This protocol is for cultured cells.
For each monoclonal antibody (MAb) marker use 1 X 105 - 1 X 107 cells per 100 µl volume:
- If adherent cells, trypsinize, scrape or treat with EDTA to get single cells suspension, otherwise skip to step 2.
- Wash cells with 2 ml COLD Phosphate Buffered Saline (PBS) or another buffer with 0.1%-5% protein (eg Bovine Serum Albumin, BSA) by centrifuging at 150-300 X g in the COLD (4°C).
- Wash once more (Optional).
- Aliquot 100 µl of 1-100 X 105 cells (use wash buffer above with 0.1% NaN3 added) in Falcon® #2052 or #2054 tubes on ice.
- Add 20 µl MAb (1-10 µg/ml final concentration, depending on Mab).
- Incubate 10 - 30 min on ice.
- Wash again as above with 2 ml cold azide buffer.
- Resuspend in 100 µl of Secondary Antibody (eg. Fluorescinated Goat anti-mouse IgG). Otherwise skip to step 7 if using directly conjugated MAb.
- Incubate 10 - 30 min on ice in DARK. Wash again with azide buffer.
- Resuspend in 100 to 500 µl COLD wash buffer and add equal volume of COLD buffered 2% para-formaldehyde (see recipe) to fix cells. OR leave in wash buffer and analyze LIVE (Add propidium iodide, 0.5 µg/ml final concentration). Ideal final concentration of cells should be 1 X 106/ml.