I am trying to do IF to U2OS cell line in 96 wells plate. during the procedure I lose many cells which dissociate off the plate. Does anyone has a tip how to stay with many cells at the end of the process?
the protocol I use (briefly)
Wash CSK buffer
fixation with 3% paraformaldehyde
permeabilize with 0.1%Nacitrate 0.1% triton-x-100
thanks
Or
Hello there. Why don't you try cold acetone for the fixation? It works for me.
I agree with Nurul, try cold acetone. Gently pipette it on the side of well as to minimize disruption to cells. Also try coating the wells with poly-L-Lysine prior to plating your cells so that they can attach better.
I have had problems with cells coming off during staining as well. The two things that helped were coating with fibronectin and using Primaria plates from Falcon (they supposedly bind cells better than most dishes). As everyone said, also try to make every wash step as gentle as possible.
thanks guys maybe I will try acetone for fixation.Does anyone have experience with poly-l ornithine? I coat the plate with poly-l ornithine but still many cells disconnect.
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