| Assay Method Information | |
| | Inhibition Assay of HCoV-OC43 |
| Description: | Human colon adenocarcinoma cell line HCT-8 (ATCC@ CCL-244, American Type Culture Collection, Manassas, VA, USA) was obtained from American Type Culture Collection (“ATCC”). It was established as stock at early passage to ensure cell line-specific characteristics. HCoV-OC43 viruses (ATCC@VR1558™, American Type Culture Collection, Manassas, VA, USA) were grown and propagated in HCT-8 cells cultured with DMEM and 2% FBS (Biological Industries, Kibbutz, Israel). The cells were seeded in a 96-well plate and then cultured in a DMEM medium containing 2% FBS. Subsequently, they were pretreated with one of Compounds 1-6 at one of five predetermined concentrations in a 5-fold dilution for 1 hour prior to HCoV-OC43 virus infection at an MOI of 0.05. The resultant supernatant at the 72 d.p.i. were subjected to an end-point assay and a TCID50 determination after 6 days to measure viral-yield inhibition. The cells (72 d.p.i.) were fixed with 80% acetone and were analyzed by an IFA assay using an antibody against HCoV-OC43 N protein. EC50 were determined accordingly. The viabilities of HCT-8 cells were also studied using CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit (“MTS”) (Promega, Madison, WI, USA). CC50 values were calculated. In addition, to demonstrate the cytopathic effect of HCT-8 infected by HCoV-OC43 at an MOI of 0.05, the cells thus treated were stained with crystal violet after fixation at 6 d.p.i. |
| Affinity data for this assay | |
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