Pierce renilla luciferase glow assay kit

Vero cells (ATCC^® CCL-81™) grown in clear
384-well plates were exposed to varying concentrations of ARN-75039 and
ARN-75041 and pseudotyped virus in assay media consisting of 50% Opti-MEM, 50%
Minimum Essential Medium (MEM; Cytiva), 1% FBS, 1% MEM non-essential amino
acids, Penicillin-Streptomycin (100 units/mL Penicillin/100 μg/mL
Streptomycin), 1 mM Sodium Pyruvate and 2 mM L-Glutamine. Each of the
pseudotyped virus preparations generated was diluted to give a similar
luminescence signal to background value of > 200. The assay volume was 50
μL/well with the final DMSO concentration at or below 1%. Control wells
were treated with assay media and 1% DMSO. Cells were incubated for 24 h at
37°C and 5% CO₂ and assessed for luciferase activity according
to the Pierce Renilla Luciferase Glow Assay Kit (Thermo Fisher
Scientific). Cells were lysed with 20 μL of lysis buffer and 5 μL
of cell lysate was transferred to an opaque white plate and mixed with 12.5
μL of coelenterazine diluted in buffer. The mixture was incubated at room
temperature for 10 min on a shaker before luminescence detection (Beckman
Coulter DTX 880 multimode detector; Beckman Coulter). Luminescence signals for
compound-treated and control wells were used to determine the antiviral activity
(percent inhibition of the luciferase signal) for each compound.