ioannap

Hi there,

I agree with all that is mentioned above regarding the speed, 5000rpm is probably too much, however one more thing you could check is that all this does not have anything to do with your 1.5ml centrifuge tubes.

Is anyone else in your lab having similar issues?
At some point we had this problem in our lab and were very confused with not getting the expected pellets.
We had recently changed the supplier we had for the tubes. We connected the dots finally and tried harvesting cells from 2 duplicate wells and pelleting them using tubes from our previous and new supplier.
There was a nicely formed pellet in the "good" tubes and almost nothing visible in the "bad" tubes. There were cells smeared on the wall of the tube, but that's not good enough really.

If in your case it has anything to do with the tubes, I would suggest -if you cannot change the tubes obviously!- to spin at lower rpm and for longer, like 6-8mins, which seemed to help a bit.

Good luck!

Thank you for your answer bob1, very much appreciated.

For 293Ts I agree - you say "Boo" and they detach..but I am growing a cell line that is very strongly adherent in normal conditions (having a hard time trypsinizing them each time).

For my current experiment I am using nocodazole, so they should be stopping at prometaphase, before they start to divide. However even though I use it in small concentrations, nocodazole is a microtubule-disrupting agent so maybe it affects structural elements/ adherence and that is why they are easily detached like this? _ although it does happen with other chemicals as well (like with minute H2O2 concentrations- for "light" DNA damage)

Maybe I could try adding complete medium after the treatment in a spare well, just to test if they get back to being attached (since cell cycle resumes) or if they are apoptotic and then they would prob not recover.