Flow Cytometry-Based Cell Sorting for Different Circulatory B Cell Populations

After performing non-specific blocking, add 1 microgram of each selection antibody per 1 x 10⁶ cells to the tube with gentle mixing for a 30-minute incubation on ice. During the last five minutes of the incubation, add 5 microliters of 7-AAD to the cells. Then, add 2 milliliters of fresh PBS to the cells, and vortex before centrifuging.

Resuspend the pellet in fresh sorting buffer at a 1 to 5 x 10⁷ cells per milliliter concentration, and filter the cells through a 40-micron cell strainer into a new 5-milliliter polystyrene tube. Then, sort the cells by flow cytometry into three 15-milliliter tubes containing 5 milliliters of fresh RPMI medium to collect the naive B cells, memory B cells, and plasmablast plasma cells.