Celltiter 96 aqueous non radioactive cell proliferation reagent

CPE reduction assays with Vero cells were performed by seeding cells at a density of 5,000 cells/well in 96-well clusters, 24 h before compound treatment and infection. The next day, 1.5-fold or 2-fold serial dilutions of the compound were added to the cells, followed by 500 PFU/well of ZIKV in a total volume of 150 µL. Each concentration was tested in quadruplicate and each assay plate contained the following controls: no cells, uninfected&untreated cells, infected&untreated cells and infected&solvent-treated cells. After 4 days, 30 µL/well of CellTiter 96® Aqueous Non-Radioactive Cell Proliferation reagent (Promega) was added. After a 3-h incubation, reactions were stopped and virus was inactivated by adding 30 µL of 37% formaldehyde, followed by measuring the absorption at 490 nm. Viability assays on uninfected cells were performed in parallel to determine the CC 50. Data were normalized to untreated uninfected cells and EC 50 and CC 50 values were calculated with Graph-Pad Prism 7 (see section 2.8).