Hi,
I'm currently seeding 293T in 6 well plate (5*10E5 / well) for transfection.
However, the condition of cells seems unable to adhere and growth well, this happened 24hrs after seeding, not even transfected. And cell died after 3 days.
On the otherhand, cells that I maintained (10E6 / 10cm plate) seems normal, from growth rate to morphology.
I have tried the same protocol 4 times, the first 2 time it works fine (cell growth properly and transfecton is successful), but the recent 2 time all failed, I also change to fresh medium, it doesn't work.
The only possible reason I could imagin is I accedentily overcrowded the cell I maintained once, would that be a possibility to my failure?
HI Zheng
As you are culturing HEK293T you must have observed that these cells are delicate and need very careful handling. The confluency of more than 70 % may lead to detachment of the cells from the surface. A temporary fix could be that you may pre-coat the cell surface with Poly-L-lysine. And always watch for cell confluency with normal or normal-like cells. Hope it helps
Hello Yuhung Zheng
This is very common. HEK293T cells are very delicate cells and they easily detach from the culture plates. I have experienced the same. Firstly, make sure that while sub-culturing/passaging, you do not leave your cells out for longer periods and give them a thorough washing. This exerts a shock/stress over the cells. Secondly, don't seed too many cells as HEK293T have the tendency to behave in this manner at higher confluency. Do not cross more than 60-70% confluency with 293T. In case you visualize the cells under a microscope or take the culture dishes out of the incubator, remember to keep them back inside without much delay since regular temperature shift and conditions results in some cellular shock. HEK293T cells grow best without antibiotics. Moreover, if they do tend to be loose, you can make use of lower serum concentrations (2-2.5%) in your media. Apart from this, try using wells coated with poly-l-lysine, it helps in cellular adhesion by enhancing the interactions between the ECM of the cell and wells.
Good luck!
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