Discussion of all aspects of cellular structure, physiology and communication.
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I'm culturing the YPEN-1, an endothelial cell line, and I've some problems to detach the cells. After trypsin passage I don't have a good cells separation, the cells detach as clumps and it is impossible to obtain a single cell suspension. So it is impossible to count the cells and to obtain a good monolayer after each passage...Sombody can help me?
There are several things you could try:
1. Hit the flasks. I'm serious. If you've never tried it, it is definitely worth to try. Nothing bad can happen, so hit away. In my experience it is best to hit it from the side, so cells won't fly into the tip of the flask.
2. Leave them with the trypsin a little longer in the incubator. Not too long though, you might end killing them.
3. How exactly do you count them? When i used to count cells i first inactivated the trypsin with growth medium, then take the whole thing and put in the centrifuge, then throw away the medium+trypsin and resuspend in a buffer such as PBS. Then you can try to further break down the clumps by rapidly pipetting in and out with an automatic pipette. I used to do this with a 100-1000 pipette, but then again i never had any problem. You could use a 20-200 pipette set on 200 because the tip is much narrower. If all else fails, that might solve your problem.
4. If the clumps are small, leave them as is. The counting chamber has a huge error anyway.
5. I just thought of this, no idea if it would work: what about putting the cells in a Corning tube and vortexing them? There might be a speed big enough to break them apart without killing them.
Anyway, let me know what you end doing to solve the problem.
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter
Thank you for your suggestions,
I’ve tried to practice them, but I didn't observe big changes…the cells are still like clumps. The only modification that worked a little more has been the addition of EDTA at the trypsin solution; I leaved the monolayer with trypsin until the cells was almost detached; at this time I removed the trypsin solution and, after adding the complete medium, I’ve started to detach the cells by pipetting up and down. Only in this case I’ve obtained a better cells suspension, even if there was still a lot of clumps that prevent the formation of a good new monolayer. I think this would be a serious problem when I’ll start with the transfection reactions.
Count is not a so big problem. The problem is that the number I obtain is different from the real number in the suspension that is altered by the clumps
thank you for your help
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