I've been using A549s for the past couple of years to transfect in my protein of interest for imaging. Recently I've started to experience issues with transfecting cells seeded on glass coverslips. The same plasmid prep, PEI stock, and seeding density results in good transfection efficiency and cell survival in wells where cells are simply seeded on the plastic, but when seeding on glass coverslips almost all the cells in the well round up and die after 24 hours.
I've not previously had this issue, and have tried a couple of different frozen stocks of A549s to ensure it's not a problem with the particular batch of cells themselves. I've used the same coverslips with HEK293T transfections and not seen this same toxicity, nor do I see A549 cell death if I'm not introducing transfection reagents. I'm loath to coat the slips with polylysine as this would interfere with some of my downstream applications. If anyone has experienced a similar issue or has any suggestions I'd be very grateful!
It may have been glass and the plastic materials surface differences playing a role here. I would think that PEI might stick to the glass, and wouldn’t washed of with media replacement. May be you can try coating the glass with collagen to see it could make difference for cell survival after the transfection.
Dear Evelyn!
It is also possible that the protein you are using for transfection inhibits A549 cell adhesion surface markers. Therefore, if possible (flow cytometry), evaluate adhesion molecules (integins, ICAMs, cadherins, etc.) in cells on plastic and on coverslips. You may need to use a different transfection protein that does not affect adhesion molecules
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