I generated stable cell lines using lentiviral transduction recently. Cells are positive as checked in FACS but I am constantly observing black dotted background in culture flask after each passage. Thinking it can be contamination I put a drop of this cuture medium on TSA plate, nothing grew on it. Do cells secrete some granules after stable transfection?
These black dotted background can be cellular debris or signs of mycoplasma contamination. Mycoplasma can be stain by DAPI or you can use the Lonza kit (chemiluminescence detection) to detect mycoplasma.
I argree with Marie-Jose - it looks like mycoplasma contamination. For detection I recommend PCR (for primers see Uphoff and Drexler 2002) and for the treatment BM cyclin from Roche. Good luck!
I don't buy the mycoplasma thing. They should overgrow your culture over time, and you know, kill stuff. So if your cells aren't sick, and don't get sick over time, I think it will be something else. It could be some effect relating to the insertion site. Do all your clones have it?
Or, it would be intriguing if it's what you're thinking. U937 does have formal "biomarker-level" granulocytic potential. But I'm not sure it can make functional granules. Did you try if they can kill microbes, like donor granulocytes do? What are you going to do with it, cell therapy? This would be cool. Keep an open mind, Scientist! :)
What i have read about mycoplasma contamination is that myco positive cells make cluster. Interesting thing is that I inserted my tool in U1 as well as in U937 and all cell lines having same dusty background. I surfed a lot over internet but could not find anything and finally posted my question here as any experienced person can tell me something about it. One technician in lab told me that sometimes cells secrete inclusion body kind thing after stable cell generation. But that was not satisfactory for me.. has anybody faced such issue with them?
What is the viability of your cells after transfection? Cell debris looks like granules...
@zdenek Andrysik: I think your point is valid that background can be debris though its size is small because yesterday i observed that few infact very few cell's membrane was nt perfectly smooth, those cells were little swelled and i could make that these cells gonna burst. Probably thats the reason I am observing dotted background. If this is the case then what i should do to prevent this????
I recommend you to test your cells in culture regularly for mycoplasma (in every defrosting mainly) when you will have spread the problem of mycoplasma, it is probably possible that the transduction of your cells leads a cellular death. You can try to eliminate the cellular debris by slow centrifugation. Usually the debris are in the supernatant and the cells at the bottom of the tube.
@Marie-José Stasia - Thanks, we have mycoplasma detection kit in our lab.. i will check.
I have experienced a similar situation recently and saw few black dots as well in my flask as the color of cell suspension was yellow and it looked over grown,my question is can RPMI be one of the reasons of contamination?
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