Evaluating MnBuOE Cytotoxicity in NSCLC

The cytotoxicity of MnBuOE per se in both NSCLC cells was evaluated by the CV staining assay as previously described [38 (link)]. Briefly, 3 × 10³ cells/well were seeded in 200 μL of complete medium in 96-well plates and incubated for 24 h at 37 °C under 5% CO₂ atmosphere. After incubation, the culture medium was changed and cells were exposed to different MnBuOE concentrations (0.5–200 μM) for 72 h in order to assess a concentration–response profile. Cisplatin (50 μM) was used as a positive control for both cell lines. After the 72 h incubation period, PBS was used to wash the cells in order to remove non-adherent cells. The viable cells were fixed with ice-cooled EtOH and stained with 0.1% CV for 15 and 5 min, respectively. The excess of the dye was then removed with tap water and stained cells were resuspended with a solution of 96% EtOH/1% acetic acid. Absorbance was measured at 595 nm with a SPECTROstar OMEGA microplate reader (BMG Labtech, Offenburg, Germany). Absorbance values of the control cells were defined as 100% cell viability. Five independent experiments were performed and six replicates were used for each condition in each independent experiment. The Motic AE2000 Inverted Phase Contrast Microscope was used to perform image acquisition with a 40× objective.