Microplate spectrophotometer

Cell viability without virus: To assess cell viability, CellTiter 96^®AQueous One Solution (Promega, Madison, WI) was used. Briefly, Vero cells were seeded into a 96-well plate at a cell density of 5 × 10⁴ per well and allowed to adhere for 24 h, followed by treatment with serially diluted selected LA for 24 h. Next, 100 μl CellTiter 96^®AQueous One Solution was added according to the manufacturer’s protocols. The absorbance was measured using a microplate spectrophotometer (Molecular Devices, San Jose, CA). Results are expressed as a % of LA-free control (mean + / − SD, n = 10).
Cell viability with virus: To assess cell viability, Vero cells were seeded into a 96-well plate at a cell density of 5 × 10⁴ per well and allowed to adhere for 24 h, followed by treatment with serially diluted selected LA and HCoV-OC43, yielding an MOI of 0.1. The cell viability was assessed using CellTiter 96^®AQueous One Solution (Promega, Madison, WI) after 24 h, following the manufacturer’s protocols. The absorbance measurements were done using a microplate spectrophotometer (Molecular Devices, San Jose, CA). Results are presented as a % of LA-free control (mean + / − SD, n = 10).

pNPP was used to test the inhibitory function of PTP1B. In a plate with or without a sample, a recombinant PTP1B enzyme (0.5 units diluted in PTP1B reaction buffer) was added. The plate was pre-incubated for 10 minutes at 37 °C before adding the substrate (2 mMpNPP). The enzymatic reaction was stopped by adding 10 M NaOH after 15 minutes of incubation at 37 °C. A microplate spectrophotometer was used to calculate the absorbance at 405 nm (Molecular Devices, Sunnyvale, CA, USA). The reference compound was ursolic acid [25 (link)]. The results were expressed as mean ± S.E.M. of three independent experiments performed in duplicate.