The field of flow cytometry has evolved over the last few years with the addition of lasers into flow cytometers; lasers allow a wider range of fluorescent dies to be used. An even more important advancement has been the number of new dies developed; these dyes enable us to do 5, 6 or even 12 simultaneous stainings. This is of critical importance when identifying and characterizing very low cell frequencies in the periphery.
The major problem to circumvent in this multiple staining flow cytometry experiments is the spectral overlap between fluorescent dies. It is well known that FITC and PE dyes overlap a lot, which can give false positive results. When the receptor targeted is highly expressed, one can simply get a cell sample and perform a single staining as a positive control which will then be used for compensation. However, not all receptors or intracellular cytokines are highly expressed and using cells might provide poor quality controls.
BD™ CompBeads are polystyrene microparticles that bind any mouse kappa light chain-bearing immunoglobin. Mixing the amount of fluorescently-labeled antibody used for your samples with one drop of CompBeads provides a bright positive control; using the CompBeads Negative control (included in the kit) provide a measure of background fluorescence. Note that only 10 minutes are needed for the antibody binding to the beads. Using the FACS-Diva software (BD), we are able to set up the compensation for up to 9 colors in just one click. Manually adjusting the compensation can be a hard task, but using the beads is helpful since the mean fluorescence ranges from 104 to 105 (except for PercP which is a dim dye). Anti-rat and anti-hamster CompBeads are also available for antibodies not of mouse origin.
The use of the CompBeads is much recommended when using tandem conjugates such as APC-Cy7 which is less stable than traditional dyes such as FITC. So far, we have used FITC, PE, PerCP, Pacific-Blue, Pacific-Orange, APC, APC-Cy7, PE-Cy7, AmCyan, PE-Cy5.5, Alexa-700 without any difficulties. The only difficulty was when using fluorescent probes which do not bind to beads but only to cells. The best examples are 7-AAD (cell death marker) and CFSE (cell proliferation marker). You will easily get a bright signal for CFSE stained cells, but to obtain a bright signal for 7-AAD one needs to induce cell death (by heating or adding H2O2) to see association of 7-AAD.
Some important recommendations: Always titrate your directly-conjugated antibodies to have the optimal experimental conditions for you (saving antibody) and for your cells. A routine check of your cytometer and settings is necessary; for this, I recommend the Sphero™ Rainbow Calibration Particles (BD cat# 559123), which enable you to check up to 8 different dyes in few minutes and very inexpensively!
BD™ CompBeads From BD Biosciences
The compensation beads from BD Biosciences are used to set up compensation controls. It is a very useful tool to have bright positive controls for polychromatic flow cytometry which requires an accurate compensation between dies. You gain time and eliminate the need for cells that are not always available when using patient’s blood.
The beads are small and need to be seen by setting a low side scatter (SSC) value; this is not useful when working with cells needing a high SSC value. When using dyes such as CFSE, you must go back to the old fashion way, using an FITC or Alexa-488 stained cell sample.
The Bottom Line
Life is much easier in the colorful world of polychromatic flow cytometry. Standardization of protocols will be required in the future and the use of compensation beads will certainly be a solution to this important issue.