Does anybody have any experience with sticky mitotic HeLa cells in the plates? I am growing many 15cm plates for collecting as much mitotic cells I can have for pull-down assay. But it seems that it's really hard to collect mitotic cells by pipetting, so I need like 5 min pipetting to get them detached from the plate. It's horrible task when I am doing this for 10 plates or even more!!!
For the drugs I am using, I am doing double thymidine block and release in Noco or Monastrol. I always see the problem with both conditions!
Does anybody know a possible reason for that? I appreciate any suggestion!
It can help to sort of beat the plates against something to dislodge the mitotic cells. I have been told by someone good at this that selecting for cells that don't adhere well when rounded up in mitosis can help. This involves using less heroic measures to shake the mitotic cells loose (and probably no drugs), growing those cells up and selecting again through several cycles to select for ones that are less adherent.
Another concern would be that the longer you have cultured cells on a plate, the more extracellular matrix the cells lay down. This matrix holds the cells on the plate better. Thus, doing the mitotic shake-off on plates that have not been growing for too many days might work better.
Thank you Prof. Deininger for your swift reply. I guess growing cell for long time might be my problem. Because I grow them for 4 days (because the double thymidine block). I can manage to make it only 3 days.
Actually, I didn't get exactly what you mean in the first part of your answer. But, I should use some drugs for the main experiment (to compare two conditions in Mass Spec.) and also THY block should be important to get nice and dense a wave of mitotic cells.
Of course I am doing mitotic shake-off, by pipetting up and down, to get what's supposed to be loose round mitotic cells. Mitotic shake-off is has never being a problem with me in 6-well or 10 cm plates.
I will try to grow cells in less days and update you. Thank you once again!
HeLa S3 cells are a bit better than some of the other HeLa strains for this. Also as Prescott says, you can just bang your dishes on the bench top, the vibrations make the mitotic cells detach.
Do you have any idea, how long hela cells take to grow at different mitotic phases?
Hi Mark
I think it's kind of challenging if I start everything again with different cell line. I just need to optimize this issue with my current cell line. Thanks for your add. Yes, I am hitting the plate against solid stuff to get them loose, but as I mentioned, it's really so hard!
I am trying now to grow them in a bit shorter time.
@Fathima, I think HeLas can go through complete cell cycle during 24 hours, mitosis takes place in the last hour.
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