| Assay Method Information | |
| | MERS-CoV nLUC in Calu-3 |
| Description: | At 48 hours prior to infection, Calu-3 2B4 cells were plated in a 96-well black-walled clear bottom plate at 5x104 cells/well. A 10 mM stock of NHC was serially diluted in 100% DMSO in 3-fold increments to obtain a ten-point dilution series. MERS-nLUC was diluted in DMEM supplemented with 10% FBS, and 1% Antibiotic-Antimycotic to achieve a multiplicity of infection (MOI) of 0.08. Cells were infected and concurrently treated with NHC in triplicate per drug dilution for 1hr, after which viral inoculum was aspirated, cultures were rinsed once and fresh medium containing drug or vehicle was added. At 48 hours post infection, nanoluciferase expression as a surrogate for virus replication was quantitated on a Spectramax plate reader (Molecular Devices) according to the manufacturer s instructions (Promega, NanoGlo). For the 100% inhibition control, diluted MERS-nLUC was exposed to short-wave UV light (UVP, LLC) for 6 min to inhibit the ability of the virus to replicate. For the 0% inhibition control, cells were infected in the presence of vehicle only. DMSO was kept constant in all conditions at 0.05%. Values from triplicate wells per condition were averaged and compared to controls to generate a percent inhibition value for each drug dilution. The IC50 value was defined as the concentration at which there was a 50% decrease in luciferase expression. Data were analyzed using GraphPad Prism 8.0. The IC50 values were calculated by non-linear regression analysis using the dose-response (variable slope) equation (four parameter logistic equation): Y = Bottom + (Top-Bottom)/(1+10^((LogIC50-X)*HillSlope)). |
| Affinity data for this assay | |
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