Flow Cytometry

Cell preparation/labeling:

1. Transfer 0.5-2 x 106 cells into a microtube. Centrifuge 5 min. at 300 x g. Discard supernatant.
2. Carefully resuspend the cell pellet with 20-100 μl ice-cold labeling buffer (PBS + 2% BSA + 0.05% NaN3).
3. Add primary antibody at the appropriate dilution in labeling buffer. Incubate on ice 30-60 min.
4. Wash the unbound antibody by filling the microtube with labeling buffer, centrifuge 5 min. at 300 x g and discard supernatant. Repeat twice.
5. Add fluorescently-labeled secondary antibody at the appropriate dilution in ice-cold labeling buffer. Incubate on ice protected from light 30-60 min.
6. Wash the unbound antibody by filling the microtube with labeling buffer, centrifuge 5 min. at 300 x g and discard supernatant. Repeat twice.
7. Resuspend the cells with ice-cold labeling buffer. Keep on ice protected from light until analyzed with a flow cytometer.
Note: If working with a fluorescently-labeled primary antibody skip steps 5 and 6 and proceed directly to step 7.