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

Samenvatting

Source: Serra S. et al. HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells. J. Vis. Exp. (2016)

The video explains a way to purify B-cells by immunomagnetic targeting and separation from peripheral blood mononuclear cells suspension. The method involve labeling unwanted cell types with specific antibodies or ligands (linked to magnetic particles) targeting specific cell surface proteins, to be depleted from the heterogenous cell suspension by magnetic separation.

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.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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