Hi Guys
For the last couple of months my lab has been riddled with infections. All cell lines. It is odd though in that the infection is only visible the day after transfection with either CaP or Lipofectamine 2000. These are not typical bacteria – they are absolutely tiny and don’t swim, but appear to wriggle. It is not Brownian motion and is definitely infection of some sort as confirmed by my confocal microscopy staining.
My working theory is that the bacteria (I feel it is mycoplasma…) are INSIDE the cells until we “stress” the cells by transfecting them, whereby the cells burst open and release the bacteria into the media. I have not seen this online so I am not sure. Has anyone seen this before?
I am aware you cannot really see mycoplasma under the light microscope but the “wriggling” things could be colonies, and my confocal appears to indicate mycoplasma infection.
We have absolutely no idea what else it could be at this stage as we have cleaned everything four times, filtered all reagents…. Everything!
Does anybody have any suggestions as to how to solve this issue? We are currently treating some cell lines with Plasmocin and any cells we “hope” are not infected are being cultured with a low dose to prevent mycoplasma infection.
It is absolutely insane how this infection won’t pass! I think it must be mycoplasma as I have started using both Gentamycin, Amp B AND Pen/Strep in my media to ensure/kill bacteria. And yet… we are always getting infections BUT only apparent following transfection. It is not the transfection reagent as I have used different brands, batches and even tested all individual reagents on cells (ie. Added a few microliters of DNA, Calcium Chloride, HBS, PBS, FBS etc…) and no infection is seen. It seems stress induces the exit of the bacteria from cells.
Has anyone seen this before?
Please help if you can!
I have worked with mycoplasma+ L2k transfected cells, we also noticed faint DAPI staining in the background. Best way to confirm mycoplasma contamination is to actually test for it. It is actually becoming quiet common depending on the region. When i was doing transfections i was confirming with 7-AAD staining using flow cytometry and we did not see significant cell death with transfected and myco + cells (but mycoplasma was inhibiting transfection). I know you started mycoplasma treatment but the biochemical/luminescent test for it is easy and has a very quick turnaround time if you cultured cells for long enough and would recommend doing this test if you want to get a more definitive answer.
Hi Michelle
Other people have seen an artefact following transient transfection that sounds very similar to what you are seeing, although the reason is not well understood - - see this thread:
https://www.researchgate.net/post/Any_suggestions_on_a_Dapi_Hoechst_artifact_with_Lipofectamine_3000_transfection
The consensus is that the material probably originates from the cells or plasmid, rather than being an infection. In any case, it is still possible to interpret experiments as long as empty vector transfection is performed as a negative control.
Thanks Alex but I don't think it's that. I haven't seen this before but have been transfecting for years.
Also, these particles are motile.
Thank you and interesting thread all the same!
We used the Lonza kit.
https://bioscience.lonza.com/lonza_bs/US/en/Cell-analysis/p/000000000000186475/MycoAlert-Mycoplasma-Detection-Kit-%2850-Tests%29https://bioscience.lonza.com/lonza_bs/US/en/Cell-analysis/p/000000000000186475/MycoAlert-Mycoplasma-Detection-Kit-%2850-Tests%29
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