Negative Immunomagnetic Selection: A Method to Purify B-cells from Peripheral Blood Mononuclear Cells
Published: April 30, 2023
Abstract
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
- Resuspend 107 PBMCs/ml in PBS 0.1 % bovine serum albumin (BSA) 2 mM EDTA, pH 7.4 (isolation buffer).
- Add 10 µg of mouse monoclonal anti-CD3, -CD14 and -CD16 primary antibodies for 107 cells and incubate for 30 min at 4 °C.
- Wash the cells with the isolation buffer and centrifuge at 500 x g for 5 min at 4 °C.
- Resuspend the cells in the isolation buffer at 107 PBMCs/ml.
- 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.
- 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.
- Remove the tube from the magnet and resuspend the washed beads in 100 µl of isolation buffer.
- Incubate 107 cells with 100 µl of magnetic beads for 30 min at 4 °C with gentle tilting and rotation.
- 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.
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
Cite This Article
Negative Immunomagnetic Selection: A Method to Purify B-cells from Peripheral Blood Mononuclear Cells. J. Vis. Exp. (Pending Publication), e20278, doi: (2023).