Negative Immunomagnetic Selection: A Method to Purify B-cells from Peripheral Blood Mononuclear Cells

Protocol

1. Purification of Leukemic B Cells by Negative Isolation

1. Resuspend 10⁷ PBMCs/ml in PBS 0.1 % bovine serum albumin (BSA) 2 mM EDTA, pH 7.4 (isolation buffer).
2. Add 10 µg of mouse monoclonal anti-CD3, -CD14 and -CD16 primary antibodies for 10⁷ cells and incubate for 30 min at 4 °C.
3. Wash the cells with the isolation buffer and centrifuge at 500 x g for 5 min at 4 °C.
4. Resuspend the cells in the isolation buffer at 10⁷ PBMCs/ml.
5. Before incubating with the cells, resuspend sheep anti-mouse IgG magnetic beads in the vial. Transfer 100 µl of beads per 10⁷ cells to a tube.
6. Add 1 mL of isolation buffer and place the tube in a magnet holder for 1 min. Discard the supernatant with a 5 ml pipette.
7. Remove the tube from the magnet and resuspend the washed beads in 100 µl of isolation buffer.
8. Incubate 10⁷ cells with 100 µl of magnetic beads for 30 min at 4 °C with gentle tilting and rotation.
9. Place the tube in the magnet holder for 2 min and transfer the supernatant with the CD19⁺/CD5⁺ unbound cells to a fresh tube with a 5 ml pipette.

Materials

Name	Company	Catalog Number	Comments
Human blood
Purified anti-CD3, -CD14, -CD16	Made in-house		Mouse monoclonal
Dynabeads sheep anti-mouse IgG	Invitrogen	11031
Phosphate-buffered saline (PBS)	Amresco	E404-200TABS	tablets
Bovine serum albumin (BSA)	ID bio	1000-70	Standard grade
Isolation buffer			PBS 0.1% BSA 2 mM EDTA, pH 7.4
DynaMag-15 Magnet	Invitrogen	12301D	Dynal magnetic bead separator