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Tranfection cell density and overgrowth


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#1 rawat

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Posted 04 December 2009 - 02:16 PM

I have been doing transfections for several months and am still unclear about overgrowth.

Lipofectamine 2000 kit suggests seeding cells to final confluency of 90-95% on day of tranfection. This is surprising to me. I will be tranfecting HEK 293 cells and other protocols online suggest a similar cell density before tranfection of this line. I am worried that if I transfect at 95% confluency, in 24-48 hours the cells will overgrow. I have been told that overgrowth leads to inconsistencies in data (and I don't understand why exactly). Would appreciate some advice.

Thanks

#2 bob1

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Posted 06 December 2009 - 03:04 PM

I transfect at whatever density I need to ensure that the cells will not be over confluent at the end of the experiment. I have also found that transfections have a greater efficiency at 70ish% confluency.

#3 giny

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Posted 08 December 2009 - 04:51 PM

Hi rawat,

I do transfection in HEK293 too. I normally seed the cells such that they reach 50% confluency on the day of transfection. After transfection, I will let them to grow for 48 hours. My boss said after seeding HEK293 for transfection, must let them to grow for more than 24 hours before transfection.

Hope this help.

#4 gabamaria

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Posted 21 April 2010 - 03:59 AM

hi,

Lipofectamine transfection needs a high confluency of the cells, because it is a big stress for the cells, so you will for sure see some cells dying after 24h, but the strongest cells will grow and divide.
just follow the instructions and see what happens.

good luck :D

Marie




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