Monday, October 27, 2003

Description
G2 Phase Block with Hoechst 33342 for Cell Synchronization

Procedure
1. Presynchronize cells at G0 by serum starvation. Add Low Serum Medium to cells at 70% confluency and allow cells to grow in this medium for 2 days. (See Hint #1)

2. Stimulate the cells to reenter the cell cycle by replacing the Low Serum Medium with Complete Medium containing 4 g/ml Aphidicolin and allow the cells to grow in this medium for 14 hr.

3. Alternatively, add Aphidicolin to exponentially growing cells to 4 g/ml for 24 hr.

4. Remove the Aphidicolin-Containing Medium and rinse the cell culture with 10 ml PBS. Replace PBS with Complete Medium with Hoechst 33342. Allow the cells to grow for 12 hr in this medium. The actual time may vary depending on the cell line used and should be determined empirically.

5. Remove the Hoechst-containing medium and rinse the cells with PBS. Replace the PBS with Complete Medium. The cells should now be synchronized in the cell cycle. This can be easily checked by Propidium Iodide staining followed by flow cytometry

Recipes
Complete Medium with Hoechst 33342 Dulbecco's Modified Eagle's Medium
0.5 to 1 g/ml Hoechst 33342 (should be determined empirically for each cell line)
100 Units/ml Penicillin
100 g/ml Streptomycin
10% (v/v) Calf Serum


Complete Medium with Aphidicolin Dulbecco's Modified Eagle's Medium
100 Units/ml Penicillin
4 g/ml Aphidicolin
100 g/ml Streptomycin
10% (v/v) Calf Serum


Low Serum Medium 20 mM HEPES
0.5% (v/v) Calf Sserum
100 Units/ml Penicillin
100 g/ml Streptomycin
DMEM


PBS pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl


Complete Medium 100 Units/ml Penicillin
Dulbecco's Modified Eagle's Medium (DMEM)
100 g/ml Streptomycin
10% (v/v) Calf Serum



Supplies


Tips
1. The amounts of Aphidicolin and Hoechst (and the times of exposure) may vary depending on the cell line you are working with. Also, different cell lines use different media, thus "Complete Medium" may be different for your cell line than what is described here.