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

Based on their cell surface receptors, human circulating B cells are classified as CD19-positive naïve B cells, CD19 and CD27-positive memory B cells, and CD19, CD27, and CD38-positive plasmablast plasma cells.

To sort these cell populations, take a purified B cell suspension. Incubate with human immunoglobulin G antibodies. The antibodies block the B cells' Fc receptors, preventing non-specific antibody binding.

Add fluorescently-labeled antibodies targeting CD19, CD27, and CD38.

The anti-CD19 antibodies bind to the CD19 receptors ubiquitously expressed on B cells.

The anti-CD27 antibodies bind to the CD27 receptors expressed on memory B cells and plasmablast plasma cells.

The anti-CD38 antibodies bind to the CD38 receptors expressed on plasmablast plasma cells.

Add a fluorescent DNA-binding dye as a dead cell marker.

Dead cells exhibit a loss of membrane integrity, allowing the cellular entry of the dye and binding to the double-stranded DNA; live cells remain unstained.

Centrifuge to remove unbound antibodies and dyes and resuspend the cells in FACS buffer. Filter the cells through a strainer to obtain a homogeneous suspension and eliminate clumps.

Using a flow cytometer, sort the dead cells from the live ones. Within the live cells, CD19, CD27, and CD38 expression distinguish naïve B cells, memory B cells, and plasmablast plasma cells.

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.