The BD® CompBeads Compensation Particles Set contains polystyrene microparticles that are used in fluorescence compensation settings for multicolor flow cytometric analyses. The beads are available as anti-mouse, anti-rat or anti-rat/hamster sets. The BD® CompBeads Compensation Particles Set provides two populations of microparticles, the species-specific Ig, κ particles, which bind any κ light chain-bearing immunoglobulin, and the negative control particles that have no binding capacity. When mixed with a fluorochrome-conjugated rat antibody, the BD® CompBeads Compensation Particles provide distinct positive and negative (background fluorescence) stained populations that can be used to set compensation levels manually or automatically using instrument setup software. Since the compensation adjustments are made using the same fluorochrome-labeled antibody to be used in the experiment (see technical data sheet), this method allows you to establish compensation corrections for spectral overlap for any combination of fluorochrome-labeled antibodies without having to use valuable tissue samples or hard-dyed beads with potentially mismatched fluorescence spectra.
We highly recommend using the BD® CompBeads Compensation Particles for experiments with tandem dye (i.e., PE-Cy7, APC-Cy7, etc.) conjugates that may have distinct spectral characteristics for each conjugate
Anti-Mouse, Anti-Rat, Anti-Rat/Hamster Ig, κ/Negative Control Compensation Particles Set Protocol
- Vortex the BD® CompBeads Particles thoroughly before use.
- Label a separate 12 x 75 mm sample tube for each fluorochrome-conjugated rat Ig, κ antibody to be used in a given experiment.
- Add 100 µL of staining buffer (e.g., BD Pharmingen™ Stain Buffer (FBS) [Cat. No. 554656] or BD Pharmingen™ Stain Buffer (BSA) [Cat. No. 554657]) to each tube.
- Add 1 full drop (approximately 60 µL) of the BD® CompBeads Negative Control Beads and 1 drop of the species-specific BD® CompBeads (Anti-Mouse, Anti-Rat or Anti-Rat/Hamster) Ig, κ Beads to each tube and vortex.
- Add 20 µL of each prediluted antibody stock (or bulk antibody diluted to a concentration optimal for staining 106 cells) to be tested on a given experiment to the appropriately labeled tube. (Make sure the antibody is deposited to the bead mixture, then vortex.)
- Incubate 15–30 minutes at room temperature. Protect from exposure to direct light.
- During the incubation of beads and antibody, set the flow cytometer instrument PMT voltage settings using the target tissue for the given experiment (e.g., whole blood, splenocytes, etc.). If you are unsure, use the BD® CompBeads Negative Control Beads as your negative reference point and proceed.
- Following the incubation step (see Step 6 above), add 2 mL staining buffer to each tube and pellet by centrifugation at 200 x g for 10 minutes.
- Discard the supernatant from each tube by careful vacuum aspiration using a fine-tip Pasteur pipette.
- Resuspend the bead pellet in each tube by adding 0.5 mL of staining buffer to each tube. Vortex thoroughly.
- Run each tube separately on the flow cytometer. Gate on the singlet bead population based on FSC (forward-light scatter) and SSC (side-light scatter) characteristics.
- Adjust flow rate to 200–300 events per second if possible.
- Create a dot plot for the given fluorochrome-conjugated antibody as appropriate.
- Place a quadrant gate such that the negative bead population is in the lower left quadrant and the positive bead population is in the upper or lower right quadrant. Adjust the compensation values until the median fluorescence intensity (MFI) of each population (as shown in the quadrant stats window) is approximately equal)..
- Repeat Steps 13 and 14 for other tubes, as necessary.
- Proceed to acquiring the actual staining experiment.
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
