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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)
Staining Tips
Determine optimal concentrations (brightest staining/lowest background) of each primary and secondary antibody by titrating, in a preliminary experiment, between 1.0 µg and 0.1 µg antibody per 100 µl wash buffer for 106 cells.
When performing multi-color labeling, directly-conjugated mAbs can be added simultaneously, rather than sequentially.
For reducing FcgII/IIIR-mediated antibody binding (or binding of SAv-PE or SAv-Cy-Chrome) which could contribute to background, the use of anti-mouse CD32/CD16 (Mouse BD Fc Block™; Cat No. 553141/553142) or anti-rat CD32 (Rat BD Fc Block™; Cat. No. 550270/550271) is recommended. BD Fc Block™ can be added to cells (~0.25 µg per million cells, 3 - 5 min, 4°C) and need not be washed out prior to addition of primary mAb. It is important to verify that no secondary reagent will bind the BD Fc Block™.
