Comparison among different MTT assay protocols in NIH3T3 fibroblasts

Method: NIH3T3 fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum. Cells were harvested by tripsinization and plated in 96-well microplates at 5 different cell densities per well (10³; 5x10³; 10⁴; 5x10⁴ and 10⁵), at 37^oC in a humidified atmosphere of 5% CO₂, for 24h. Cells were, therefore, subjected to the reduction of dimetiltiazol difenil Tetrazolium (MTT) for 4h. Thereafter, 4 different methods for extracting the product (formazan) were tested: MTT1 – removal of MTT and dissolution in 200µL of 50% ethanol and 1% acetic acid. MTT2 – addition of 10µL MTT and 160µL de MEM without phenol. After 4h, dissolution in 50µL of 20% SDS and 0.01 M HCl. MTT3 – removal of MTT and dissolution in 200µL of DMSO. MTT4- removal of MTT and dissolution in 200µL of pure isopropanol. Absorbance values were measured using spectrophotometer at 550nm. The data were analyzed by 2-way ANOVA/Bonferronis test (p<0.05).

Result: The absorbance average varied according to the number of cells and experimental groups: MTT1 (-0.01 to 0.13), MTT2 (-0.03 to 0.22), MTT3 (0.76 to 1.31) and MTT4 (0.66 to 1.04). MTT1 and MTT2 only presented absorbance higher than 0.13 at density above of 5x10⁴ cells. The best relationship between cell density and absorbance values was found in MTT3 and MTT4.

Conclusion: The protocols that presented the best dose-response were MTT3 and MTT4, which will be used in cytotoxicity assay using NIH3T3 fibroblast density of 5x10³.