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My adherent cells are detaching


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#1 PurpleKnight

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Posted 17 November 2009 - 04:42 PM

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 ÁM 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!

#2 miBunny

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Posted 17 November 2009 - 05:37 PM

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.

#3 PurpleKnight

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Posted 17 November 2009 - 07:18 PM

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.


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!




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