B cell enrichment via Dynabeads

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