Isolation and Purification of B Cells from Human Peripheral Blood
내레이션 대본
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.
