Skip to main content Skip to navigation

Procedure for Manual Setting of Compensation for Multi-Color Flow Cytometric Analysis




When performing simultaneous, multi-color, immunofluorescence analysis using a flow cytometer, intrinsic spectral overlap of the different fluorochromes used, if uncorrected, will lead to emission of a given fluorochrome into an 'inappropriate' detector. Such a lack of compensation for this spectral overlap can lead to misinterpretation of data from false positive populations and artifactual populations on multi-color contour plots. However, through the use of appropriate single and double stained control samples, compensation for spectral overlap can be accomplished by electronic subtraction of unwanted signal, and accurate flow cytometric analysis of multi-color stained cells can be successfully achieved.

Monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC), R-phychoerythrin (PE), and PE-Cy5 can be used to analyze multiple antigenic determinants within a cell population. Such an analysis can be accomplished with a single excitation wavelength (488 nm), using a 15 mWatt argon ion laser. FITC emission is measured as a green signal (530 nm peak fluorescence) by the FL1 detector, PE is measured as an orange signal (575 nm peak fluorescence) by the FL2 detector, and PE-Cy5 is measured as a violet signal (peak fluorescence 670 nm) by the FL3 detector. (For further information, please refer to "Fluorochrome Absorption and Emission Spectra." or visit our Fluorescence Spectrum Viewer. However, a significant amount of orange fluorescence is present in the FITC emission, some green fluorescence is present in the R-PE emission, and so on. This spectral overlap, if uncorrected, will lead to a fluorochrome signal being picked up by an inappropriate detector. Compensation is the process of correcting for this overlap; it is the electronic subtraction of unwanted signal to remove the effects of spectral spillover. Through compensation, the fluorescence measurement of a cell sample stained with one fluorochrome is electronically forced to be identical to that of the unstained cells, with regard to the two remaining, inappropriate detectors.Lack of compensation or an improper compensation set-up can yield false positives and artifactual histogram shapes. For example, if fluorochrome-stained samples are undercompensated during multi-color analysis, a false double positive population might be perceived on the 2-color contour plot, and the data misinterpreted. To prevent such compensation-related artifacts in three-color immunofluorescence staining, it is important to set the compensation for a multi-color analysis using single color and then dual color controls. That is, set compensation based on staining cells with each antibody fluorochrome individually. Next, fine-tune compensation set up with 2-color stained controls. For this 2-color control step, staining exclusive positive populations and a negative population is the most helpful for adjusting fluorescence signal along the appropriate axes. In order to standardize results and monitor instrument performance over time, color compensation should be initially set each day using fluorescence reference standards, and appropriate automatic calibration/compensation software.




  1. Perform instrument calibration/standardization procedures each day according to the established protocols of your laboratory.
  2. Run an unstained (autofluorescence control) cell sample. Adjust FSC (forward scatter) and SSC (side scatter) detector settings so that cells of interest are displayed on scale and can be gated as desired.
  3. While gating on the cells of interest, adjust FL1, FL2, and FL3 detector settings so that autofluorescence background is roughly within the first decade of the log scale of the fluorescence intensity histogram. Compare auto-fluorescence control (unstained cells) with stained cell positive controls to confirm that the stained cells are on scale for each parameter.
  4. Set compensation based on running cells stained with each antibody-fluorochrome conjugate individually (single stain). While monitoring 2-color dot plots, adjust compensation settings so that positively stained cells are directly in line with the unstained background cells and parallel with the appropriate axis. For example, with a PE-conjugated mAb stained cell population, adjust the FL1-%FL2 setting up or down so that the FL2 positive population is vertically aligned with the FL2 negative population (i.e., on the FL2 vs. FL1 dot plot). Then, with the FITC-conjugated mAb stained cell population, adjust the FL2-%FL1 setting up or down so that the FL1 population is horizontally aligned with the FL1 negative population (i.e., on the FL2 vs. FL1 dot plot). And so on for the third color, adjusting both FL2-%FL3 and FL3-%FL2.
  5. Next, fine tune compensation by running 2-color control cell samples stained with 1) FITC and PE mAbs and 2) PE and PE-Cy5 mAbs. It is most helpful to choose control mAbs which stain mutually exclusive cell populations. Alternatively, 2-color compensation adjustment can be performed on a tube of cells derived from mixing two different single stain controls: e.g., mix aliquots of the FITC and PE labeled cells. This would obviate the need for mutually exclusive cell markers. Each population of fluorochrome labeled cells should be contained within the appropriate quadrant. On some flow cytometers, FL1 and FL3 cannot be directly compensated against each other. Hence, a FITC/PE-Cy5 control would not be helpful for 2-color compensation set-up.
  6. Check the 3-color stained cell population; the previous steps should have compensated the signals sufficiently so that no further adjustment is necessary for this sample.
  7. Start collecting control and sample data and save.
  8. Compensation settings may need to be readjusted for each multi-color staining experiment. Therefore, for each multi-color experiment, single and dual color staining controls are needed so that steps 3 - 7 can be repeated. To minimize the amount of adjustment between sets of reagents, use the brightest staining reagent/cells of each fluorochrome for initial compensation set-up.


Useful Reagents


Calibrite beads (cat. no. 340486, 340487, 349502, 340497, 345036)BD recommends using the calibrite beads for flow cytometer daily calibration. This will check the laser alignment, and allow you adjust the Fluorescence compensation settings for multicolor flow cytometric analyses. The calibrite beads are available as 2 or 3 color-set: Unlabeled, FITC, PE, or Unlabeled, FITC, PE, PerCP. and you can add APC or PerCP-Cy5.5 to the 3-4 colors set. These products are found on the web by typing "calibrite" into the search engine.Compbeads (cat. no. 552843, 552844, 552845)BD recommends using the Compbeads if using tandem dyes (ie, PE-Cy7, APC-Cy7, etc.), The compbeads are to optimize fluorescence compensation settings for multicolor flow cytometric analyses. These are highly recommended for use in all experiments with all fluorescences, especially using tandem dye (ie, PE-Cy7, APC-Cy7, etc.) conjugates, which may have distinct spectral characteristics for each conjugate. These products are found on the web by typing "compbead" into the search engine.Rainbow beadsTo check calibration on your machine, you can look at intensity, mean fluorescence, and number of peaks. The Rainbow beads are single bead population particles that are dyed to either single fluorescent intensity or several different fluorescent intensities. They are available with single or multiple fluorophores. These products are found on the web by typing "rainbow" into the search engine.


Useful Links


Multicolor Flow Cytometry


Fluorescence Reference Chart


Fluorescence Spectrum Viewer


Webinar: The Digital Flow Cytometer: Performing Instrument Characterization for Optimal Setup. Presented by Joe Trotter, November 2006

APO-BRDU and APO-DIRECT are trademarks of Phoenix Flow Systems.