B cell enrichment via Dynabeads¶
Contributed by Martin Fitzpatrick, University of Birmingham, United Kingdom
B cell enrichment using Invitrogen Dynabeads magnetic separation
- Count cells in PBMC sample for enrichment.
B cells constitute ~5% of total PBMC population.
- Calculate total Dynabeads for use at 4x B cell number (4 beads/cell)
- Wash x2: Centrifuge cells 1200rpm 5 minutes, resuspend cells in serum-free RPMI.
After final wash, resuspend in 100ul per 10^6 beads.
- Add B cell specific beads at number required. Stock at 4x10^8 beads/ml.
- Agitate tube at 4’C for 30 minutes; e.g. place on roller in cold-room
- Resuspend in 4mls cold RPMI + 1% FCS. Place tube into magnet for selection.
Wait for 4 minutes, then remove supernatant to fresh tube (B cell depleted PBMCs). Repeat x5
- Resuspend beads in 500ul RPMI + 1% FCS
- Add 20ul detachabeads (10ul/25ul beads used)
- Agitate at room temperature for 45 minutes; e.g. roller.
- Resuspend in 4mls RPMI + 1% FCS. Place tube into magnet for selection.
Wait for 4 minutes, then remove supernatant to fresh tube (B cells). Repeat x3