Dear all, i was confounded by a phenomenon i witnessed under my light microscope - that is, when i tried mixing two different cell types together, i started to observe dark aggregates forming over time. What are the possible explanations for this? Thank you for your kind responses :)
Hi Justin,
can you attach your picture here to get a good feedback. And you need to explain very well on what type of cells are working because many cells have different physical and biochemical characteristics. let us see the picture and inspect them. good luck.
Hi Justin,
Like Mebrahtu said we need a picture to know more about the aggregates. How ever when the cells forms clusters or growing over on each other, then under the microscope, the light scattering of dense cell wall and membrane will look like black spots. If you upload your images we can clarify your doubly easily
Regards
Sundarajan K
Hello Justin,
I have seen such problems with few cultures in past. If they are small black dots everywhere outside and inside cells, I would suggest you to check for mycoplasma contamination which is often case associated with black dots. Since mycoplasma grow slow, so cell growth might not seem practically hampered but gets slow. You can check it by commonly available kits and even by DAPI. There might be other reasons like over-trypsinization and media compatibility in co-culture but Myco-contam seems most probable to me here.
Hope it helps!
Ajay
Dear Ajay, I highly doubt that it is a contamination issue here because i have repeated the same experiment many times and i happen to get the same results. I am not sure if black aggregates form as a result of the compatibility or the non-compatibility of the two cell lines and thus the aggregates. Some cell types, when mixed form homogenous and indistinguishable cultures as compared to this one. Or could it possibility be a result of some secreted matrix from the cells?
Hello Justin,
Have you tried to repeat your experiment with fresh cell stocks ? Did you still got black dots? Have you really tested for Mycoplasma and are you 100% sure that its not Mycoplasma contamination? Again there can be possibility of secreted matrix also but I doubt that you will obtain that in the form of black dots. In continuation of the answers by other people, I would like to ask you to add some photographs of your cells so that I can have a look and rule out the possibility. Because I know how the Mycoplasma contamination looks like and sometimes you can passage your cells multiple times and it will just seem perfect but still having active and slow proliferating Mycoplasma. Its time that you need some confirmatory tests for contamination/matrix deposition and also try to play with media since I don't know exactly what sort of media you are using to do co-culture experiments.
Hope it helps!
Ajay
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