Posted 28 June 2012 - 10:24 AM
I have a question regarding cell culture.
I am growing adherent cells and after treating them with chemicals f.i. to induce cell cycle arrest, I can see that some cells remain adherent and some go into suspension.
Trypan blue assay shows that the floating ones are not dead. So are they still alive but in the apoptotic pathway or can cell cycle arrest have an effect in their adherence maybe?? And generally if adherent cells go into suspension are they necessarily dead/ going to die??
Western shows different results in the adherent and floating cells so I am not sure what to trust.
Any suggestions or ideas would be very welcome!
Thanks a lot!!
Posted 28 June 2012 - 01:41 PM
Posted 01 July 2012 - 05:00 AM
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.
Posted 01 July 2012 - 01:39 PM
Posted 04 July 2012 - 06:26 AM