I have 4T1 cell line and it is contaminated with Mycoplasma .I passed it through nude mice subcutaneous injection and cultutered tumor cell and tested for mycoplasma presence and found it's positive with mycoplasma.
Pleased suggest me how to get ride of Mycoplasma for the cell line.
Hi.
You won´t get rid of mycoplasma but you can treat your cells with commercial antibiotics to reduce dramatically the infection but as far as I know you won´t get rid of it.
If you intended to perform in vitro experiments you can use antibiotics, then you let your cells 2 weeks in treatment and then you check again for myc infection, you probably will see that mycoplasma has disappeared, and then it would be ok to do in vitro experiments. Keep on mind that if you remove the antibiotic treatment mycoplasma will appear again.
As you are using cells to perform in vivo experiments I would recommend to star again with mycoplasma free cells, it is widely reported that cells infected with mycoplasma behave differently and you won’t be able to interpret your in vivo results in a clean way.
Best.
Hi,
As Carlos said it's rather difficult to get read off the fungus contamination. But you may try combination of these three drugs: 0.5mL of Gentamicin of 50mg/mL + 1mL Kanamycin of 50mg/mL + 1mL of Amphotericin of 5mg/mL. It's a dose for 0.5L of medium. Put this mixture instead of the standard antibiotic supplementation that you use.
Good luck!
For mycoplasma infection you should use Ciprofloxacin Hydrochloride (Cipro). You can buy it commercially for the elimination of mycoplasma infection. Below are links to some publications which analyzed its use in cel culture. Or you can use other drugs (link from Invivogen). But we used Cipro for our unique cel line.
However, if you can defrost or obtain mycoplasma-free cells it is better to switch to them.
https://www.ncbi.nlm.nih.gov/pubmed/3282011
http://link.springer.com/article/10.1007/BF00365081
http://www.invivogen.com/review-plasmocin
There are three actions to be taken to eliminate mycoplasmas from a cell.
First, find the origin of the contamination: this very often comes from the culture medium, such as the culture serum and the trypsin used to detach the cells in culture.
Second, using an effective anti-mycoplasma, such as ciprofloxacin or tylosin, both are effective but toxic at the useful dose.
MRA and Plasmocine are less toxic, but they are also less effective.
Tiamulin remains the best compromise between toxicity, efficacy and cost.
Finally, the cell line can be decontaminated with IP injection on mice, but using an immunocompetent mouse, such as Balb / c, and not a nude mouse. However, care must be taken not to derive the cell because you can recover natural cells from the mouse. This latter practice, although very effective, is difficult to accept by the ethics committee which manages laboratory animal facilities and is regularly rejected because in vitro decontamination techniques can be used and can replace this rather barbaric procedure.
Thanks Pin for your valuable tips
Could you share some references about IP injection in mice please
There is not really a recent article on the subject, because from an ethical point of view this practice is no longer acceptable to reviewers.
It is a practice that is transmitted from one laboratory to another.
In fact, the procedure is the same as for the production of monoclonal antibodies in the form of ascites:
1-sensitization of the mouse, by injection in IP of 0.5 ml / mouse of Pristane.
2- after 5 to 7 days, IP injection of the hybridoma, between 10,000 and 1 million cells under 0.5 ml of PBS.
3- as soon as the belly of the mouse begins to grow, between 7 and 9 days, recover the ascites sterile with an 18-20G needle, spin and recover the cellular pellet containing the hybridomas.
4) culturing the hybridomas in culture medium containing an anti mycoplasma. The possible presence of red blood cells is not inconvenient.
After a few days of growth, proceed to the cloning of the hybridoma to ensure its stability and specificity.
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