2.2. MTT Assay

The effect of salamandrin-I treatment on HL-60 growth (proliferation and/or viability) was assessed by measuring the 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma Aldrich, St. Louis, MO, USA) dye absorbance of the cells [26]. For this, a total of 5 × 10⁴ cells were seeded into 96-well plates containing 100 µL of the culture medium. The cells were treated with different concentrations of salamandrin-I (0, 1, 5, 10, 25, 50, and 100 µM) for 48 h. After this period, the cells were incubated with 10 µL of MTT (5 mg/mL) for 4 h. The supernatant was removed after centrifuging of the plates at 400× g for 10 min. The reaction product was solubilized by the addition of DMSO (Sigma Aldrich, St. Louis, MO, USA). The optical density was read on a Multiskan FC Microplate Photometer (Thermo Scientific, Waltham, MA, USA) at 570 nm. The IC₂₅ and IC₅₀ values were calculated using nonlinear regression analysis in GraphPad Prism 9 (GraphPad Software, Inc., San Diego, CA, USA).