Note: BD Horizon™ V500 and AmCyan conjugated reagents can show significant differences in emission spectrum on stained cells and when captured on BD™ CompBeads. Thus, spillover values for these dyes evaluated with BD™ CompBeads may not provide correct compensation for cells. Therefore, single stained cellular controls are recommended to set up compensation for AmCyan and BD Horizon™ V500 reagents. BD Horizon™ V500-C has been modified to minimize these spectral differences and BD™ CompBeads may be used to determine spillover values for RUO antibodies conjugated to BD Horizon™ V500-C.
Without affecting compensation function, some lots may profile as a bi-modal histogram, which may be possible due to inherent light scatter and/or residual aggregation of the compensation particles. Optimization of instrument voltage or gating conditions may be helpful for improving histogram visualization.
This BD™ CompBead Plus Set has been tested with mouse Ig antibodies conjugated to various fluorochromes and analyzed using a BD FACS brand flow cytometer to ensure specificity and reactivity of the particles. See the specific instructions below on the use of the BD™ CompBead Plus Set:
1. Vortex BD™ CompBead Plus thoroughly before use.
2. Label a separate 12 x 75 mm sample tube for each flurochrome-conjugated mouse Ig, κ antibody to be used on a given experiment.
3. Add 100 µl of staining buffer [e.g., BD Pharmingen™ Stain (FBS), Cat. No. 554656 or BD Pharmingen™ Stain (BSA), Cat. No. 554657] to each tube.
4. Add 1 full drop (approximately 60 µl) of the BD™ CompBead Plus Negative Control (BSA) and 1 drop of the BD™ CompBead Plus Anti-Mouse Ig, κ beads to each tube and vortex.
5. Add 20 µl of each prediluted antibody stock (diluted to a concentration optimal for staining 10^6 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.)
6. Incubate 15 - 30 minutes at room temperature. Protect from exposure to direct light.
7. During the incubation of beads and antibody, set the flow cytometer instrument PMT voltage settings using the target tissue for the given experiment (eg, whole blood, splenocytes, etc). If you are unsure, use the BD™ CompBead Plus Negative Control (BSA) beads as your negative reference point and proceed.
8. 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.
9. Discard supernatant from each tube by careful vacuum aspiration using a fine-tip Pasteur pipette.
10. Resuspend bead pellet in each tube by adding 0.5 ml of staining buffer to each tube. Vortex thoroughly.
11. 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.
12. Adjust flow rate to 200 - 300 events per second if possible.
13. Use the compensation tool in BD FACSDiva™ software to calculate the compensation for your experiment, or proceed to step 14 to manually adjust compensation values.
14. To manually set compensation values, create a dot plot for the given fluorochrome-conjugated antibody as appropriate [i.e., to set compensation for a fluorescein (FITC)-conjugated antibody, use an FL1 vs. FL2 dot plot].
15. 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, and adjust the compensation values until the median fluorescence intensity (MFI) of each population (as shown in the quadrant stats window) is approximately equal (i.e., for FL2 -%FL1, the FL2 MFI of both bead populations should be approximately equal when properly compensated).
16. Repeat Steps 14 and 15 for other tubes, as necessary.
17. Proceed to acquiring the actual staining experiment.