluorescence spillover from the fluorochrome is detected in a second channel. 
  2. The cell population expresses two markers that are stained with different fluorochromes emitting into these two channels.

To exclude double-positive cells from being measured during compensation, the compensation control should be stained with a single color only.

Tip 2: Samples must contain a negative and a positive cell population for median comparison.

To set compensation correctly, the median fluorescence intensities (MFIs) of negative and positive cells in the spillover channel must be compared. After adjusting the compensation factor, the MFIs should be around the same value.

Tip 3: Negative and positive cells must have the same level of autofluorescence.

If there is a difference in autofluorescence, samples might be overcompensated in the experiment. Compensation is a powerful method for correcting spectral overlap, but it is not appropriate for adapting differences in autofluorescence.

Tip 4: Surrogate markers can be used for compensation (e.g. for rare cell applications).

It is not necessary to stain the same marker in control sample and experimental sample. Different markers can be used for compensation. This is particularly important when the positive cells are rare and proper compensation would require the acquisition of a large number of cells.

Tip 5: Use bright markers for setting compensation.

If an alternative marker is used for setting compensation, it is crucial that the marker in the compensation control is at least as bright as the marker in the experimental sample. Otherwise, compensation might not be adjusted properly. In general, dim compensation controls are not suitable for determining precise compensation values.

Tip 6: Compensation control samples must contain the same fluorochrome as the experimental sample.

While it is not that important to use exactly the same cells in the compensation control samples, it is vitally important to use exactly the same fluorochrome. FITC, for example, should not be used to compensate for GFP. Different shapes of fluorochrome emission spectra result in differences in the magnitude of spectral overlap and therefore require different compensation.

Tip 7: Tandem fluorochrome conjugates require lot-specific compensation.

Tandem fluorochromes are indispensable for multicolor applications. Their large Stokes shift expands the variety of fluorescence channels that can be used in an experiment. However, the saturation of the donor fluorochromes with acceptor molecules can differ from lot to lot. This can result in differences in the emission behavior of the donor fluorochrome. Therefore, different compensation values may be required for different antibody lots. This means that exactly the same antibody conjugate lot has to be used for compensation samples and experimental samples.

Tip 8: Compensation beads can be used instead of cells.

Compensation beads are "artificial cells" that can be loaded with fluorochrome-antibody conjugates. They can then be used to set compensation. They fulfill many of the requirements outlined above and are therefore universal and excellent compensation controls:

  • negative and positive beads have the same autofluorescence 
  • the fluorescence emission of these beads is very bright
  • the capture beads can be loaded with the same fluorochrome conjugate lot used in the experiment, thus enabling lot-specific compensation

A number of kits are available from Miltenyi Biotec that enable straightforward compensation. The kits are based on beads coated with antibodies against the kappa light chain of the labeling antibodies. Therefore, the beads can bind to the labeling antibodies and mimic cells that are positive for the fluorochromes conjugated to the antibody. The kits also contain beads that are not coated with antibody, thus resembling negative cells. The beads allow for lot-specific compensation of tandem fluorochromes as they can be loaded with exactly the same lot:

Contact your local MACSQuant Specialist

If you want to learn more about compensation or other topics in flow cytometry, feel free to contact your local MACSQuant Specialist here.