Black optical bottom plates

Neutralization assays were performed as previously described [37 (link)]. Briefly, HEp-2 cells were seeded at a density of 2.4 × 10⁴ cells/well in 384-well black optical bottom plates from ThermoFisher (Cat#142761). Serial 2-fold dilutions were performed on samples, either NHP sera or monoclonal antibodies, in a final volume of 40 µL. The starting dilution was 1:10 for sera and 1:10, 1:200, or 1:500 for the monoclonal antibodies. An equal volume of recombinant mKate-RSV subtype A (strain A2) was added to the diluted samples and incubated at 37 °C for 1 h [38 (link)]. After incubation, 50 µL of each diluted sample-virus mixture was added to the HEp-2 cells and incubated for 37 °C for 24 h. Fluorescence endpoints were recorded at 24 h using excitation at 588 nm and emission at 635 nm (SpectraMax M2e, Molecular Devices, San Jose, CA, USA). The IC₅₀ titer for each sample was determined using a four-parameter non-linear regression curve fit with GraphPad Prism version 7 (GraphPad Software Inc., San Diego, CA, USA), which was then used to calculate the IC₅₀ concentration of each monoclonal antibody or NHP sera.

Oxidative stress in live cultures was assessed with CellROX Green (Life Technologies, #C10444), which remains non‐fluorescent until oxidized by intracellular ROS. The fluorescent signal intensity is proportional to the levels of intracellular free radicals. Cultures were plated in black optical bottom plates (Thermo Fisher, #NUN165305) and incubated in CellROX Green for 30 min after treatment, followed by 2× PBS washes prior to imaging. Images were captured on an Incucyte Zoom in phase contrast and green at 20× magnification (0.61 μm/pixel resolution) with three images per well. Mean Green Intensity was normalized to culture confluence for each image.