Please find the following protocol:

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. he 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. 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 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.


Solutions:

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

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

Low Serum Medium
20 mM HEPES
0.5% (v/v) Calf Sserum
100 Units/ml Penicillin
100 μg/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 μg/ml Streptomycin
10% (v/v) Calf Serum


Bioreagents and Chemicals:

DMSO
Potassium Phosphate, Monobasic
Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
Streptomycin
Penicillin
Hoechst 33342
Aphidicolin
Calf Serum
Dulbecco's Modified Eagle's Medium (DMEM)
Propidium Iodide


Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p9005

Hi,
I was wondering what percentage of cells hare synchronized ater the Hoechst addition?
Also if I were to use NIH3T3 and Hydroxyurea, what changes would you suggest be made?

gurunatc

trook said:

Please find the following protocol: 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. he 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. 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 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. Solutions: Complete Medium with Hoechst 33342 Dulbecco's Modified Eagle's Medium 0.5 to 1 μg/ml Hoechst 33342 (should be determined empirically for each cell line) 100 Units/ml Penicillin 100 μg/ml Streptomycin 10% (v/v) Calf Serum Complete Medium with Aphidicolin Dulbecco's Modified Eagle's Medium 100 Units/ml Penicillin 4 μg/ml Aphidicolin 100 μg/ml Streptomycin 10% (v/v) Calf Serum Low Serum Medium 20 mM HEPES 0.5% (v/v) Calf Sserum 100 Units/ml Penicillin 100 μg/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 μg/ml Streptomycin 10% (v/v) Calf Serum Bioreagents and Chemicals: DMSO Potassium Phosphate, Monobasic Sodium Phosphate, Dibasic Potassium Chloride Sodium Chloride Streptomycin Penicillin Hoechst 33342 Aphidicolin Calf Serum Dulbecco's Modified Eagle's Medium (DMEM) Propidium Iodide Reference Link: http://www.bio.com/protocolstools/protocol.jhtml?id=p9005

A very stupid question.. Addition of the Hoechst 33342 in culture alters the radioresistance of the cells considerably, so won't this protocol change the nature of the cells one is working with ?
But I suppose that's only in the case of cancer-therapy studies that one cannot follow this protocol.