Isolation and Purification of B Cells from Human Peripheral Blood

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board

1. Isolation and Purification of Human Peripheral Blood B Cells

1. Draw ~ 10 mL of blood from the median cubital vein (in the cubital fossa anterior to the elbow) into a 15-mL tube containing K₂EDTA (1.5 to 2.0 mg/mL blood) and immediately invert the tube several times to prevent clot formation.
2. Add 35 mL of autoclaved (121 °C, 15 min) red blood cell (RBC) lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, and 1 mM EDTA; pH 7.4) to the tube containing the fresh blood sample (≥ 3:1 vol/vol) and incubate at room temperature (RT) for no longer than 5 min.
   NOTE: The appearance of light transmission through the tube indicates the completion of RBC lysis.
3. Centrifuge at 600 x g at RT for 5 min. Ensure that the pellet is white in color.
4. Resuspend the pellet with 10 mL of autoclaved phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, and 1.47 mM KH₂PO₄; pH 7.4) and centrifuge as in step 1.3.
5. Discard the supernatant, resuspend the pellet with 10 mL of RPMI 1640 medium (supplements: 10% fetal calf serum, 100 U/mL penicillin/streptomycin, 0.25 µg/mL amphotericin B, and 2 mM L-glutamine), and then plate the cells into a 10-cm culture dish (10 mL of blood per dish). Place the dish in a 37 °C incubator with 5% CO₂ for 30 min.
6. Gently swirl the culture dish a few times and place the culture medium (suspension cells) into 15-mL plastic conical tubes. Discard the adherent cells (mostly macrophages) on the culture dishes.
7. Centrifuge at 600 x g at RT for 5 min. Discard the supernatant.
8. Resuspend the pellet with 1 mL of RPMI 1640 medium. Count the cell number with a hemocytometer or an automated cell counter.
   NOTE: The viability of isolated leukocytes is normally greater than 90% by trypan blue exclusion.
9. Centrifuge as in step 1.7 and resuspend the cells in ~ 200 µL of cold PBS buffer (0.5% bovine serum albumin (BSA) and 2 mM EDTA) at the concentration of 5 – 10 x 10⁶ cells/mL.
10. Add 5 µL of biotinylated anti-human Ab cocktail specific to blood cells (for the negative selection of B cells) per 10⁶ cells and incubate on ice for 30 min.
    NOTE: The anti-human Ab cocktail should at least include Abs specific to CD2 (or CD3), CD14, and CD16.
11. Add a 10-fold excess volume of sterile PBS to the cells, centrifuge at 600 x g for 5 min, and discard the supernatant.
12. Add equal amounts of streptavidin-conjugated microbeads (5 µL per 10⁶ cells) to the pellet and mix thoroughly.
13. Incubate on ice for 30 min in a 15-mL plastic conical tube.
14. Add 2 mL of PBS buffer into the tube.
15. Place the tube into a magnetic stand and incubate at RT for 8 min. The brown microbeads will gradually attach to the tube wall next to the magnet.
16. With the tube remaining in the magnetic stand, carefully transfer the supernatant into a new sterile 15-mL tube.
17. Repeat steps 1.14 to 1.16 and combine the two supernatants that contain the untouched B cells. Discard the microbeads.
18. Centrifuge at 600 x g at RT for 5 min. Discard the supernatant.
19. Resuspend the pellet in RPMI 1640 medium for downstream experiments.
    NOTE: Typically, 1 – 5 x 10⁵ B cells with a purity greater than 95% from 10 mL of peripheral blood can be isolated.