My adherent cells are detaching - (Nov/17/2009 )

Hi,
For an iodide uptake assay, I need to incubate adherent cells for 1 hour at 37C in HBSS with 10 mM Hepes and 10 �M NaI.
At the end of the incubation period, all the cells are round and detach leaving me with very little cells after washing if any at all. I don't know (yet) if the cells that detach are dead or not.
I've tried adding 5% BSA to the buffer but it doesn't help. My HBSS is calcium and magnesium free but I doubt this can have such a dramatic effect (and this is currently being tested!)?
Any idea of what to try next? I would rather not coat the plate with polylysine if at all possible !
Thanks in advance for your input!

Cheers!

Is there any reason you can't throw in some calcium? My guess is that the cells are detaching due to a lack of calcium. An alternative protocol for removing adherant cells is too just use EDTA to remove the divalent cations.

miBunny on Nov 17 2009, 06:37 PM said:

No there is no reason at all! I usually use HBSS with no calcium/magnesium (cells tend to clump with the regular HBSS (the one with Ca, Mg). I just got some HBSS with Ca and Mg today so I'll run another test tomorrow! My cells usually don't come off that easily with trypsin so I was not expecting them to detach with just HBSS ...!
Thanks for your input!
Cheers!