Hi all,
Recently we are trying to do telomere qFISH in human iPSC to see if the telomere become shorter after drug treatment. We used colcemid to arrest hiPSC in metaphase so we can easily see the telomere signal, however, the results are not so good.
Here's the protocol we used:
1. treat colcemid for 16hrs (100 ng/ml in medium) (also tried nocodazole but the results are the same)
2. trypsinize the hiPSC
3. treat hypotonic buffer for 15 min in 37 degree (75 mM KCL buffer)
4. fixation (MetOH: acetic acid = 3:1)
5. drop the cells from high to make cells burst
This protocol worked fine in other cell types but not hiPSC, we found that hiPSC always stuck together and make them difficult to burst, so I want to ask if anyone has a good protocol that worked in human iPSC?
Thank you very much for your reply.
Best,
Eric
I've done FISH with mESCs but not with hiPSCs.
One trick I was taught was to add a few drops of the fixative to the hypotonic buffer during that incubation, I am not really sure why but this seems to help stop cells from clumping up during this phase. You also might want to to try inverting the tubes every few minutes during the 15 minute KCl incubation if you are not doing that already.
Do you think they are still clumped up after trypsinization or are they clumping up afterwards?
Hi Stephan, thank for your suggestion, we add 3 drops of fixative "after" hypotonic buffer incubation, maybe we can try to add fixative "during" the incubation. I think they clump up afterwards during trypsinization, and we think maybe add EDTA to prevent they clumping up. I also did qFISH in miPSC and worked really well so I think it's because of the property of hiPSC?
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