Wst 1 assay

Cell proliferation was measured by water-soluble tetrazolium (WST)-1 assay (Roche). Mouse keratinocytes were cultured at a density of 5.0 × 10⁴ cells/well in a 96-well microplate. At 0, 1, 2, and 4 days after seeding, the WST-1 reagent was added to the cultures and cells were incubated for 4 h. For cytotoxic assay, the cells were seeded at a density of 3.0 × 10⁵ cells/well in a 24-well microplate and incubated for 24 h. The cells were exposed for 24 h to DMBA (Sigma-Aldrich) or dimethyl sulfoxide (DMSO, Wako, Osaka, Japan) as a control, or exposed for 48 h to cisplatin (CDDP, Bristol-Myers Squibb, New York, NY) or PBS as a control. WST-1 reagents were added to the cultures and cells were incubated for 4 h before absorbance measurement. Absorbance at 450 nm and 650 nm was measured using a microplate reader, Powerscan 4 (DS Pharma Biomedical, Osaka, Japan). The measured absorbance (A450–A650) is directly proportional to the number of living cells. Note that the cells were pretreated with 10 ng/ml TGF-β1 (R&D Systems, Minneapolis, MN), 10 ng/ml SB431542 (TGFBRI inhibitor, Selleck Chemicals, Houston, TX) or PBS as a control for 24 h before incubation with DMBA.

WST-1 assay (Roche, Basel, Switzerland) was used to monitor cell proliferation (Chou et al., 2014 (link)); cells were trypsinized and resuspended in culture medium, then plated at 5 × 10³ cells per well in 96-well plates and incubated overnight. After plant extracts treatment for 48 h, the cells were incubated with 10μl WST-1 reagent for 2 h. The cell viability was quantified by multi-well spectrophotometry (Anthos, Biochrom, Cambridge, UK). The absorbance at 450 nm was monitored, and the reference wavelength was set at 620 nm.

Cell proliferation was assessed by Wst-1 assay following the manufacturer's instructions (Roche) and performed at the same time and with the same preparation of cells as the one used for the invasion assay. Briefly, 3 × 10⁴ SKOV3 cells were seeded in 96-well plates and incubated in 100 μl of medium for 24h at 37°C in a CO₂ (5%) incubator. The incubation culture medium was then removed and replaced by 100 μl of medium containing 5% FBS (as control) or 5% ascites for 48 hours before being replaced with medium containing 10% Wst-1 reagent for 30 minutes. Microplate was then read at 450 nm.

To assess the viability of A549 cells, a WST-1 assay (Roche, Mannheim, Germany) was performed according to the manufacturer's instructions. A549 cells were seeded on 96-well plates (1.0 × 10⁴ cells/well) and incubated at 37°C for 12 h. The cells received C-ion irradiation (0, 2 or 8 Gy) or continuous exposure to Y-27632 (0.01, 0.1, 1, 10 or 50 µM). After incubating for 24, 48, 72 or 120 h, 20 µl of WST-1 reagent was applied to each well. After incubating for an additional 3 h at 37°C, absorption of 450 nm light was measured using a Multiskan FC (Thermo, Helsinki, Finland).

Cell viability was evaluated by using the water soluble tetrazolium salts (WST-1) assay (Roche, Mannheim, Germany, 5%) or the CellTiter-Glo Luminescent viability assay (Promega, USA) following manufacturer’s instructions.

Cytotoxicity of selected compounds was evaluated in MRC5 cells using MTT assay. Briefly, cells were treated with compounds for 48 h. Culture media was replaced and cells were incubated with MTT (1.2 mM, Life Technologies, Eugene, OR, USA) for 2 h. Formazan crystals were dissolved by addition of acidic isopropanol and cell viability was measured by a spectrophotometer (Bio-Rad, Hercules, CA, USA). Cytotoxicity of compounds on Vero E6 cells was measured using several cytotoxic assays, including a WST-1 assay (Roche, Basel, Switzerland). The cytotoxic concentration 50 (CC50) is the concentration of compound that reduced cell viability by 50%. This was calculated using four-parameter logistic regression (IC50 Toolkit IC50.org).
Selectivity index (SI) was calculated by dividing CC50 by EC50 for each compound and virus tested. The selectivity index is an indicator that measures the window between cytotoxicity and antiviral activity. A high selectivity index reflects a low toxicity at concentrations that show an effective antiviral activity. It also indicates that there is an important range of concentrations showing an effective antiviral effect with minimal toxicity. Conversely, a low selectivity index would indicate an important toxicity for concentrations with effective antiviral activity.