Hi all
I'm looking for a range of reagents (eg Nocodazole) or methods (eg Mitotic shake off) to block/stall cell lines grown in culture in G2-M phases of the cell cycle.
Ideally I'd want to block the cells so that, once synchronised, I can perform an assay that lasts 6h then release the block (so that the cells continue cycling). I'd also (in a perfect world) want the drug/intervention to be as non-toxic to the cells as is practical.
Many thanks in anticipation.
Al
Hi Alasdair,
To my knowledge, very few options (nocodazole) are available to arrest cells in G2/M phase. But, thymidine block might be of your interest.
I have done this successfully to synchronize leukemic cell lines. The cells are blocked in G1/S transition but they will resume cell cycle synchronously after you wash away thymidine. The release duration required to reach different phase is quite reproducible.
Thymidine is not really 'drug' unlike aphidicolin, nocodazole, mimosine etc. However, optimization is required to determine whether a single or double block is required and at what dose is optimum to minimize toxicity due to prolonged incubation. I have done single and double block on different cell lines and the dose ranges from 0.75 to 2 mM.
Attached along is a cell line blocked with single treatment of thymidine for 21 hours (S0) and released afterwards. AS- asynchronous.
Hi Alasdair,
Just posted a very similar answer to this on another question thread on RG!
One alternative to chemical synchronisations (which obviously induce some pretty big changes in the cell phenotype/morphology) is to synchronise with a non-chemical method, such as elutriation centrifugation. We do this regularly in our lab, and it allows you to separate cells on the basis of size (including distinguishing G1, S and G2/M populations) without any chemical modifications.
If you're interested, check out, for example, Banfalvi et al 2011:
http://www.ncbi.nlm.nih.gov/pubmed/21755439
A big advantage of this is that you can divide activity replicating cells without arresting them; if you're looking to perform some incubation/treatment during the G2/M arrest, this method might not be appropriate - but just thought I'd mention it as an interesting alternative!
Let me know if you'd like more details/are interested in trying it,
Regards,
Dan
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