Hi guys,
I am trying to culture mouse embryonic stem cells ( JM8A3; C57BL/6N ) on coverslips coated with 0.1% gelatin for confocal microscopy. The cells grow well but once I start washing the media with PBS, the cells detach from the coverslip. Anyone can suggest a way to fix that?
what is the best way to grow mESC for confocal studies?
Thank you in advance
If you're washing before you fix them just skip that step - remove the media and go straight to fixing. If it's for some other purpose and can't be avoided try adding and removing the PBS really gently.
I'm working with iPS cells and have this problem all the time. I found a good solution. Give up on trying to stain the cells on glass at all - you will just waste time and your nerves. Wash them off with PBS into a 1.5mL eppendorf tube and do the staining inside the tube. It works just fine. After fixing the cells you can centrifuge the tube in table-top rotator (very small one), so the procedure becomes MUCH easier. After you finish the staining, just add 100 microliters of PBS+DAPI into the tube, resuspend the cells and put on fresh glass as a drop. Then go and image.
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