Dear Colleagues,
I am performing an MTT assay with a primar culture of murine peritoneal macrophages and a plant extract dissolved in DMSO. After the treatment, all treated wells appear contaminated, looking very cloudy. After incubation with MTT, all wells show a very intense purple.
The DMSO, the culture medium and the MTT used have been filtered and still look the same.
I believe that is not a good idea to filter the extract because some component can be retained.
Could the contamination come from the cells?
Would anyone have any suggestions for this case?
Appreciate.
You did not mention that control wells are free of contamination or not. If not then your extract are the source of contamination. In that case, you have to do the extraction in aseptic condition and need to do all the calculations, dilutions in clean bench to avoid the contamination...
GOOD LUCK!
Dear Caio,
I think the best way is to cultivate your MTT solution in a microbial medium like nutrient broth if it is ok you can culture your working cells and passage them for a week and see if there is any contamination. Mycoplasma is very usual contamination and you can avoid that by adding gentamicin or Mycoplasma removal agent. Hope it would be of help.
Good Luck
In addition to the two responses above. It won't be out of place to filter your extract with a 0.45uM filter especially since it is soluble in DMSO. If there are any insoluble debris after dissolving the extract with DMSO, then it means the extract is not fully soluble in the solvent. Also try doing further serial dilutions of the extract keeping in mind that the DMSO concentration in the cells should not exceed the recommended levels.
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