RSV plaque forming assay

The RSV plaque forming assay was performed as described (Z. Wen, D. Casimiro, and D. DiStefano. Unpublished conference presentation). Briefly, HEp2 cells at concentration of 1.2×10⁶ cells / ml in EMEM medium (EMEM supplemented with 2% FBS and 2mM glutamine) were seeded at 50 μl per well into round-bottom 96 well plates. Seventy-five microliters of viral inoculum or 2-fold serially diluted samples were added to each well. Samples were mixed well with cells and then incubated for 1 hour at 37°C before the plate was centrifuged at 300×g for 10 minutes for better host cell settlement. One hundred fifty microliters of EMEM medium supplemented with 1% methycellulose (Sigma-Aldrich) was overlaid in each well to prevent viral spread to neighboring cells. Plates were incubated at 37°C with 5% CO2 for 3 days. Cells were then washed with phosphate buffered saline (PBS) and fixed with ice-cold 80% acetone (Sigma-Aldrich) for 10–20 minutes. The plate was then allowed to dry for 20 min before washing with PBS supplemented with 0.05% tween (PBST). Fixed cells were stained with an in-house mouse anti-RSV F antibody (1.25 μg/ml) and an anti-nucleoprotein monoclonal antibody (1.25 μg/ml). These two antibodies were incubated with the fixed cells for 1 hour before anti-mouse IgG Alex488 conjugated secondary antibody (Invitrogen) was added (1:500 diluted). Unbound secondary antibody was washed off after 1 hour of incubation. Plates were analyzed for image capturing and automated counting by EnSight imager reader 2.02 (PerkinElmer).