1. Seed 10- or 15-cm plates with cells in media supplemented
with dialyzed serum (regular FBS fine) 24 h before treatment.
(Seed enough cells so that you end up with approximately 2.0x106
cells per plate at harvest time, but prevent contact inhibition.)
2.
Treat cells as desired. Minimize exposure to light from this
point on.
3. At the desired time point(s), pulse-label the cells with
10 micromolar BrdU for 30 minutes from a 100X stock solution
(1 mM or 0.307 mg per ml PBS). Alternatively, continuously-label
cells with 65 micromolar BrdU from a 100X stock solution (6.5
mM or 2 mg per ml PBS).
4. Aspirate medium and wash cells once with PBS-. Trypsinize
cells, minimizing the time in trypsin. Transfer cells to a
15-ml centrifuge tube and spin at ~1,000 rpm for 3 minutes.
Aspirate supernatant and re-suspend cells in 200 microliters
PBS-.
5. Add 5 ml fresh ice cold 70% EtOH dropwise while vortexing
the cells at setting #4. Allow cells to fix overnight. (Fixed
cells should not be stored more than a week.)
6. Start boiling water bath. Centrifuge cells and aspirate
supernatant. Resuspend cells in 1 ml cold 0.1M HCl/0.5% Triton
X-100. Incubate for 10 minutes on ice. Add 5 ml dH20. Centrifuge
cells and aspirate supernatant.
7. Resuspend cells in 2 ml dH2O. Place in boiling water bath
for 10 minutes, then quickly transfer to ice for 5 minutes
to cool.
8. Add 5 ml PBS-/0.5% Triton X-100. Centrifuge cells, aspirate
supernatant, and resuspend cells.
9. Make 5 microgram/ml
anti-BrdU-FITC solution (100 microliters per sample): (1:20
dilution of 100 microgram/ml stock diluted with 0.1% BSA in
PBS-)
10. Resuspend cells in 100 microliters antibody solution.
Incubate 30 mintues at room temperature in the dark, mixing
every 5 to 10 minutes.
11. Cut filter squares and syringes. Make PI stain (5 micrograms/ml
PI + 200 micrograms/ml RNase): (12.5 microliters 2 mg/ml PI
+ 100 microliters 10 mg/ml RNase + 4.9 ml PBS-)
12. Add 5 ml PBS- to cells. Centrifuge and aspirate supernatant.
13. Resuspend pellet in 250-500 microliters PI stain, depending
on number of cells (optimally, each sample will be at a final
concentration of 1x106 cells per ml).
14. Filter final solution through mesh filter on 1 cc syringes
into Falcon 2052 tubes. Store at 4C for at least 10 minutes,
but not more than a couple hours
.
15. FACScan.
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