[Fwd: Re: help! 293F cell line]

Miguel Garcia Toscano mtoscano at ugr.es
Tue Jan 22 11:57:27 EST 2008


Hi

293F cells are easily to be transfected (with GenJet, Lipofectamine 
2000...), but to obtain a high quality transfection the cells need to be 
very healthy. If you do not watch in your media any traces of 
contamination under the microscope ( yeast can be seen and bacteria 
turns the clear media to a cloudy one) may be what you have in your 
cultures is mycoplasm. This is not detected unless you perform a PCR or 
another detection method. This contamination is very common in any lab, 
difficult to solve once you have the problem, and very annoying. To 
avoid mycoplasm contamination you must use a hood only for cell line 
cultures, do not use primary cells or another source of contamination. 
Use very clean material, use lysol to spread to any instrument (pippets, 
tips...), you must be very careful.
I use to culture 293T cells (is the same line) DMEM media, 10% FBS, at 
37C and 10% CO2. I use Triple at 0.1 times concentrated (Like-trypsin. 
gibco) to detach the cells and I recommend to use sterile commercial PBS 
mycoplasm tested.

Hoping you are lucky!!

Migue

-- 
Miguel Garcia Toscano, PhD
Temple University
School of Medicine
Medical Research Building
Department of Physiology
3420 N. Broad St. Philadelphia
PA. 19140
Phone: 215-707-4336
Fax: 215-707-4003

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