This answer is from my personal knowledge and opinion and is NOT an official statement or recommendation from the US  FDA. 

Mycoplasma infections of cell lines and the effects on MANY cellular pathways are well documented in the older literature. There are even books about this, but they are all over 10 years old, as are most of the published papers on this topic.    Your only really viable option is to discard the infected cells and start fresh with KNOWN clean cells or restart primary cultures. Primary cultures, when collected asceptically from a 'sterile site',  and all equipment,  medium etc is sterile, are rarely naturally  infected with mycoplasmas.   The infection comes during the lab phase of the work, not from the host.  (Obviously, this is not necessarily true for non-sterile sites, such as  tissue from mucosal surfaces).  Curing the cell lines is probably not an option for epigenetic studies because of the intense chemical decontamination of the cells, which may kill the mycoplasmas, but will also affect the cells.  In essesnce, you have no way of knowing if the cells you end up with are the same as the ones you started with.  BTW, most of the antimicrobial agents used in cell cutture are bacteriostatic, not bacteriocidal, so they slow the growth of mycoplasmas, but don't kill them. They just make them harder to detect.  This is why most people familiar with mycoplasma infections of cells recommend that you use no antimicrobials in the medium and test, test, and re- test the cell cultures on a very regular basis. If they are very valuable, freeze back aliquots of the clean cells every so often so you can go back to that passage number.  If the cells turn up positive, at least you have limited the effect of the infection to a short time period and can go back to an earlier time point in the experiments and start again.   Rapid tests like DAPI staining and PCR work well only if there are lots of mycoplasmas around (i.e., about 1000 per ml is usually the lower limit of detection).  In my experiece with such things, if you remove the antimicrobials from the cell culture medium, it usually becomes immediately apparent that mycoplasmas are not the only problem...the cultures also are infected with 'normal' bacteria, which are much easier to detect because the medium becomes a turbid soup of bacterial culture.  That usually is the clue to just discard them and start over.

We treat our Chinese hamster ovary cell lines with Normocin and Plasmocin, both are  available from InvivoGen:

http://www.invivogen.com/mycoplasma

Hi, I don't know exactly how micoplasma could interfere with developmental pathways in cells..but if they're contaminated, I wouldn't use them, mainly because I would expect an increase in cell suffering and cell mortality in such cultures (if they're already few, it's even more likely that it happens). Also, you risk to contaminate other cells you (or others in your lab) are working with!

Are you sure they got contaminated? You could use a kit to detect micoplasma in your culture to check it!

Dear All,

Thank you for your valuable answers and suggestions. I did the detection test using the Mycoprobe Kit, from R&D Systems. Its based on absorbance principle (O.D.), different from a PCR, and known to be much more sensitive. I would do a run again, reason being even the negative control in the kit gave a positive O.D. value.

Passaging mESCs upto weeks can't be an option as I have to keep the passage numbers as low as possible to study the epigenetic changes initiated as embryo level, from which the cell lines have been derived.

Its quite a scary thought to throw everything away as its my one year's work.

I would do a PCR based assay as well, and probably go back and test earlier passages to confirm the results. 

Thank you once again!

Dear Terry,

Thanks for your reply. I have a couple of questions in the same regard.

1.Did the treatment work on all cell lines?

2. Did you check if they had any possible effects on your cells? Genotypic/ Karyotypic etc.?

3. Do you recommend or have you tried including these as a part of your routine culture media?

4. which method of detection do you use, before/ after treatment?

Thanks!

This answer is from my personal knowledge and opinion and is NOT an official statement or recommendation from the US  FDA. 

Mycoplasma infections of cell lines and the effects on MANY cellular pathways are well documented in the older literature. There are even books about this, but they are all over 10 years old, as are most of the published papers on this topic.    Your only really viable option is to discard the infected cells and start fresh with KNOWN clean cells or restart primary cultures. Primary cultures, when collected asceptically from a 'sterile site',  and all equipment,  medium etc is sterile, are rarely naturally  infected with mycoplasmas.   The infection comes during the lab phase of the work, not from the host.  (Obviously, this is not necessarily true for non-sterile sites, such as  tissue from mucosal surfaces).  Curing the cell lines is probably not an option for epigenetic studies because of the intense chemical decontamination of the cells, which may kill the mycoplasmas, but will also affect the cells.  In essesnce, you have no way of knowing if the cells you end up with are the same as the ones you started with.  BTW, most of the antimicrobial agents used in cell cutture are bacteriostatic, not bacteriocidal, so they slow the growth of mycoplasmas, but don't kill them. They just make them harder to detect.  This is why most people familiar with mycoplasma infections of cells recommend that you use no antimicrobials in the medium and test, test, and re- test the cell cultures on a very regular basis. If they are very valuable, freeze back aliquots of the clean cells every so often so you can go back to that passage number.  If the cells turn up positive, at least you have limited the effect of the infection to a short time period and can go back to an earlier time point in the experiments and start again.   Rapid tests like DAPI staining and PCR work well only if there are lots of mycoplasmas around (i.e., about 1000 per ml is usually the lower limit of detection).  In my experiece with such things, if you remove the antimicrobials from the cell culture medium, it usually becomes immediately apparent that mycoplasmas are not the only problem...the cultures also are infected with 'normal' bacteria, which are much easier to detect because the medium becomes a turbid soup of bacterial culture.  That usually is the clue to just discard them and start over.

Hi Pooja,

If you are considering treating for mycoplasma, I have treated cells with the following for effective Mycoplasma removal using http://www.abdserotec.com/mycoplasma-removal-agent-accessory-reagent-buf035.html. It takes culturing the cells with these for a week, but we used it for 10 days. Let the cells grow in culture for another 10 days and then check for Mycoplasma. Also, take a sample from a well that is 70 - 90% confluent and the media has been on the cells for at least 2 days, if it is a fresh media change, mycoplasma cannot be detected.

Dear Kavita,

Thanks for your reply! Could you please tell me which cells did you treat with this agent? Do you also use it as a regular ingredient in your media? Di you also check cells after 1 week, as they claim on their website?

Also, if you could tell me which technique you use for detection?

Many thanks!

Hi Pooja,

I have treated both, murine primary neural cells and human fetal stem cells with this, but as I earlier pointed out, the treatment time was increased to 10 days instead of 7 days. No, I do not use this as a regular ingredient in my media. It is finally antibiotics and not only does it slow down cell growth, but you could end up dealing with antibiotic resistance, for say, a future contamination. Yes, I checked the cells but after 10 days. I used the Lonza mycoplasma kit for detection, using a plate reader with OD as the measuring parameter.

Hope it works!

I have 39 years experience of cell culture and mycoplasma testing and I would 100% support what Maureen Davidson said. I have personally helped people decontaminate cells from mycoplasma after caving into a lot of begging and whimpering. It worked but quite frankly it was waste of time as, (mentioned by Maureen), the phenotype was altered. 

The inescapable fact to remember is that detection of mycoplasma is always behind any spread. If you think you have cleaned it up and you haven't, then you will have a lot of angry co-inhabitants on your case. Mycoplasma is not only destructive to cells but also Dept strategy as its real or potential presence gets the blame for anything slightly untoward. If you must go head then total quarantine of all equipment and reagents is vital. If that cannot be guaranteed for the decontamination and subsequent 6-8 week QC period, then do not even start. A years work down the drain might sound bad but it can get a lot worse. You best strategy is to take all suspect material to the autoclave. 

Again after 37.5 years experience doing cell culture I totally agree with both Jim and Maureen. Mycoplasma contamination affect all cell activity and you will not revert the cells back to the "normal" phenotype. Also we use solid agar detection as the method of choice along with Dapi staining. Solid agar is the Gold standard test and has been for the past 50 years. It is very sensitive but to get a definitive negative the test takes 4 weeks.

The ATCC have not used PCR testing for many years as this is known to give many false negatives and I have personal experience of this. 

The old expression is: "If in doubt chuck them out"