While performing cell viability assay using MTT in fish cell lines grown in L-15 media without CO2 at 28 degrees, I found a highly faint purple colour in control cells (after solubilising formazan with DMSO)which was difficult to observed with the naked eye. I had seeded 20,000 cells/well in 96 well plate which was 80-85% confluent and cells were healthy at the time of adding MTT in serum-free media for 4 hours. Although microplate reader could detect the difference in absorbances between control and treatments, I could not figure out the reason for the extremely faint purple colour of formazan. Do fish cell lines have extremely low expression of NAD(P)H-dependent oxidoreductases (mitochondrial succinate dehydrogenase ) which reduces MTT to insoluble formazan.
Suhad Mohammed Thank you for sharing the article, but it does not explain the reasons for getting such low absorbance. However, I also got absorbance in the range of 0.09-0.13(comparable to fig.1 of this article) with two fish cell lines(not Zebrafish) with 20,000 cells while we used to get 3-5 fold more absorbances with only 4000-5000 cells of some commonly studied cancer cell lines cultured for 24 h even with less confluency. So i was interested in knowing the reasons for this high disparity in absorbances.
Hi Abhinav,
As you should already know, MTT assay is representative of cell metabolic activity and not of cell number so seeding higher cell number wouldn't essentially increase absorbance. L-15, which you used in your cell culture, contains little or no glucose. If you add glucose to your cell culture or use a glucose media like MEM, DMEM, MCDB, RPMI 1640, DMEM: HAM'S F12, Fischer's media, IMDM etc. it will increase the reaction rate and give stronger signals of absorbance in MTT assay. L-15 contains galactose instead of glucose, that's why you are getting a low signal and less formazan crystal formation because cell's metabolic activity is low.
Hope it helps!
Ajay
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