Hi, I have started to optimize my cell line for Lentiviral transduction and I found that my cells were positive for mycoplasma contamination again although this was a new batch of cells. I will be starting to treat the cells with Plasmocin in a day or two. However, since I have already started my optimization of Lentiviral transduction for shRNAs, I would like to know if my optimized values will be affected by mycoplasma infection or not?
Mycoplasma is a real pain! It would be convinience treat teh cells first. Even simple drugs can affect cell metabolism.
Hi Marisa.. Thank you.. Yes I am treating the cells that I have not transduced today but I have already started the optimization last week for few cells.. I wanted to know if I should proceed with the optimization or just leave it.. Will mycoplasma infection affect my values such as puromycin concentration to kill the non-transduced cells, Multiplicity of Infection etc.. If they will affect, I wont continue with that experiment..
Mycoplasma contamination has no effect on lentiviral transfection. After transfection, I came across mycoplasma contamination (from the source) however, treated with ciprofloxacin hcl. In these days, before the transfection, I treat the cells with cipro (as a precaution).
Hi Venkatesh.. Thank you for the reply. And that is so good to hear.. And one question,there will be atleast some metabolic effect when you are treating with ciprofloxacin right? For now I am just optimizing some factors for the cell line I am using.. But in future, I would be analyzing the transfection by metabolomics too and hence my question above!!
The effect of mycoplasma infection on transduction can depend on cell type and the extent of infection. Specially if the infection affects the cell growth, then it may influence the transduction and transgene expression too.
Thanks for the reply Mohsen.. Fortunately my cells are growing well without any problem..
Ciprofloxacin inhibits DNA polymerase. Since mycoplasma has no cell wall, pen/amp has no effect on them. However, Cipro effectively inhibits myco. There is a need to expose the cells with cipro at least two times to confirm the removal of mycoplasma. This need to be checked with by PCR as well as DAPI.
I am not sure about the impact of Ciprofloxacin on mammalian cells (as Cipro is against bacterial DNA pol). However, once the Cipro media is removed, there may not be any major effect. Once after ensuring the removal of mycoplasma, further cell culture can be done with media containing Pen/Strep.
That is cool.. Thats a pretty useful information. I will look into the detailed procedures and I will try that out. Is it possible for you to send me a link or an article with respect to this? Thanks again..
I would absolutely discard anything that has been infected with Mycoplasma because the infection of course does affect your cell gene expression pattern. You might treat them if they are not replaceable but it is likely your results downstream will differ from cells that were never infected. In that case reproducibility of your experiments is at risk and retraction of your results if published can happen in the worst case.
Mycoplasma contamination can be transferred in liquid nitrogen from what I read, so if your vials are all contaminated, it is either from the initial batch of cells or maybe through the liquid nitrogen tank. I have had Mycoplasma once in my career and it was through a special cell line from another lab. Clean working and very strict safety precautions allowed me thankfully to avoid contaminating anything else and we got rid of the cells quickly. I would urge you to do the same if you can afford it at all!
For the question about transfection: do you mean transfection for lentiviral packaging or lentiviral transduction? Colleagues have told me it affects transfection of 293T clearly so if they have trouble with that always immediately check for mycoplasma first. As I said I have no experience myself but there is enough literature out there to support that notion.
https://www.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/transfection/ for example.
Hello Jung. Thank you for the response. Yes, I also got this cell line from another lab the new batch of cells were not growing and was dead. So, I have no other choice than to treat the one I have and I am treating them with Plasmocin. I have discarded all my plates except the two T75 flasks which I am treating.
With respect to transfection, I was talking about lentiviral transduction. I was optimizing the MOI and Puromycin kill-curve for this cell line and this was the fourth time. The result was not as expected as the efficiency of transduction was 0.1% for most of the conditions (Even at an MOI of 2). Almost all the cells were dead (I treated them with Puro to make sure the non-transduced ones die). So, I guess as you suggested and as it is given in the link, I might have to start the optimization again after removing the contamination!! This is really annoying!!.. Thanks anyways, it was a worthy information.
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