Isolation and Flow Cytometry Analysis of Lung Cells

For flow cytometry analysis, lungs were collected at day 1 after RSV or mock infection and digested with collagenase, as previously described (24 (link), 25 (link), 27 (link), 28 (link)). Cells were passed through nylon mesh to get a single cell suspension, and incubated with anti-FcγRIII/FcγRII mAb (anti-mouse CD16/CD32; BD Biosciences, San Diego, CA, USA) to reduce nonspecific binding, for 30 min at 4°C. After washing, cells were stained with the following anti-mouse antibodies: anti-F4/80 APC, anti-CD11b PerCP-Cy5.5, and anti-Ly6G FITC (for neutrophils). After incubation for 30 min at 4°C with the antibodies, cells were washed and then fixed in 200 µl of 1% paraformaldehyde in PBS. All the antibodies were purchased from BD Pharmingen, San Jose, CA, USA, except anti-F4/80 which was obtained from ebioscience, San Diego, CA, USA. Corresponding isotype Abs were used as controls. Cells were acquired with a FACS Canto flow cytometer equipped with Cell Quest software (both from Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). Analysis was performed by using the FlowJo Software (Tree Star, NJ, USA).