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293T cell contamination post-transfection


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#1 labstud4

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Posted 31 August 2012 - 09:52 AM

I am producing lentiviruses using HEK 293T cells, and 24h post-transfection (Lipo2000 and OPTI-MEM) with my gene of interest, VSVg, and CMV helper plasmids, my cells appear to be contaminated. Under a microscope, I can see tiny black dots in the space surrounding the cells that do not float and are seemingly attached to the plate. The media remains clear. At 48h post-transfection, the black dots seem to have grown in number, yet the media still remains clear. After harvesting the viral supernatant, I will filter using 0.2um and then aliquot for infection. Does anybody know where this contamination comes from? Is this contamination--could these dots be granules secreted by the cells due to the stress caused by transfection?

Edited by labstud4, 31 August 2012 - 09:54 AM.


#2 Curtis

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Posted 01 September 2012 - 03:40 PM

there are different types of bacterial contamination, the most common is cocci contamination, or at least they are common in South East Asia, I'm not sure. they usually group together and you must be able to see slight movement if you look closer. cocci can be anywhere in the flask, they can float or attach to cells or even be at a location that there is no cell. bacilli usually move faster, I've seen bacilli that move really fast in contaminated flasks. cells don't secret granules that big, and I think it is very unlikely to see granules formed by your lipofectamin, though I had such issues when I used very high concentration of Fugene6 or when I transfected with calcium phosphate method. Most likely what you have is contamination, and there is nothing you can do, if you tell your supervisor he will ask you to throw your flasks away. you can't publish data with contaminated cells. throw them away immediately. The contamination can come from different sources, even your breath or your forearm hair, you might never know. ...you may also need to check your cells for mycoplasma.




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