We have used TransIT-X2 (Mirus) for years to transfect different cell lines, but suddenly I am having a lot of trouble with my HEK 293 MSR cells becoming very round and dying/detaching soon after transfection
I plate the cells into a 6-well plate, transfect the cells when they are 40-50% confluent - 100ul OptiMEM, 2ul (1ug) DNA, 3ul TransIT-X2, incubate 20-30 mins at RT and mix dropwise into media in well.
The cells start to look rounded in the 6-well plate after 18-24 hours. After 24 hours, I trypsinise the cells and plate them onto sterile coverslips with fresh media for experiments, but when I come back to them 12 hours later, they're very unhappy/ dead with most of them floating, round and in clumps in the media. We have always used this protocol and have used HEK 293 MSR cells in the past a lot and this has never happened before.
I have changed:
the media (and the FBS in the media)
the PBS
the TransIT-X2
the OptiMEM
the cells
made fresh DNA with high quality Qiagen miniprep kits
all to no avail...
It happens no matter the DNA used, including empty vector, and different vectors!
Any ideas? We are stumped.
Thanks in advance.
Hi Catherine
Something that occurs to me is that you could try to change the trypsin by a Cell Dissociation Buffer, enzyme-free, it could be less aggressive for HEK, I have worked with that solution and it works very well,
regards.
Thank you, Victor. I have tried Tryp-LE as well as trypsin. It still occurs. It is primarily transfected cells that round up, no matter how little transfection reagent (or which) and dna I use. Over the weekend and yesterday, we looked at the cells under x40 magnification and they actually didn’t look very healthy - perhaps like mycoplasma or some sort of contamination, despite treating with mycoplasma antibiotics - perhaps a resistant contamination. We have tried to source cells from a previous batch, so hopefully these will be healthier and solve this issue, as we seem to have tried everything else (new cells were previously from the same 2 batches)
I had this issue before, so I tried type I collagen coating and that fixed all of my issues.
Good luck!!!!
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