Mlx luminometer

The IL-6 level was determined by a chemiluminescent immunoassay using the Access Immunoassay Systems (Beckman Coulter, Pasadena, CA, USA). Levels of IL-10 were measured by a chemiluminescent immunoenzymatic assay using an MLX™ luminometer (Dynex Technologies, Chantilly, VA, USA). CRP was measured using an AU 5800 analyzer (Beckman Coulter). ESR was determined using the SEDI SYSTEM 1 analyzer (Becton Dickinson, New Jersey, USA).

Cells in 96-well plates were washed once with PBS prior to infections with the different viruses at the appropriate MOI in PBS supplemented with 1 mM Ca²⁺ and Mg²⁺, 0.3% bovine serum albumin (BSA), and 1% P/S. After 1 h at 37°C, the inoculum was removed, cells were washed once with PBS, and postinfection medium was added: DMEM supplemented with 0.1% FBS, 0.3% BSA, 20 nM HEPES, 1% P/S, and 1 μg/ml TPCK-treated trypsin, together with 6 μM Renilla luciferase substrate (EnduRen live-cell substrate; Promega). Where noted, the virus inoculum was preincubated with the indicated concentrations of monoclonal anti-H5 antibody (no. 200-301-976; Rockland) or polyclonal anti-H5 serum (no. 2705; BEI Resources) at 4°C for 1 h before infection of cells. Also where noted, the indicated concentrations of baloxavir acid (no. HY-109025A; MedChemExpress) or S20 fusion inhibitor (58 (link)) (ChemBridge) were added to the cells following infection. Real-time luminescence measurements were taken at various time points using an EnVision multilabel reader (Perkin Elmer) or a Dynex MLX luminometer (Dynex Technologies). All experiments included infection of multiple wells for technical replicates, and each experiment was independently performed at least 3 times. Mock-infected wells, otherwise treated identically, acted as negative controls for background luminescence from the reagents.

IAV strain A/WSN/33 (WSN) was grown on Madin-Darby canine kidney (MDCK) cells, and IAV reporter virus WSN-Ren was propagated on MDCK-HA cells overexpressing the HA protein of strain A/WSN/33 (20 (link), 36 (link)). Cells were infected at the indicated multiplicity of infection (MOI) in PBS for infection (PBSi) (Dulbecco’s phosphate-buffered saline [DPBS; Thermo Fisher Scientific] supplemented with 0.3% bovine serum albumin [BSA] [Sigma-Aldrich], 1 mM Ca²⁺ Mg²⁺, 100 units/mL penicillin, and 100 μg/mL streptomycin [Thermo Fisher Scientific]) for 1 h at 37°C or for 1 h on ice for synchronized infection. After removing the inoculum and washing the cells with DPBS, they were overlaid with postinfectious DMEM (piDMEM) (DMEM [Thermo Fisher Scientific] supplemented with 0.1% FBS [Thermo Fisher Scientific], 0.3% BSA [Sigma-Aldrich], 20 mM HEPES [Thermo Fisher Scientific], 100 units/mL penicillin, and 100 μg/mL streptomycin [Thermo Fisher Scientific] containing 1 μg/mL TPCK [tosylsulfonyl phenylalanyl chloromethyl ketone]-treated trypsin [Sigma-Aldrich]) for the indicated times. For WSN-Ren infections, piDMEM was supplemented with 6 μM Renilla luciferase substrate (EnduRen live-cell substrate; Promega) and real-time luminescence measurements were taken at the indicated time points using an EnVision multilabel reader (Perkin Elmer) or a Dynex MLx luminometer (Dynex Technologies).

IAV strain A/WSN/33 (WSN) was grown on Madin-Darby canine kidney (MDCK) cells, and IAV reporter virus WSN-Ren was propagated on MDCK-HA cells overexpressing the HA protein of strain A/WSN/33 (20 (link), 36 (link)). Cells were infected at the indicated multiplicity of infection (MOI) in PBS for infection (PBSi) (Dulbecco’s phosphate-buffered saline [DPBS; Thermo Fisher Scientific] supplemented with 0.3% bovine serum albumin [BSA] [Sigma-Aldrich], 1 mM Ca²⁺ Mg²⁺, 100 units/mL penicillin, and 100 μg/mL streptomycin [Thermo Fisher Scientific]) for 1 h at 37°C or for 1 h on ice for synchronized infection. After removing the inoculum and washing the cells with DPBS, they were overlaid with postinfectious DMEM (piDMEM) (DMEM [Thermo Fisher Scientific] supplemented with 0.1% FBS [Thermo Fisher Scientific], 0.3% BSA [Sigma-Aldrich], 20 mM HEPES [Thermo Fisher Scientific], 100 units/mL penicillin, and 100 μg/mL streptomycin [Thermo Fisher Scientific] containing 1 μg/mL TPCK [tosylsulfonyl phenylalanyl chloromethyl ketone]-treated trypsin [Sigma-Aldrich]) for the indicated times. For WSN-Ren infections, piDMEM was supplemented with 6 μM Renilla luciferase substrate (EnduRen live-cell substrate; Promega) and real-time luminescence measurements were taken at the indicated time points using an EnVision multilabel reader (Perkin Elmer) or a Dynex MLx luminometer (Dynex Technologies).