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problems with 293T cells after medium removal!!! - (Dec/09/2010 )

hi!!!!
I have a very big problem with my 293T cells in culture!!!!!
I use them for simple transfection by using CaCl reagent, so i need to change the medium 2h before the transfection, but after the "very gently" change with fresh medium, 293t strart to detach and to become rounded.
this is a problem appeared in the last 2mounths!!
I changed the fbs, the flask, I tested them for mycoplasm, I used a low passage batch of cells...what else??
please help me!!
thanks in advance!!

-calimero-

calimero on Thu Dec 9 17:52:04 2010 said:


hi!!!!
I have a very big problem with my 293T cells in culture!!!!!
I use them for simple transfection by using CaCl reagent, so i need to change the medium 2h before the transfection, but after the "very gently" change with fresh medium, 293t strart to detach and to become rounded.
this is a problem appeared in the last 2mounths!!
I changed the fbs, the flask, I tested them for mycoplasm, I used a low passage batch of cells...what else??
please help me!!
thanks in advance!!



I have this problem with my 293 cells too. So I modify the protocol and seed less number of cells than recommended in the protocol. Some times I seed 2 or 3 6 well plates with different seeding density and pick the best one for my transfection. This is optimizing the protocol right?

-SciCell-

THANK YOU FOR your answer...but I think that, in my case, number of cells is not my problem.I tried different concentration of cells with the same result!!
sigh!!!

-calimero-

hi!!!!
I have a very big problem with my 293T cells in culture!!!!!
I use them for simple transfection by using CaCl reagent, so i need to change the medium 2h before the transfection, but after the "very gently" change with fresh medium, 293t strart to detach and to become rounded.
this is a problem appeared in the last 2mounths!!
I changed the fbs, the flask, I tested them for mycoplasm, I used a low passage batch of cells...what else??
please help me!!
thanks in advance!!

 

Hi

 

I am having the same problem as you. I have to make some transfections but my reagent is limited. I don't get to get it wrong. And I have to change medium before transfection and the same thing happens every time. Cells don't seem contaminated, they are growing at good rate, but every time I change medium some detach, and after half an hour the rest become rounded and the "boundaries" between several cells becomes blurry. I thought it was because of PBS, but just changing medium, very carefully, without PBS wahing, produced the same results.

 

I am afraid that if I use this cells the experiment will be ruined, and I could pretty-please ask for more cells, but I have no more myself, no one else in this lab works with them. This is the only mention I have ever seen/heard of the problem. Could you tell me if you solved it and how?

 

Thank you very much.

 

I hope you can aswer. :)

-Sofia Ruiz-

Have you (all) pre warmed your medium before you do the changes?  Is the medium buffered appropriately (i.e. the phenol red hasn't gone purple/pink)?

-bob1-

I found when transfecting dozens of plates of these I could only take a stack or two out of the incubator at a time. As they are only 'semi-adherant' you must be very careful with temp changes. Replace with 37deg media and you should be fine. Also gently apply fresh medium to the sides of the wells, not directly!

 

Mind you, I just plated them ~16hr before and removed media/replaced when transfecting. I don't think an additional change is necessary. Even comparing transfection efficiencies between 50-90% confluency I had little detachment.

-Osibisa-

HEK cells are well known to have this problem…..we've had success utilizing coated dishes, either poly-d lysine of collagen.  good  luck!

-Tom M-