Table 3.

Flow cytometry protocol – 2 hour prep, 1.5–4 hour protocol.

Thaw cells at 37°C until almost unfrozen and add to DMEM/F12 plus 20% FBS and 100U/mL Penicillin, 100μg/mL Streptomycin, 0.25μg/mL Amphotericin B. Spin and wash twice with PBS. Spin and resuspend in 1ml PBS. Add 1μL (or according to manufacturer’s protocol) of amine reactive viability dye and incubate for 30 minutes at 4°C. Spin and wash once with a cell-staining buffer (eg. PBS with 2% FBS and 0.1% sodium azide, or a commercially available alternative). Resuspend in 100μL and add Fc receptor blocking antibody according to manufacturer’s instructions for 5min at RT. Split each sample in two (one to be stained and the other to remain unstained, approximately 5 million to 500,000 cells each) and bring to 100μL in cell staining buffer. Either aliquot approximately 100,000 cells or a commercially available compensation bead as recommended by the manufacturer for single antibody compensation. Add all antibodies to the cells set aside to be stained, and each antibody to the single antibody compensation tubes. Incubate in dark on ice for 15–30min with occasional shaking. Spin and wash once in cell staining buffer. Spin and resuspend in 500μL-1mL cell staining buffer and filter through 25–40μm strainer into tube for flow cytometry. At this point cells can be fixed, permiabilized and stained if antibodies for internal proteins are to be detected. Store on ice and in the dark until flow cytometry.