M. Fero — 6/01 and D.B. — 12/98
- Harvest 106 cells/sample with PBS/FBS into a 15 mL conical tube spin at 1300 RPM. Wash by resuspending cells in 1.5 mL of PBS/FBS by spinning at 3000 RPM in a microcentrifuge.
- Incubate with 100 µL (use Pharmacia antibodies at 1:500) of primary cell surface antibody in PBS/FBS for 30 min. (Keep in dark if it is labeled). Wash with PBS/FBS.
- If the primary antibody is unlabled incubate with FITC or PE-conjugated secondary antibody if necessary for 30'. Wash in PBS/FBS.
- Fix cells with 1 mL of 0.5% Paraformaldehyde in PBS for 5 min. r.t and mix several times. Wash with PBS/FBS.
- Fix cells in 1.5 mL of 50% EtOH. Incubate 30 min. Spin.
- Wash in 1 mL of 1 mL of Hypotonic solution, spin. Wash with PBS, spin.
- Incubate in 100 µL of primary (intracellular) antibody in PBS/FBS for >30 min. Wash with PBS/FBS.
- For an unlabled rabbit primary antibody add 0.5 mL biotin-conjugated anti-rabbit secondary antibody (1:10,000) for 30 min. Wash in PBS/FBS.
- For biotinylated primary antibodies incubate in 100 µL Steptavidin-TriColor (1:100 in PBS/FBS), 30 min. Wash with PBS/FBS.
- For DNA content add 500 µL of 0.5 µg/mL Höchst 33342 (LSR, FL-5), or 0.5 µg/mL DAPI (LSR, FL-5), or PI (propidium iodide, 10 µg/mL)
- PBS/FBS: 0.5% heat inactivated (56°C, 30') FBS in PBS.
- 0.5% Paraformaldehyde in PBS
- 50% EtOH
- Hypotonic Solution: 10 mM HEPES, 0.55% NaCl, 0.1% NaN3, 4% h.i.FBS.
- FITC (FL-1) or PE (FL-2) conjugated cell surface primary antibody
- Primary nuclear antibody
- Biotinylated secondary (Goat anti-rabbit, Caltag L42015)
- Streptavidin-TriColor (Caltag, SA1006) (FL-3)
- Höchst 33342 (or DAPI) 0.5 µg/mL for DNA staining.