Hello everyone!
I knocked a gene into a cancer cell line by lentiviral transfection. The expression level of this gene in the overexpressing cells is almost 300 times than that of wild-type cells through qPCR and WB. However, I found that the overexpressed cells seemed to be circular and could not retain their original shape after using 3.7% formaldehyde or 2.5% glutaraldehyde for 10 minutes. But the wild type cells look normal and do not change their shape after fixation. Has anyone encountered the same situation? or Does anyone know the cause of this phenomenon?
Pictures are in the attachment.
Thank you all...
do you observe cell rounding on live cells? or is it just after fixation?
if cells are already rounded and probably loosely attached to the petri dish, maybe they are somehow blocked in M phase...this can be seen on DIC image or by staining with dapi...alternatively, they have disrupted /Tub /actin cytosqueleton...check this by IF anti tub or phalloidin staining..
Dear Dr Fesquet,
No, i have not seen this, sorry to report.
Very interesting though, thank you for sharing your question.
If you get a chance, please answer, is fixation solution room temp, or 4C? I have used cold (-20C) MeOH to fix monolayer cells for staining and counting, it works very well.
What is the cancer cell line (Hek, HeLa, 3T3)?
Some genes, when overexpressed to this much more than wild-type can be toxic. Like GFP...
Best Wishes,
Charles A
Didier Fesquet
Thank you very much for your answer. Cells were not rounded before fixation. and I also considered IF staining before. But cells are easily washed away in the process of IF staining because the cells are detached after fixation.
Charles D Anderson
Thank you for your answer.
The fixation solution is in the room temp. the cancer cell line is a breast cancer cell.
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