Staining for BrdUrd with PI + FITC (4h for 10 samples)
2 X106 cells optimal. Can do with less. More than 4 X106 cells is too many.
1. Trypsinize, count and fix cells in cold 70% EtOH. Approximately 2X10 6/ml.
2. Centrifuge at 1100 rpm 8 minutes, room temperature.
Wash 1X with PBS or HBSS
Centrifuge as above.
3. Resuspend in 1 ml PBS containing 0.5 mg/ml ribonuclease A. Incubate 30
min at 37oC. Add 3 ml PBS to each sample. Centrifuge as above.
4. Resuspend (vortex pellet) in 1 ml 0.1N HCl containing 0.7% Triton X-100.
Incubate 10 min on ice. Add 3 ml PBS. Centrifuge as above. Decant liquid.
Turn tubes upside down to drain.
5. Resuspend in 1 ml sterile distilled H20. Puncture caps of tubes for venting.
Incubate 15 min in boiling water bath.
6. Immediately put in ice-water bath and chill 15 min.
7. Add 3 ml PBS containing 0.5% Tween 20. Centrifuge as above.
8. Remove supernatant. Vortex. Resuspend in 100ul of HBT (PBS+).5%Tween
20 +5% serum or BSA). Transfer to 1.5 ml ufuge tube containing 100ul of
antiBrdU (Pharmingen) diluted 1:100 in HBT. (The dilution of primary and/or
secondary antibody may need to be adjusted to diminish background.)
Incubate 30 min at room temperature. Add 1 ml HBT. Centrifuge at 3200 rpm
for 2 min. If the pellet is loose and you must respin, do not use a higher speed.
Remove supernatant.
9. Add 100ul of 1:20 dilution of FITC-conjugated antibody (sigma F0257 FITC
conjugated antimouse IgG). Incubate 30 minutes at room temperature. Add 1 ml
HBT. Spin as in #8. Remove supernatant.
10. Resuspend in 0.5 ml propidium iodide solution (0.018mg/ml in PBS). Keep
in dark.