I lose cells after4% PFA fixation at room temp for 15 mins, cultured in serum free media. I tried PLL coating but it doesn't seem to help much. I tried titrating PFA cocentration and time but nothing seems to work so far.
Hi are you saying you are fixating your cells using 4% PFA WITH serum free media? May I know why are you adding media together during fixation? The point of fixation is to fix the cells, the media supposedly plays no roles here. Also if you are doing that it might just cause the PFA to be diluted and just be toxic to your cells, which may cause it to detach.
I am culturing cells in serum free media before fixation. I do fixation in 4 percent PFA in PBS.
I see. Sorry for the misunderstanding. Maybe you can check for the pH of the PFA if it’s from an older batch. Or perhaps you can try a different fixation methods. Maybe methanol or ethanol? I heard that you can get better architecture using these, but not sure if it’ll solve the issue you’re having.