MTT assay for cell proliferation and sensitivity to erlotinib

NIH-3T3 cells stably harboring vector alone, or EGFR WT and mutants (2 × 10³ cells) were plated into 96-well plates in triplicate and were allowed to adhere overnight. The cells were then treated with DMEM containing 4% FBS with or without 50 ng/ml EGF (Sigma) and replaced every 2 days. Cell proliferation was determined every day, for 5 days, by replacing culture media with 0.5 mg/ml MTT reagent (AppliChem, Darmstadt, Germany) and incubating at 37 °C for 4 h. The reagent was then removed and cell-crystals were dissolved in 150 µl DMSO and the absorbance was measured at 570 nM. Cell proliferation curves of these NIH-3T3 cells were based on the mean of the absorbance from triplicate wells, then means ± SEM from the three independent experiments were calculated and plotted as fold increase of cell proliferation compared to day 0, using GraphPad Prism 6 program.

To test sensitivity to erlotinib, 4 × 10³ NIH-3T3 cells stably harboring vector alone, EGFR WT and mutants were seeded into 96-well plates in triplicate overnight. These cells were then treated with erlotinib (Santa Cruz Biotechnology Inc., CA, USA) at various concentrations from 0.0001 to 5 µM for 72 h by replacing new media with erlotinib every 2 days. Viability of the cells treated with erlotinib, as an indicator of erlotinib sensitivity, was determined by MTT assay. Cell viability curves of cells treated with erlotinib were based on the mean of absorbance from triplicate wells, then means ± SEM from the three independent experiments were plotted as percentage of control without erlotinib at the end of 72 h, using GraphPad Prism 6 program.