I just started my staining/ imaging studies on HT22 cell line,
For that I was fixing the cells after the drug treatment.
As soon as I added 4%PFA soln, to the 8 well chamber slide, the cells got detached and started floating.
I could not find any logical cause,
if anyone can suggest to me a solution, that will be really helpful.
Thanks
Hi Tushar, PFA crosslinks primary amines. My thoughts are that either the cells are not firmly attached in the first place, or you are not washing away the serum containing media with PBS before adding the PFA..
Hi, I guess you might want to repeat the experiment to see if this anomaly persists. As far as I know, PFA should not affect the cell attachment. However, you can alter the concentration by reducing it by 1%. The drug might also react with the PFA. Please look out for a few articles that have shown reaction between PFA and the drug you are using.
one simple possibility is that when you added the pfa, the sheer force of addition of a liquid detached the cells from the surface. Once the cells have been killed by the pfa, they are more likely and more easily detached
I don't know why your cells are floating. But I would like to suggest try Sigma's PFA instead of Merck's PFA.
Dear Tushar,
I am facing this exact same problem at present. have you had any luck?
In my case I wash before fixation with warm (37ºC) PBS and the cells look fine after washing. So it is only after I add 4% PFA that the cells start detaching.
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