Methyl cellulose overlay media

Human RSV strain A2 (ATCC, Manassas, VA; #VR-1540) was infected at a multiplicity of 0.1 into Hep2 cells. The virus was allowed to grow for 5 days at 37°C in a 5% CO₂ atmosphere. The infected Hep2 monolayers were collected and the virus was released by sonication. Cell debris was removed by centrifugation at 2500 g for 5 minutes at 4°C. Virus was collected by centrifuging the supernatant for 2 hours at 22000×g at 4°C. Virus were suspended in culture media and snap frozen and maintained at −80°C. Infectious virus titers were determined on Hep2 cells by performing serial dilutions of the RSV stocks and counting infected cells stained for indirect immunofluorescence with an RSV F-specific monoclonal antibody (Abcam, Cambridge, MA). Additionally, plaque assays were performed as previously described [25] on Hep2 cells using methyl cellulose overlay media (R&D Systems) and staining with 0.5 mg/ml thiazolyl blue tetrazolium bromide (MTT; Sigma Aldrich) solution in PBS for 3 hours at 37°C. Non-infected Hep2 cell cultures were processed in the same manner as RSV infected cells and the resulting sample collection was used as a mock control.