Pe conjugated anti siglec f

The right apical lobes were fixed in 4% paraformaldehyde/PBS overnight before paraffin embedding. Tissue sections were collected and analyzed for the level of mucus in airways by PAS (Periodic acid Schiff) staining. For lymphocyte assays, lungs were cut into small pieces and digested for 20 min at 37 °C with collagenase (20 μg/mL, Sigma Aldrich), Dispase II (1 mg/mL, Roche) and DNase I (25 μg/mL, NEB). After enzymatic digestion, tissues were further dissociated by mechanical passage through a wire mesh. Cell pellets were collected by centrifugation. Cells were re-suspended in red blood cell lysis buffer (Sigma Aldrich) for 5 minutes, washed with PBS, stained with antibodies, and analyzed on a BD FACS Aria II flow cytometer. Antibodies included APC-conjugated anti-CD11c (eBioscience), PE-conjugated anti-Siglec-F (BD Bioscience), and PE-Cy7-conjugated anti-CD45 (Biolegend). All antibodies were used at 1:100. Data were analyzed using FlowJo software (Tree Star, Ashland, OR).

For flow cytometry analysis, cell suspensions were incubated in FACS staining buffer (PBS containing 1% BSA) and subsequently stained for 30 min at 4 °C with an optimized concentration of antibodies (BD Bioscience, Franklin Lakes, NJ, USA): PE-conjugated anti-CD3, PerCPCy5.5-conjugated anti-CD8, PE Cy7-conjugated anti-CD4, PerCPCy5.5-conjugated anti-CD45, APC-conjugated anti-CD11c and PE-conjugated anti-SiglecF to determine cell types in the BAL. Cells were acquired on an LSRII flow cytometer (BD Bioscience) and the data were analyzed using the FlowJo software (v 7.6.5; Ashland, OR, USA). Based on surface marker expression, six different cell types were identified: CD45⁺ (total leukocytes), CD45⁺SiglecF⁺CD11c^low (eosinophils), CD45⁺SiglecF⁺CD11c^high (alveolar macrophages), CD45⁺CD3⁺ (total T cells), CD4 T cells (CD45⁺CD3⁺CD4⁺) and CD8 T cells (CD45⁺CD3⁺CD8⁺).

Leukocytes from the lungs were enumerated using flow cytometry. Lungs were excised from mice, diced and digested with 40 μg/ml Liberase (Roche Diagnostics) in Iscove’s modified Dulbecco’s medium (IMDM, Sigma) and then passed through a 70-μm cell strainer (BD Bioscience) and washed with IMDM with 5% FCS. RBCs were lysed for 5 min in RBC lysis buffer (Sigma). Cell counts were performed on a Brightline hemocytometer (Hausser Scientific) with trypan blue exclusion. Cells were seeded in 96-well U-bottom plates and incubated with Fc block (BD), followed by staining with Zombie UV™ Fixable Viability kit (Biolegend) and fluorochrome-labelled antibodies (Biolegend unless otherwise specified): BUV395-cojugated anti-CD8 (BD), BV785-conjugated anti-I-A/I-E, BV711-conjugated anti-CD64, BV650-conjugated anti-CD11c, BV510-conjugated anti-CD3, BV421-conjugated anti-NK1.1, PerCpCy5.5-conjugated anti-Ly6C, FITC-conjugated anti-CD4, PECy7-conjugated anti-CD11b, PE-Texas Red-conjugated anti-F4/80, PE-conjugated anti-Siglec F, APCCy7-conjugated anti-CD19, AF700-conjugated anti-Ly6G, AF467-conjugated anti-MRP14 (BD). Cells were washed with PBS and assessed using Fortessa X20 (BD). Analysis was performed on FlowJo (Treestar).