Stain Compensation Bead Protocol

How to Stain Compensation Beads

There are at least 9 different vendors who provides Anti body capture beads a.k.a Compensation beads. All beads perform equally in analyzer, but some beads are made of highly dense polymer which made them difficult to run in sorters. For sorter we recommend NOT to use Ultra Comp, One Comp or Beads from Slingshot Bio.

Some manufacturers provide one vial that has a mixture of blank and Ab capture bead (type 1), some manufacturer provides two separate vial one for blank another for ab capture (type 2). In case of analyzer generally, for type 2 use ONLY the Ab capture bead for all preparation, use it also for universal negative (no stain).

1. One drop of bead is more than enough to create 2 tubes even 3 tubes. Add required number of drops in the unstained tube, add required amount of stain buffer.
2. Vortex briefly
3. Distribute equal volume (100 µl) in FACS tubes
4. Stain with Ab amount (defined from titration)
5. Stain for 15 min, dark, RT
6. Wash with 3 ml stain buffer or PBS, 500 g, 5 min
7. Decant carefully resuspend in 200 µl of stain buffer
8. Keep in ice, dark until use

Prepare fresh beads every day, DO NOT reuse stained beads even it is within 24 hours. It can seriously jeopardize unmixing/compensation and cannot be recovered.

DO NOT use brilliant stain buffer with beads, as these beads are essentially plastic, they will chemically react with this buffer