Isolation and Purification of B Cells from Human Peripheral Blood

To perform immunomagnetic negative separation of B cells, collect human peripheral blood in an anticoagulant-containing tube to prevent blood clotting. Add red blood cell, RBC, lysis buffer to lyse the RBCs without affecting other blood cells.

Centrifuge to sediment the intact, denser blood cells from the less dense lysed RBCs, which remain in the supernatant. Discard the supernatant. Resuspend the pellet in fresh medium.

Transfer the cells to a culture plate. Incubate to allow the macrophages and monocytes to adhere to the plate while the non-adherent cells, including B cells, remain in suspension.

Collect the cells in suspension into a fresh tube. Centrifuge to pellet the cells and resuspend them in a buffer.

Add a cocktail solution containing biotinylated antibodies specific for blood cells other than B cells. The antibodies bind to antigens on the surface of non-B cells, leaving the B cells unlabeled.

Centrifuge. Discard the supernatant containing unbound antibodies.

Add a solution of streptavidin-conjugated magnetic beads. During incubation, the streptavidin on the microbeads binds tightly to the biotinylated antibodies bound to non-B cells, forming complexes.

Place the tube in a magnetic field to adhere the magnetic beads-bound non-B cells to the tube walls, leaving the B cells in suspension. Transfer the supernatant containing pure B cells to a tube.

Centrifuge and resuspend the B cells in medium for further downstream analysis.

Immediately after obtaining a 10-milliliter blood sample from the median cubital vein into a 15-milliliter tube containing potassium-supplemented EDTA, invert the tube several times to prevent clot formation. Then, add 35 milliliters of autoclaved red blood cell lysis buffer to the cells for a 5-minute incubation at room temperature.

When light can be observed through the tube, centrifuge the blood and confirm the presence of a white pellet. Remove the residual lysis buffer with 10 milliliters of autoclave to PBS, and resuspend the pellet in 10 milliliters of RPMI 1640 medium.

Plate the cells in a 10-centimeter culture dish for a 30-minute incubation at 37 degrees Celsius and 5% carbon dioxide. Then, gently swirl the culture dish a few times and transfer the floating cells into a plastic 15-milliliter conical tube.

After two washes by centrifugation, resuspend the pellet at a 5 to 10 x 10⁶ cells per milliliter concentration in 200 microliters of cold PBS buffer, and add 5 microliters of biotinylated anti-human antibody cocktail specific for blood cells per 1 x 10⁶ cells.

After a 30-minute incubation on ice, wash the cells in a 10-fold excess volume of sterile PBS, and resuspend the pellet in 5 microliters of streptavidin-conjugated microbeads per 1 x 10⁶ cells for a 30-minute incubation on ice. At the end of the incubation, add 2 milliliters of PBS buffer to the cells and place the tube onto a magnetic stand for 8 minutes at room temperature.

When the microbeads have attached to the wall of the tube, carefully transfer the supernatant into a new conical tube, and add 2 more milliliters of PBS buffer to the cells. After the second supernatant collection, collect the pooled cells by centrifugation, and transfer the cells to a 5-milliliter polystyrene tube in fresh PBS buffer at a 1 x 10⁷ cells per milliliter concentration.