I am currently working on cytotoxic test involving K562 (leukemia cell lines), in this experiment, it involved the use of MTT to test for the cell viability. However, I encountered a problem here, which is upon the addition of MTT and incubation of 4 hours (37 degree celsius, 5% CO2), the wells added with plant extracts (which to be tested if they have effect on the cell viability) and MTT become very dark purple, which is very different from my control, 5-Fluorouracil, where all remained yellow. Hence the problem here is, supposedly all of them should remain yellow until the addition of DMSO right? But how come the wells added with plant extracts turned purple but control remained yellow? The higher the concentration of plants extract, the higher the intensity of the purple colour. I have confirmed that it was not due to contamination. Can any experts help?
Did you try incubating your plant drugs with MTT w/o cells? Some drugs can interact with MTT giving false results In that case you might want to try a different assay for cell viability. . Also make sure you did not dissolve your drug in DMSO at a very high concentration. Less than 0.05-0.1% final concentration is ideal. As an initial screening you can even try Trypan Blue assay to determine if there is any effect at all. Other thing you can consider is to run a cell density curve to make sure the OD you got is within the acceptable linearity.
Dear Suraj George, thanks for the reply.
My plant drugs are not dissolved in DMSO because all of them are soluble in water.
Anywhere, I've tried incubate the plant drugs with MTT and medium w/o cells, upon 4 hrs of incubation, the solution turned slight brownish and the intensity increased with increasing concentration of plant drugs. The medium for all wells was removed prior to the addition of DMSO and no pellets were precipitated after centrifugation (for the plants drugs with MTT and medium w/o cells). There are pellets for others with cells.
So, does the changes of colour for the MTT and plants drug with medium interfere and contribute to the false result?
Is there anywhere that I can improve on the cytotoxic assay or eliminate the possible reaction of MTT and my plant drugs?
I value and appreciate any suggestions/ways which may contribute to a better and more accurate cytotoxic result.
The possible cause may be MTT is being reduced by the test substance or materials which are generated in the culture media during the assay. possible solution is before adding the MTT solution, change the culture media to remove the test substance or materials in the culture media.
Yes MTT can interact with some plant drugs leading to erroneous results. It may be difficult to eliminate this reaction with MTT (if present) as you do not know what are the different chemicals present in the drugs and they all can interact.
I can think of two possible suggestions:
1. Try a different assay other than MTT like BrdU or Thymidine. Check other papers to see what assays were used with plant drug which you use.
2. Run a simple trypan blue assay at various concentrations and see whether your plant drug has any significant effect. I am telling this because you mentioned your control cells were yellow and treated cells were more purple. Indeed that has to be reverse in the case of MTT assay.
- Badges
- Science topic