Harvest cells from tissue, preparing a single cell suspension. Red blood cells may be removed by lysis or density gradient: Red blood cells from murine peripheral blood or a spleen cell suspension can be lysed using BD Biosciences Pharmingen's PharM Lyse™ (Cat. No. 35221E) solution. Add 2.0 ml of 1X Lysing Solution to the spleen cell suspension or per 200 µl of murine peripheral blood. Gently vortex immediately after adding the lysing solution. Incubate at room temperature, protected from light, for 15 min. Centrifuge 200 x g for 5 min. Carefully aspirate and dispose of supernatant, without disturbing the pellet. Resuspend pellet in 1X cold wash buffer (PBS/0.1% NaN3/1.0% fetal bovine serum). Centrifuge at 350 x g for 5 min. Finally, resuspend cell pellet to a 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 Staining Tips) in wash buffer and deliver to the wells of a U-bottom microtiter plate in a volume of 50 µl.
Deliver 106 cells in 50 µl to each well 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 (or 3X if a biotin-conjugated primary antibody is used). After each centrifugation, 350 x g for 5 min, aspirate wells or flick plate to remove supernatant. Vortex gently or tap plate to loosen pellet prior to adding next wash or diluted secondary reagent.
If a second-step reagent is needed, resuspend cell pellet in 100 µl of appropriately diluted secondary reagent (e.g., fluorochrome-conjugated avidin, streptavidin, anti-Ig allotype, anti-Ig isotype, polyclonal anti-Ig). For example, dilute antibody to ~1 µg per 100 µl in wash buffer and add this to each well containing the loosened cell pellet.
Incubate at 4°C for 20-40 min in the dark.
Wash 2X with 200 µl wash buffer, as in Step 5. Use 100 µl wash buffer to transfer cell pellets to 0.4 ml aliquots of wash buffer (final concentration ~106 cells in 0.5 ml) in tubes appropriate for flow cytometer. Acquire sample data on flow cytometer as soon as possible after staining. (Please see Staining Tip 5.)
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 instrument set-up, please see description in "Procedure for Setting Compensation for Multi-Color Flow Cytometric Analysis".
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™. Please see description in "The Uses of BD Fc Block™ in Immunophenotyping of Mouse and Rat Leukocytes".
For very low-density cell surface markers (e.g.,cytokine receptors), a three-step protocol may amplify the staining: use purified primary antibody (steps 2-4 of above procedure), biotinylated anti-Ig for the 2nd step (steps 6-7, above), and fluorochrome-conjugated avidin or streptavidin as the 3rd step (repeat steps 6-7). We find that SAv-PE and SAv-BD Cy-Chrome™ are "brighter" than FITC conjugates and may provide even better discrimination of low-density antigens, especially in the presence of BD Fc Block™, for mouse cells. (Please see Staining Tip 3.)
We have found that freshly-isolated leukocytes and cell lines may wait for analysis in wash buffer at 4°C, without fixation, for up to 18 hr post-staining, without loss of viability. Activated lymphocytes may lose viability rapidly, and data should be collected within 5 hr post-staining. To preserve cell integrity beyond these time limits, paraformaldehyde fixation may be necessary; however, it is possible that the quality of staining may be diminished by such fixation. We do not recommend fixation of stained cells, except when the possibility of exposure to biohazardous material exists.
Every experiment must include controls. Negative controls are samples` of the same cell population treated exactly as the test sample, but with the omission or modification of one of the staining steps. Examples of negative controls are unstained cells, cells exposed to the 2nd step reagent alone, or cells exposed to isotype controls which are the same isotype and format (e.g.,purified, biotin or fluorochrome) as the primary antibody and titrated in parallel. For multi-color staining, single-color stained controls should be included. To identify markers on an unknown or novel cell type, positive controls (i.e., cells which are known to express the antigen of interest) should be included in each experiment and should be handled exactly as the test samples.
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