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HeLa culture synchronization


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#1 modon001

modon001

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Posted 08 March 2010 - 01:39 PM

Ok, hopefully this is the right place for this question. I have a protocol to synchronize HeLa cell cultures at the G1/S boundary, and then a release/capture at metaphase. It's basically a double-thymidine block, followed by release and capture/treatment with nocodazole.

I'm hoping to find someone who's done this, or something similar, because I have some questions.

Ultimately, I want dishes of cells where a large majority of cells are in Mphase - want to lyse them and do coIP.

But I feel like I need to show my method is working. I've found some papers that show synchronization with FACS, but I don't have ready access to reagents and a FACS machine. So I was wondering...

1) Can I do this with cells cultured on coverslips, and show by immunocytochemistry that more of the cells are in Mphase?
If so, are there antibodies or stains that work better than others?
How can I tell the difference between Mphase cells and dead cells? (I tried with DAPI to mark DNA, and Phalloidin, and I had some trouble)

2) Will the Mphase cells come up off the plate while I'm washing with PBS during cell harvest/lysis?

3) Any other tips?

Thanks for your patience, and any help you can give. This is a new thing for me.




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