I have transfected HEK293 cells to express Protein-GFP conjugate, transfection rate was high (>90%), but the cell morphology changed, many of the cells appear round, but not floating, and they are fluorescent (GFP filter). The cells were transfected using CaCl2+BBS, two days ago.
So, is the morphology change normal?
Thank you.
Need further conditions about the cell confluency and how much plasmid transfected.
No more than 10ug for the 100-mm plate, and calibrate the respective plasmid according to the ratio of their diameter for different size of the 6/12/24 well plate you used.
Be careful about the endotoxin that might be existing in your plasmid aliquot.
I'd like to refresh the medium 6-8 h post Ca2+ transfection to relieve the toxicity.
Generally, 293T become round when they are feeling 'unhappy', try to make them happy then.
I used 10ug for 50 mm plate, about 40% confluency. The cells are regular HEK293, not 293T, thanks!
If the your protein is toxic to cells at a certain expression level and your expression level is high enough to reach it in a sizeable percent of cells after the transient transfection, then you can have plenty of round HEK cells due to this toxicity at high expression levels.
The other cells - with a lower expression - will have a proper shape. Eventually the unhealthy round cells will die.
So, this change in morphology is a normal response to toxicity but you probably wouldn't want to use this cell population to study other biological questions.
This can be encountered even with "plain" cytosolic fluorescent proteins under a CMV promoter if they are somewhat toxic on their own at high protein levels or if they have tags/truncations that make them more toxic. Further, unlike cytosolic constructs, for ER-targeted (membrane-tethered or secreted) constructs such toxicity is very common even at low expression levels (probably, it's easy to overload this compartment), so for many purposes it may be necessary to select stable transformants without toxicity.
An illustrative example below: a non-toxic, a somewhat toxic and a quite toxic cytosolic versions of mVenus2 (derivative of Aequorea victoria GFP) under a CMV promoter, 2 days after the transfection into HEK293T (=tsA201) cells. 100 ng pDNA (XtremeGene 9 reagent) per 500 ul media in wells of a 24-well plate. Also, one day after the transfection (not shown) the difference is clear but less pronounced between the first two of them, it builds up over time as protein levels rise in individual transfected cells
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