The acetone was removed from cells, after which 96-well plates were left to dry in oven at 60°C for 30 min. Then, 100 μL of 0.4% (w/v) SRB in 1% acetic acid (v/v) was added to each well and incubated at room temperature for 30 min. Unbound INCB018424 ic50 SRB was removed by washing the plates five times with 1% acetic acid (v/v), and the plates were then left to dry in an oven. After drying

for 1 day, cell morphology was assessed under a microscope at 4 × 10 magnification (AXIOVERT10; Zeiss, Göttingen, Deutschland) and images were acquired. Fixed SRB in wells was solubilized with 100 μL of unbuffered Tris-base solution (10 mM), and plates were incubated at room temperature for 30 min. Absorbance in each well was read at 540 nm using a VERSAmax microplate reader (Molecular Devices, Palo Alto, CA, USA) and a reference absorbance of 620 nm. The antiviral activity of each test compound in CVB3- or EV71-infected

cells was calculated as a percentage of the corresponding untreated control. The antiviral activity of seven ginsenosides against HRV3 was determined using a Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega, Madison, Wisconsin, USA). The Cell Titer-Glo Reagent induces cell lysis and the generation of luminescence proportional to the amount of ATP present in cells. The resulting luminescence intensity is measured using a luminometer (Molecular Devices) according to the manufacturer’s instructions. Briefly, HeLa cells were seeded RG7204 supplier onto a 96-well culture plate, after which 0.09 mL of diluted HRV3 suspension containing CCID50 of the virus stock, and 0.01 mL culture medium supplemented with 20 mM MgCl2 and the appropriate concentration of ginsenosides, was added to the cells. The antiviral activity of each test material was determined using a concentration series of 0.1 μg/mL, 1 μg/mL, 10 μg/mL, and 100 μg/mL. Culture plates were incubated at 37°C in 5% CO2. After 48 h, 100 μL of Cell Titer-Glo reagent was added to each well, and the plate was incubated at room Adenylyl cyclase temperature for 10 min. The resulting luminescence was measured and the percentage cell viability was calculated as described

for the antiviral activity assays. Cell morphology was assessed as described for the SRB assay. To measure cytotoxicity, cells were seeded onto a 96-well culture plate at a density of 2 × 104 cells/well. The following day, the culture medium containing serially diluted compounds was added to the cells and incubated for 48 h, after which the culture medium was removed and cells were washed with PBS. The next step was conducted as described above for the antiviral activity assay. To calculate the CC50 values, the data were expressed as percentages relative to controls, and CC50 values were obtained from the resulting dose–response curves. Differences across more than three groups were analyzed using one-way analysis of variance (Graphpad PRISM, version 5.01, San Diego, CA, USA).