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Stably transfected cells start dying


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#1 biomart

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Posted 05 February 2015 - 12:01 AM

Hi!

I transfected J82 bladder cancer cells with pmep4 vector, and thereafter performed selection with 300 ug/ml G418 (the optimal concentration had been titrated beforehand). During the first week, I saw several isolated colonies that were growing well, but starting a bit after two weeks, most colonies, that had earlier been growing during selection, started dying. As of now, I only have two colonies left, and these are neither growing nor dying. The only remarkable thing about these cells is that they seem pretty big. As concerns bacteria, the cells are mycoplasma checked, and there are no signs of any other contamination. Three days ago, I changed medium to G418-free to see whether that would make the cells grow, but nothing has happened. Any suggestions as to what could have gone wrong, what I can do to save the cells and any additional things I should do during a new transfection?



#2 bob1

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Posted 05 February 2015 - 01:06 PM

It sounds like the cells are senescent (irreversable), which means they will not divide any further, but should be able to survive passaging, though this won't do anything to get them to grow.  You need to start again unfortunately.



#3 kelly1

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Posted 06 February 2015 - 07:34 PM

Isn't there any thing that I can do to save the cell? 



#4 bob1

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Posted 07 February 2015 - 12:26 AM

If they are senescent, then no, there is no way to reverse this process.  If they are not senescent, you can try adding more FBS, lowering the selection antibiotic amount, handling gently, feeding cells with conditioned medium... etc.



#5 CandyTon

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Posted 09 February 2015 - 07:42 PM

Hi biomart! Have you solved the problem yet? cause I, too, have similar problem in my lab. If you get a solution, could you share that with us? Thanks a lot. smile.png



#6 2444256703

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Posted 10 February 2015 - 10:39 PM

Maybe you can just buy these cells you need, many companies do provide quite good transfected stable cells with a reasonable price.



#7 Badette

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Posted 18 February 2015 - 07:42 AM

I had the same problem and I trypsinized my colonies and put them in a new well, with 20% FCS in the culture medium... It worked and 2 weeks later I went back to a 10% FCS culture medium.






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