One glo buffer

HEp-2 cells seeded in 12-well plates were infected with recRSV-fireSMASh in the absence or presence of L1502Q, H1632Q, or Y1631H substitution in L, at a multiplicity of infection of 0.1 in three independent replicates. Cells were scrapped and harvested every 12 hours for 4 days following infection. After virus release through freeze-thaw and clarification, viral titers were determined through TCID₅₀ titration with firefly luciferase bioluminescence as the readout, using One-GLO buffer (Promega, catalog no. E6130) and a Synergy H1 (BioTek) plate reader.

Compound stocks were prepared in dimethyl sulfoxide (DMSO) and, upon dilution in cell culture medium, reached in all wells a final DMSO concentration of 0.1%. For luciferase-based dose-response assays, HEp-2 or primary HAE cells were seeded a day before to reach 50% confluence in 96-well white plates. Threefold serial dilutions of compounds were prepared in triplicate using an automated Nimbus liquid handler (Hamilton) and transferred to the cells. Immediately after addition of compound, cells were infected with recRSV-fireSMASh. Each plate contained four wells each of positive and negative control (medium containing 100 μM cycloheximide or vehicle, respectively). Luciferase activities were determined at 48 hours after transfection using One-GLO buffer (Promega, catalog no. E6130) and a Synergy H1 (BioTek) plate reader. Normalized luciferase activities were analyzed with the following formula: % inhibition = (Signal_sample – Signal_min)/(Signal_max – Signal_min) × 100, and dose-response curves were further analyzed by normalized nonlinear regression with variable slope to determine EC₅₀ and 95% confidence intervals (CIs) with Prism 9.0.1 for MacOS (GraphPad).