4.6. Rapamycin Treatment and Assessment of Cellular Metabolic Activity

Metabolic activity of cultured primary and metastatic mammary gland tumor cells, treated or not with rapamycin, was determined by the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Life Technologies Corporation, Carlsbad, CA, USA) which is yellow in formazan (violet). This conversion indicates the number of viable cells. The shade of violet is directly proportional to the concentration of viable cells in the culture plate.

Tumor cells were transferred to 96-well plates (10.000 cells/well) and after 24 h of incubation in MEGM™ culture medium, rapamycin (diluted in DMSO) was added in 4 concentrations (9, 10, 11 and 12 µM). Rapamycin concentrations were chosen based on previous studies in breast tumor cells [9,10]. After 24, 48 and 72 h, MTT (0.5 mg/mL in DPBS) was added and incubated at 37 °C for 4 h and then, MTT salt were dissolved with DMSO. Analysis of the results was carried out 10 min after the addition of DMSO, in a microplate reader (Biochrom Asys Expert Plus Microplate Reader, Biochrom Ltd, Harvard Bioscience, Holliston, MA, USA), at 550 nm absorbance. MTT was carried out in six repetitions for each rapamycin concentration. A control with DMSO, at the same concentration as the highest dose of rapamycin, was used to ensure that there was no effect of the product on cell proliferation.

Based on test results, maximum inhibitory concentration (IC₅₀) of cell viability was determined using the formula: Cell viability % = (absorbance of the treated sample−blank absorbance)/(absorbance control cells DMSO—blank absorbance) × 100; whereas the blank was DPBS and cells in the control group did not came in contact with rapamycin.