Unfortunately mycoplasma infection is known to be associated with chromosomal instability and has been shown to affect many key genes in oncogenesis such as p53 and ras. Even if you “clean-up” your cells irreversible damage may have already been done to them. The results obtained from these cells might not be indicative of the cell line or the cells in general. Even if the mycoplasma has not altered your cells, the treatments to remove it may have. I agree with the majority of people hear, get new cells. It is sad to say, but that is the best course.

Mycoplasma infection is a severe problem in cell culture and is extremely difficult to removed. The best solution is to discard the infected lines and get fresh frozen samples from ATCC.

An alternate treatment is to passage the cells through mice if possible, though you risk picking up xenotropic retroviruses when you pass human cell lines in mice. Antibiotics that claim to cure mycoplasma tend to control it rather than cure it - in my experience, they are ineffective.

The infected lines must be removed from your incubator and put into isolator boxes, as they can cross-contaminate uninfected lines.

Thank you Gary ,

But unfortunately i had taken different frozen cells but all cells was contaminated.....can you tell me is this contamination will affect my further experiment??

Biological Industries produce antibiotic cocktails for use to eliminate mycoplasma. http://www.bioind.com/Media/Doc/Mycoplasma_Biological_Industries.pdf

I have used these successfully with other myeloid lines in the past. You must make sure that the mycoplasma has completely gone before you use the cells experimentally. Otherwise you have no idea how much influence the mycoplasma is having on your results. It will also make publication more difficult if you're not strictly working with something that is purely one cell type. Also, yes be sure to decontaminate your working space: hood, water bath and incubator and separate your cells from others.

If you can't get rid of the infection then I agree with Dr Gilmore and I would purchase new lines from ATCC (this will also help with publication as they are mycoplasma tested as well as phenotyped etc).

Mycoplasmic contamination can drastically alter the way your cells grow and how they respond to external stresses. It is a complicating factor in any analysis, and will make your results questionable for any resultant publication - the reviewers would make you replicate the results with uninfected lines before you could publish it.

I've used the ELISA test, but the most effective test is Vero cells. The problem with the Vero cell assay is you have to culture the test cells for a week in the absence of any antibiotics.

I strongly recommend getting rid of the contamination any way you can - though as I said, the best way is to get fresh cultures from ATCC. If the antibiotic cocktail does the trick, by all means, use it.

Mycoplasma can come from other infected lines, and it can be present in batches of fetal bovine serum. I always purchase fetal bovine serum that has been screened for mycoplasma.

Unfortunately mycoplasma infection is known to be associated with chromosomal instability and has been shown to affect many key genes in oncogenesis such as p53 and ras. Even if you “clean-up” your cells irreversible damage may have already been done to them. The results obtained from these cells might not be indicative of the cell line or the cells in general. Even if the mycoplasma has not altered your cells, the treatments to remove it may have. I agree with the majority of people hear, get new cells. It is sad to say, but that is the best course.

Note that althought ATCC tests for mycoplasma contamination, many of the cell lines in ATCC's database are positive for different strains. At one time it was estimated between 35 and 80% of all cell lines, even those from ATCC, contained mycoplasmas or ureaplasmas. Also note that while antibotics work for some mycoplasma spp, there are multiple stpecies of mycoplasma that contaminate cell cultures. Having worked with Mycoplasma for years, I can tell you they are amazing organisms with interesting biology that are extremely difficult to remove from cell cultures.

We do a PCR based myoplasma analysis. The easiest solution is, discard the cells and get new myocoplams free cells. If you really need them treat them with Plasmosin. We used the protocol of the supplier and in our hand this worked. The cells are and remained myocoplasm negative. We reduce our myocoplasm infection to virtually zero, by physically separating the isolation of primary rat/mouse cardiomyocytes from the transfection lab. We now have three separate culture rooms, ultra clean (one for expanding cell lines and stem cells, one for transfection on cell lines and one for primary cultures). People that use one or two rooms have to start in the most clean room and not the other way arround.

Thanks to all for your valuable suggestions.

Whenever i get any doubt i will again be here.

Thanx again

If you think your cabinets are contaminated, you can clean them completely using a formaldehyde bomb or more modern using H2O2 decontamination. Our institute has this equippement, but you can also ask the company that delivered the cabinets. After each culture session we clean the inside of the cabinets with Alconox (decontamination soap) solution, then with 70% ethanol, and the we leave the cabinet turning with out use for 10 minutes. ( first the soap is important and also no higher dan 70% ethanol otherwise it will not killed the bugs). We are very strict on this, and have veritually no contamination anymore.

Thanks a lot Maurice, i will try it

Plasmocin is a safe anti-mycoplasma agent that you could use on routine basis, it would take about 3 weeks to get the contamination under control. Good Luck.

I´m interested in Maurice van den Hoff´s suggestion about cabinet decontamination usign H2O2. Where can I get the protocol?? Thanks!

We get Mycoplasma-EX spray from PromoKine to disinfect surfaces after 70% EtOH. We were not able to use formaldehyde bombs in our lab set up. Regular use has dramatically reduced cross-contamination issues, and increased prevention.

Also, plasmocin will only suppress the infection, and I have had many lines come back positive after the removal of the drug. The best bet is to go back to a clean vial, or buy new cells. It's tragic, but you save money and time in the long run by doing better more reliable experiments.

Good luck!

I agree with a few comments above that it is best to discard the contaminated cells and get fresh stocks (make sure to change medium, trypsin, PBS, etc as well). However, in case of primary cells (not in your case and just for information) it is not possible to get another stock and so plasmocin, in our hands, has worked in the past. All the best.

Another problem with mycoplasm contamination is that it alters the disponibility and proportion of the recepetors (cytokines, growth factors, etc..) that the cell line express in its surface, what makes the response to treatments questionable. I would buy another ATCC fresh vial of this linage. You're gonna save time and money.