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transfection problem---SOS!


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5 replies to this topic

#1 littlecell

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Posted 04 March 2005 - 08:26 AM

i am a new one in transfection. according to many protocols for transfection, the cells should be passaged one day before to get a confluence of 70-90%. i am wroking on the baldder tumor cell line, but it grows very slow. the cells growed for more than 3 days and were almost 100% confluence before transfection. the regeant used for transfection was Lipofetamine 2000 and i used GFP plasmid to try my first transfection. but i didn't got any positive GFP cell. would anyone tell me why the cells should be passaged before transfection? if the cells were split, they would grow more and express more GFP because of high protein synthesis level, right? but my cells were almost 100% confluent, they would grows very slow. so GFP wasn't prone to be expressed by the inactive cells (even if they were tumor cells ). thanks your instruction very much!

Edited by littlecell, 04 March 2005 - 08:26 AM.


#2 neuromatt

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Posted 04 March 2005 - 08:51 AM

Not really an answer but... I routinely use lipofectamine 2000 to transfect a variety of cell lines and find that I get the best transfection effciency at high cell confluency(90%), unlike standard lipofectamine which works best at ~60% confluency. Regarding slow growth could you not seed at an initial density high enough to give you 90% in one day?

It's also worth noting that transfection effciency and conditions vary between cell lines for example a neuroblastoma cell line I use requires about 10 times as much DNA and more lipfectamine 2000 than HEK293s. It might be worth titrating the DNA and lipofectamine amounts to see what happens

Hope this helps

#3 postdoc2130

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Posted 06 March 2005 - 05:39 PM

Seed your cells at different density (to reach 50, 75, 90% the next day) the day before transfection and see the effect on GFP expression.

#4 bob1

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Posted 06 March 2005 - 08:03 PM

Hi

I think the answer to the initial question is that the cells should be passaged so as to get actively growing/dividing cells as these are more likely to take up the DNA and express it. If you have confluent or nearly confluent cultures then the cells will be starting to or more likely to senesce which means that none of your DNA will be taken up or expressed!

I too am fairly new in this area, but this is the explanation I have been given by others for splitting the cells before transfection.

Good Luck
Bob

#5 fred_33

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Posted 07 March 2005 - 12:55 AM

hi
post doc2130 and BOB1 are right.
For best resutls cells should be active (growing/dividing) and as you need to test transfection conditions regarding confluency, you should seed plates with different conditions and test them. But as neuromatt said, these will partially depend on the transfection agent you use.

Fred

Edited by fred_33, 07 March 2005 - 12:56 AM.


#6 littlecell

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Posted 08 March 2005 - 07:45 AM

thanks you three guys's information and advice very much! i will split my cells before transfection.




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