Infinite m1000pro multiplate reader

Cells were seeded into 96-well flat-bottom plates at 5 × 10³ cells/well and incubated overnight. The medium was removed and replaced with 90 µL serum-free medium, 10 µL of 10x concentrated peptide solutions in PBS were added. PBS was added as blank and 0.1% (v/v) Triton X-100 was used to establish 100% of cell death. After 2 h incubation at 37 °C, 10 µL of filtered 0.05% (w/v) resazurin solution was added to each well [70 (link)]. Resazurin is converted to the pink and fluorescent compound resorufin by viable cells [78 ]. After incubation overnight, the fluorescence intensity (λ_ex = 565 nm and λ_em = 584 nm) was measured with the Tecan infinite M1000Pro multiplate reader (Männedorf, Switzerland).
The selectivity was calculated through the activity-toxicity index (ATI) [46 (link)]. ATI = MHC/MCC50, with MHC being the minimal concentration necessary to induce 10% or 50% cell death in human red blood cells and MCC50 the median of cytotoxic concentrations (CC50) of all tested melanoma cell lines.

Cell viability, cytotoxicity, and apoptosis of infected HepG2 cells in response to different concentrations of YK51 compound were analyzed using ApoTox-Glo™ Triplex Assay kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. Briefly, HepG2 cells were seeded overnight in 96-well optical bottom plate (Nunc™, Rochester, NY, USA) at a density of 1 × 10⁴ cells per well and then inoculated with DENV2 at the optimized MOI of 15. The viral inoculum was removed after 2 h of infection and the cells were further incubated in DMEM maintenance medium (2% FBS) containing 2, 5, and 10 µg/ml of YK51 compound respectively for 24 and 48 h at 37 °C . Control cells were cultured in parallel and processed in the same manner. Thereafter, viability/cytotoxicity reagent was added to each well of 96-well plate and incubated at 37 °C for 30–180 min. Fluorescence was measured at 400 nm excitation/505 nm emission for viability and 485 nm excitation/520 nm emission for cytotoxicity using Infinite^® M1000 PRO multiplate reader (Tecan, Männedorf, Switzerland). Caspase-Glo 3/7 reagent was then added to each well and incubated at room temperature for 30–180 min. Luminescence was measured at 1.0 s using luminescence protocol. Cell viability and apoptosis of DENV2-infected HepG2 cells at various MOI values and time points were also evaluated using the same assay.