Cell Viability Assay Protocol Using XTT

Cell viability was assessed using the Cell Proliferation kit II (XTT) (Roche Diagnostics, Merck, Darmstadt, Germany) as previously described [59 (link)]. In this assay, the tetrazolium salt 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) was cleaved by viable cells in order to form an orange formazan dye that is then quantified photometrically at 450 nm. Briefly, the cells (4 × 10⁵ cells/mL) were cultured in 96-well plates for 24 h. The culture medium was then replaced by medium containing serial dilutions of the 3CLpro inhibitors, and the cells were incubated for 24, 48, 72, and 96 h. Then, XTT was added to each well and the plates were incubated for 2 h. Optical density was measured at 450 nm (reference wavelength—650 nm) using a Multiskan GO plate reader (Thermo Scientific Instruments, Waltham, MA, USA). For the quantifications, the background levels of media without cultured cells were subtracted.