Rat anti mouse cd16 32 clone 93

The right lung lobes were finely diced and incubated with collagenase IV (2 mg/mL) at 37 °C in RPMI media containing 5% fetal bovine serum (FBS) for 30 min, with gentle shaking every 5 min. The lung digests were filtered through a 70 μm cell strainer, washed, centrifuged, and cell pellets resuspended in 100 μL of Ca⁺⁺/Mg⁺⁺ free PBS containing 2% FBS and 1 mM EDTA. Cells were then treated with 0.25 μg rat anti-mouse CD16/32 (clone 93; BioLegend, San Diego, CA) for 5 min at room temperature to block nonspecific binding. An EasySep PE Positive Selection Kit (Stem Cell Technologies) was used to isolate F4/80⁺ macrophages from lung digests following the manufacturer’s directions. Briefly, the filtered lung digest was incubated with PE conjugated anti-F4/80 antibody (1.5 μg/mL) for 15 min at room temperature. A Selection Cocktail (100 μL/mL) was added to the cell suspension followed by Magnetic Particles (50 μL/mL) and incubation for 10 min at room temperature. The samples were then diluted with PBS containing 2% FBS and 1 mM EDTA and F4/80⁺ cells isolated by magnetic separation.

To quantify neutrophils, we pelleted nasal lavage samples from individual pups at 500 × g for 5 min and resuspended them in PBS containing 1% bovine serum albumin (BSA). 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 BD LSR II flow cytometer and analyzed with BD FACSDiva software.