Cell cycle arrest - adherent cells going into suspension - (Jun/28/2012 )
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!!
It depends a bit on which part of the cell cycle you are arresting them at - if you arrest them during DNA division they are probably more weakly attached than at other points in the cell cycle. Some adherent cell lines, such as 293 are poorly attached anyway, so treating them with drugs or conditions that are not conducive to growth can cause them to detach and still be alive.
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.
Do you have a vehicle control? It could be that the DMSO is affecting the cells rather than the nocodazole.
Cell biology is my area of expertise. First, and this may sound strange, define what you want to mean by "dead". Not as simple as it sounds. Do you want dead to mean can't proceed through the cell cycle? Do you want dead to mean de-polorized mitochrondiral membranes? Do you want dead to mean that the cytoplasmic membrane is not intact? In all these examples the cells will not form colonies but the last is the only case that trypan will show dead. These folks published a nice paper comparing different methods to measure death: Weyermann J, Lochmann D, Zimmer A (2005). A practical note on the use of cytotoxicity assays. Int J Pharm 288: 369-376. In a practical sense if your adhearant cells are floating then they are biologically dead. Adhearent cells floating 4 to 6 hours (or longer) after treatment will never reattach and divide. I am also not surprized that they look biochemically different than the ones still adhearent. If at all possible a colony formation type assay will give the most unequivacal results. Also, as noted by others, do not forget a vehical control sample. Good luck.