Hi, I am performing UV irradiation studies on human fibroblasts. I want to perform IF after UV exposure and a short recovery time, which would follow pre - extraction at 4 degrees and PFA 4% fixation, with the corresponding washing steps in between.
Unfortunately I found that after UV exposure of 100J/m2, cells are very sensitive to detachment and basically observed that i lost almost all my cells after the pre extraction step (not checked in between, and the non UV irradiated cells were ok).
I would say that the pre extraction buffer (* 25mM HEPES (pH 7.5) 5 mL, 50mM NaCl 10 mL, 1mM EDTA 400 uL, 3 mM MgCl2 600 uL, 300mM sucrose, 0.5 % (v/v) triton X-100) is doing at least most of the magic although I cannot discard that they are already detaching with simple PBS.
My questions are, did anyone perform a similar experiment? any recommendation?
I thought on trying to coat coverslips before seeding them. Also, this UV dose is for sure ok for Hela but, are fibroblasts more sensitive maybe? any less harsh standard dose?
Thanks!
Hello,
Cell surface receptors are sensitive to UVI, depending on the UV-dose. If you irradiate them in colorless media you should diminish the dose of UV. In my opinion. is't better to do various experiments with different doses. So you will find more sutable (or the best) dose for your experiment. Also you may try to use plastic for plasement of fibroblasts. Finally, you should remember that intracelluar events depend on the absorbed dose of UVI and UV wavelength..
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