In vitro methodological standardization of MTT colorimetric assay for evaluation of formazan activity and drug dosage

Abstract

Background Aiming to develop essential chemotherapeutic drugs to control various types of cancer, one of the primary tests is the tetrazolium reduction method. The present study aims to standardize the electromagnetic spectrum that best discriminates the absorbance and to evaluate the drug dosage of single and daily doses during the cell viability and toxicity analysis by the tetrazolium reduction method, using the ethanol extract of pequi peel in canine osteosarcoma cells. Results The wavelength of 532 nm showed the best results. We found that as the treatment time increased, the formazan conversion reduced. After 72 hours of treatment, we observed clear discrimination of dose-dependent data, with up to five discriminants within 72 hours with a change in absorbance from 0.554 to 0.064 A. The wavelength of 570 nm it was not ideal since we could not discern the difference in the spectral reflectance of the treatments and, therefore, show no statistical difference among treatments. We found no statistical difference for the coefficient of variation at wavelengths of 532 and 570nm, which were 12.77% and 8.80% respectively. Conclusions The wavelength of 532nm best discriminated the absorbance, as it presented better ability to congregate the treatment groups, greater variation between the discriminant and lower coefficient of variation, during the colorimetric test to evaluate the cellular metabolism. Moreover, the ethanol extract of pequi peel in canine osteosarcoma cells showed a statistically equal effect of a single dose administration to the dose reapplied every 24 hours.