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Procedure
Harvest cells from tissue, preparing a single cell suspension to a final concentration of 2 x 107 cells/ml (i.e., 106 cells per 50 µl).
Dilute primary mAbs (e.g., unconjugated, biotinylated, or fluorochrome-conjugated mAbs) to predetermined optimal concentrations (see Below) in wash buffer (PBS/0.1% NaN3/1.0% fetal bovine serum) and transfer a volume of 50 µl to a flow tube.
Transfer 106 cells in 50 µl to each tube already containing 50 µl of mAb (or 50 µl wash buffer for negative controls). Mix by gently vortexing or tapping.
Incubate at 4°C for 20-40 min in the dark.
Wash 2X with 200 µl wash buffer. After each centrifugation, 350 x g for 5 min, remove the supernatant. Vortex gently or tap the tube to loosen pellet prior to adding next wash. Use 500 µl wash buffer to resuspend the cell pellets (final concentration ~106 cells in 0.5 ml).
Acquire sample data on flow cytometer as soon as possible after staining. (Please see Staining Tips below)
