Metaphase Block for Cell Synchronization

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Tony Rook
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Metaphase Block for Cell Synchronization

Please find the following protocol:

Metaphase Block for Cell Synchronization

Procedure:

1. Remove the medium from an exponentially-growing cell culture and rinse it with 10 ml of PBS. 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. You usually need to grow several plates of cells in order to collect enough mitotic cells for an experiment.

2. Replace the PBS with Complete Medium with Nocodazole and allow the culture to incubate at 37°C for 12 hr.

3. Shake off loosely attached, rounded mitotic cells by gently knocking the plate and pipetting medium over the cell layer a few times.

4. Collect the cell-containing medium and place it in a sterile 50 ml polypropylene tube and pellet the cells by centrifugation at 1000 X g for 10 min. Remove the supernatant (the cell pellet may be small) and resuspend the cells in 15 ml of PBS.

5. Repellet the cells at 1000 X g for 10 min and remove the PBS. Resuspend the cells in complete medium and proceed with experiment or return the cells to cell culture flask and incubate at 37°C.

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

Nocodazole Stock (10,000 X)
6 g/ml in DMSO (CAUTION Biohazard!)

Complete Medium with Nocodazole
100 Units/ml Penicillin
Dulbecco's Modified Eagle's Medium (DMEM)
100 μg/ml Streptomycin
10% (v/v) Calf Serum
600 ng/ml Nocodazole (diluted from Nocodazole stock)

Bioreagents and Chemicals:

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

Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p9006