Quantifying Nasopharyngeal Neutrophils in Pups

Neutrophils present in the nasopharynx were quantified as previously described (18 (link)). Briefly, nasal lavage samples from individual pups were pelleted by centrifugation at 500 × g for 5 min. Samples were resuspended in 50 μl ACK lysis buffer (Thermo Fisher Scientific) and incubated for 5 min at room temperature. Afterwards, 200 μl of PBS was added to the samples, and cells were pelleted as described above and resuspended in PBS containing 1% bovine serum albumin (BSA). Samples were stained with a LIVE/DEAD Fixable Aqua dead cell stain kit (Invitrogen, Thermo Fisher Scientific). Next, samples were blocked with a 1:200 dilution of a rat anti-mouse CD16/32 (clone 93; BioLegend). Cells were stained for 30 min at 4°C with fluorophore-conjugated antibodies (diluted 1:150) against the following surface markers: CD11b-V450 (BD), Ly6G-peridinin chlorophyll protein (PerCP)-Cy5.5 (BD), and CD45-allophycocyanin (APC)-Cy7 (BD). Samples were run on a BD LSR II flow cytometer and analyzed with BD FACSDiva software.