HEK293 Cell Cytotoxicity Assay Using Resazurin

The HEK293 cell cytotoxicity assay was carried out as described previously [24 (link)], modified slightly with the use of resazurin. Briefly, HEK293 cells (ATCC) were added to sterile polystyrene black/clear bottomed 384-well plates (Greiner, USA) at 4 × 10³ cells per well, in 55 µL of high glucose DMEM supplemented with 10% FBS. Following 24 h incubation at 37 °C in 5% CO₂, under humidified growth conditions, 5 µL of pre-diluted compound was added with a MiniTrak (PerkinElmer, Waltham, MA, USA). Final assay concentrations of Pathogen Box compounds ranged from 79.4 µM to 4.0 × 10⁻³ µM. Plates were incubated for 68 h, then 10 µL of 490 µM of resazurin was added to give a final concentration of 70 µM. Plates were incubated for a further 4 h under normal growth conditions giving a total compound incubation of 72 h. Fluorescence (Ex/Em 535/590 nm) was then determined on an EnSight plate reader (PerkinElmer, Waltham, MA, USA). Positive and negative controls used were as the same as those in the T.b. brucei assay; 4 µM puromycin was a positive control and 0.4% DMSO served as a negative control.