| Assay Method Information | |
| | Antiviral assay |
| Description: | VeroE6 cells and TMPRSS2-overexpressing VeroE6 (VeroE6TMPRSS2) cells were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan). VeroE6 cells were maintained in Dulbecco s modified Eagle s medium (d-MEM) supplemented with 10% fetal bovine serum (FCS), 100 μg/ml of penicillin, and 100 μg/ml of streptomycin. VeroE6TMPRSS2 cells were maintained in d-MEM as reported (ref.1) in the presence of 1 mg/ml of G418. SARS-CoV-2 strain JPN/TY/WK-521 (SARS-CoV-2WK-521) was obtained from the National Institute of Infectious Diseases (Tokyo, Japan).Antiviral assay was carried out as described recently (ref 1): Cells were seeded in a 96-well plate (2x104 cells/well) and incubated. After 24 h, virus was inoculated into cells at multiplicity of infection (MOI) of 0.05. After an additional 72 h, cell culture supernatants were harvested and viral RNA was extracted using a QIAamp viral RNA minikit (Qiagen, Hilden, Germany), and quantitative RT-PCR (RT-qPCR) was then performed using One Step PrimeScript III RT-qPCR mix (TaKaRa Bio, Shiga, Japan) following the instructions of the manufacturers. The primers and probe used for detecting SARS-CoV-2 envelope (6) were 5=-ACT TCT TTT TCT TGC TTT CGT GGT-3= (forward), 5=-GCA GCA GTA CGC ACA CAA TC-3= (reverse), and 5=-FAM-CTA GTT ACA CTA GCC ATC CTT ACT GC-black hole quencher 1 (BHQ1)-3= (probe). To determine the cytotoxicity of each compound, cells were seeded in a 96-well plate (2_104 cells/well). One day later, various concentrations of each compound were added, and cells were incubated for additional 3 days. The 50% cytotoxic concentrations (CC50) values were determined using the WST-8 assay and Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). |
| Affinity data for this assay | |
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