Fluorescence compensation in flow cytometry

Compensation in flow cytometry

The following document describes why compensation is required for flow cytometry and how to apply it. Please refer to the manufacturer’s instructions and software manual for a more detailed compensation procedure for the instrument you are using.

What is compensation?

All fluorochromes have excitation and emission spectra. The excitation spectrum is a range of light wavelengths that add energy to a fluorochrome, causing it to emit light in another range of wavelengths, the emission spectrum. Within a flow cytometer, the appropriate ranges of excitation and emission wavelengths are selected by bandpass filters. However, when emission spectra overlap, fluorescence from more than one fluorochrome may be detected. To correct for this spectral overlap, a process of fluorescence compensation is used. This ensures that the fluorescence detected in a particular detector derivesis soley from the fluorochrome that is being measured.

In the example below, following excitation with 488 nm light, PE emission is largely detected in the detector specific for PE but the emission tail lies within the range of the bandpass filter used for detection of PE-Cy5. This will be seen as “false positive” signals in the PE-Cy5 channel and fluorescence compensation is needed to correct for this overlap.

Excitation and Emission Spectral Profiles

Example summary:

  1. Run a sample stained only with a PE-labeled antibody. Observe the signal in both PE and PE-Cy5 channels.
  2. Adjust the compensation settings until no PE signal is seen in the PE-CY5 channel (see the procedure below).

Essentially what the software is now doing in the PE-Cy5 channel would be the following:           

(PE-Cy5 + PE  overlap) – (PE  overlap) = accurate PE-Cy5  results

General procedure:

The procedure for setting correct fluorescence compensation is essentially the same on any cytometer but there are differences between the various available makes and models which means it is difficult to provide a ‘one size fits all’ protocol.  However, the following guidelines should be suitable in most cases. We would recommend to review the flow cytometer manufacturer’s instructions for more information and specific guidelines for compensation using your particular machine.

  1. Ensure that the cytometer is performing within specification using standard beads.
  2. Set voltages for fluorescence channels using an unstained sample. Forward scatter and side scatter should be adjusted so that the cell population is clearly delineated.  Dead cells, clumps and debris should be excluded from further analysis.
  3. There are several rules that should be followed in order for correct compensation to be set. Compensation controls should contain both a positive and a negative population.  The positive should be at least as bright as anything that will be encountered in the experiment and should form at least 10% of the population.  The autofluorescence of the positive population before staining should be the same as the negative control. If this is not possible using a biological system, consider the use of compensation beads to set compensation.
  4. Compensation is correctly set when the median of the negative population is equal to the median of the positive population in the spillover channel.  For example, if using a positive FITC-stained sample to apply compensation to the PE channel, the median of the negative and the FITC-positive populations should be equal in the PE channel. On a FITC vs. PE dot plot, place a box around the negative population and another around the positive population. In a statistics box, adjust the PE-%FITC level until the PE medians are equal. With modern digital systems the use of a bi-exponential display is advised.
  5. Repeat for all channels into which FITC leaks.
  6. Repeat for all single color controls.
  7. Compensation levels may be set live (either manually or via software “wizards”) or this can be performed offline. In all cases, though, the single color control settings should be saved to allow post-acquisition compensation or adjustment to be performed.

It is also important to remember that the controls used to set compensation are used solely for that purpose.  They should not automatically be used to set the negative level of fluorescence.

Tips for compensation:

  1. For each of the fluorochromes used in the test samples, a single color positive control is required for compensation.
  2. Each control should ideally be made up of a population of negative or unstained cells and a population of single color control positive cells. Both the positive and the negative cells should have the same level of background autofluorescence. Ideally,  they will be the same type of cell.
  3. As a general rule, compensate with the fluorochromes from the far-red end of the spectrum (higher wavelength), step-wise down to those fluorochromes at the lower end of the spectrum (lower wavelength).  Do not forget to check the compensation in all channels. Although most of the overlap will occur in the range on the higher wavelength side of the peak emission, due to the shape of the spectral emission curve, there may also be some bleed through/ overlap to  the lower wavelength range, below the peak emission. This will become more of an issue as more fluorochromes are used.
  4. Compensation can be difficult if there is a wide range of fluorescence intensities in negative populations, which in turn can compromise sensitivity.
  5. Some fluorochrome combinations should be avoided if possible (e.g., APC and PE-Cy5), given the high degree of emission overlap.
  6. Ideally, the positive and negative populations in the control samples should both be large, well defined and well separated. However, if you are looking for a rare population or have a very limited number of cells this might not be possible. There are some ways to overcome this problem:

a. Use different cells for your compensation controls. For example, if the test samples are primary tissue in limited supply, find a cell line that expresses the markers of interest  and use these for the compensation.

b. If a marker of interest is rare or possibly absent in the control cells, it is better to use a different antibody, directed against a more common marker, but carrying the same fluorochrome as will be used in the test. CD45 antibodies are often used for this purpose when studying rare lymphocyte markers. It is important to note however that exactly the same fluorochrome should be used. Tandem dyes (e.g., PE-Cy5, APC-Cy7) are more problematical due to variations in chemical conjugation; if a tandem conjugate is being used, e.g. PE-Cy5, the same batch of tandem should be used.

c. Use compensation beads. If the markers of interest are rare or absent on/in the control cells, you can still use the antibody if you substitute compensation beads for the cells. Compensation beads are polystyrene micro particles that bind to rat or mouse light chain Ig antibodies. (Check which ones are correct for your antibodies). Beads with no binding capacity are also available and can be used as a negative control to determine background fluorescence, ensuring that clear positive and negative populations are present in all compensation controls.

Useful links:

Mario Roederer

Document prepared in kind conjunction with Derek Davies, FACS Laboratory, London Research Institute
Cancer Research UK, 44 Lincoln’s Inn Fields, London, UK