Which solvent blank (Media + MTT + Solubilizer ?) should I subtract from the sample to get the corrected absorbance? Does negative cytotoxicity make sense?
Hello Vinayak
The corrected absorbance of the test sample can be obtained after substracting from the absorbance reading of the blank.
In MTT assay we need to have blank and controls.
The blank is usually only medium without any cells along with MTT and the solubilizing solution (either 0.04 N HCL in isopropanol or DMSO).
Controls in MTT assay include positive control which is a known cell line treated with the drug along with MTT and solubilizing solution. The negative control is the cells without treatment along with MTT and solubilizing solution.
The negative readings very close to zero would mean that the test compound kills 100% of the cells in the well. I hope you have treated the blank and the sample wells under the same experimental conditions i.e., added the cell culture medium to both the blank wells and sample wells at the same time before the MTT assay.
I hope this is helpful.
Best Wishes.
Hello, Vinayak!
It can be added that negative cytotoxicity in MTT is observed when the optical density in the experimental wells treated with the preparations is higher than in the control (medium with clays and a solubilizer). This makes sense when evaluating the effect of test substances on cell proliferation or on the activity of mitochondrial dehydrogenases, which metabolize MTT.
At the same time, negative cytotoxicity can be observed as an analysis error (due to insufficient dissolution of dye grains (farmazan) by the coubilizer, insufficient pipetting, small sample volume, chemical structure, and high doses of drugs that can form a precipitate and shield the dye from the solubilizer).
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