I am performing tests by adding increasing concentrations of drugs to my cell culture media (using DMSO as a vehicle), but in the higher concentrations especially I see these large dark cloud appearing around my aggregates. It seems to correlate with increasing drug concentrations, so I thought it might be to do with a stress response or cell death but can't find any literature to help. I don't think it's infection as it doesn't resemble any typical infections, and it doesn't look like the drug precipitate as I've seen that before (this one I'm not 100% sure about). It seems to closely correlate with the toxicity results I get from the LDH assay too. Cell line is HepG2 Any ideas?
for me it looks like a bac contamination... to exclude this, you may want to filter your DMSO stock through 200um just before adding.
Hi Ben,
It seems to me that it is a huge particle of tissue including thousands of cells. To deal with this issue you can change your cell purification procedure for instance using of micro filters or long-term vortex to separate cells from tissue perfectly.
Alex Lyakhovich thanks for your answer, I do actually sterile filter my media after being made up with DMSO in it, so don’t think that’s an infection risk. Of course it could be infected through other means but we looked at the cloud under phase contrast too and it didn’t look like bacteria. Still I’ll take extra care in my next repeat and see what happens.
Milad Esmaeilbeigi thanks for the suggestion, however these cells aren’t isolated from tissue so I don’t think it would be that, they are routinely passaged in 2D before putting into hanging drop cultures. The mystery continues...
ah.. ok.. is it increased in mass upon culturing? did you have a chance to stain it with some dyes to compare morphology with regular cells?
Hi Ben Allcock ,
It is difficult to tell from the image without seeing it in relation to the other dosed samples. However, it resembles what we classify as a type of flocculent precipitation that is distinct from the crystalline precipitation that is more common at high doses. 150 mM is a fairly high working concentration for any compound, so I would expect to see some precipitate at or near that dose level in many test compounds. If you are seeing increases in LDH without any significant reduction in cell viability than the precipitation could be interfering with your luminescence readout.
Best,
Nick
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