G1/S Phase Block for Cell Synchronization

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Tony Rook
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G1/S Phase Block for Cell Synchronization

Please find the following protocol:

G1/S Phase Block 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. The amount of aphidicolin (and the time 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. This protocol was developed to work with Swiss 3T3 cells.

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

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

4. Remove the Aphidicolin-Containing Medium and rinse the cell culture with 10 ml PBS. Replace PBS with Complete Medium (without Aphidicolin). The cells should now be synchronized in the cell cycle. This can be easily checked by Propidium Iodide staining followed by flow cytometry.

Solutions:

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 μg/ml Streptomycin
10% (v/v) Calf Serum

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

Bioreagents and Chemicals

Potassium Phosphate, Monobasic
Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
Dulbecco's Modified Eagle's Medium (DMEM)
DMSO
Propidium Iodide
Streptomycin
Aphidicolin
Calf Serum
Penicillin

Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p979