B cell enrichment via DynabeadsΒΆ

Contributed by Martin Fitzpatrick, University of Birmingham, United Kingdom

B cell enrichment using Invitrogen Dynabeads magnetic separation

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  • 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