The purpose of this protocol is to coat silicon beads with various molecules. The beads are then mixed with immune cells and can trigger activation of various processes. In this protocol the beads are being coated with B7 protein and 2C11 antibody.
- Dilute beads to 10e7 beads/ml. Count beads to be sure that dilution is correct. Adjust volume as needed.
- Add 0.5μg of 2C11 antibody per ml of beads and mix immediately by vortexing. Mix beads in the cold room by rotating for 20 minutes.
- Add 0.5μg B7 per ml of beads (10μl of B7 at 58.5μg/ml to 1ml of beads). Incubate for an additional 15 minutes in the cold.
- Add equal volume of 1% BSA to beads and mix an additional 30 minutes in the cold.
- Spin beads and resuspend in RP10 medium. Count and adjust volume to give a concentration of 2 x 10e7 beads/ml
- Remove small aliquots of beads (10e5 or fewer) to stain with appropriate antibody to verify that beads were succesfully coated.
- Beads can be stored overnight at 4ºC at this point.