How to remove mycoplasma contamination in cell lines? - (Aug/09/2012 )

Hi everyone,
The lab I am working in has encountered a major issue: mycoplasma in some cell lines. We can toss most of the affected cell lines because we can purchase them from ATCC, but we have several stable cell lines that people are not willing to throw away. Has anyone had luck eliminating mycoplasma in cell lines like HeLa, U2OS, and HEK293 cells? If so, could you please share how you were able to achieve that and what reagent(s) you used?
Much appreciated,
CellBiologist

I can't say it's impossible but it's extremely difficult and you must be lucky. I have noticed that people who use pen/strep are more likely to get it. There is a guide at Roche Applied Science website that suggests using high concentration of gentamicin. You might need to find that. But still I doubt it will be entirely effective.

how do you know you have it? by DAPI staining?

How we know we have it is somewhat controversial. We have not seen anything by DAPI staining, but when using a PCR kit, some of our stable lines are positive. Some people in lab think that the PCR kit is too sensitive; others think that if it is positive, there is mycoplasma. I know that ATCC no longer uses PCR to detect mycoplasma, but they sell a kit that does (which is what we used). Have you had any experience with detection of mycoplasma by PCR?
Thanks,
CellBiologist

yes, they are multiplex PCR kits and they are very sensitive. They detect many kinds of mycoplasma.

Hi Curtis,
I am not familiar with which species of mycoplasma are problematic for mammalian cells. Do you think that the multiplex PCR detects mycoplasma species which are not problematic for cell culture? Any insight is appreciated.
Thanks

The kits are very sensitive but not very specific IIRC. Most species of mycoplasma are problematic for cell culture. The problem with the kits are both false positives and false negatives; the false +ves are usally due to contamination of the reagents with previous PCR products from testing. False negatives are usually due to inhibitors in the medium.

You should be doing both soft agar assays and Hoescht 33258 staining (DAPI isn't sensitive enough) to detect mycoplasma.