Anti mouse cd16 32 mab 93

Mouse BMDCs were dispensed into 12-well plates at a concentration of 5 × 10⁵ cells/well and incubated with rLdpA (1 μg/mL), chitin (50 μg/mL), rLdpA–chitin complex (rLdpA, 1 μg/mL; chitin, 50 μg/mL), or lipopolysaccharide (LPS, 10 ng/mL) (eBioscience, San Diego, CA, USA) for 16 h. Cells were suspended in FACS buffer (2% heat-inactivated FBS and 1 mM EDTA in PBS, pH 7.4). Fc receptors were blocked with anti-mouse CD16/32 mAb (93; eBioscience). Cells were stained with a mixture of fluorochrome-conjugated monoclonal antibodies (mAbs) to CD11c (N418; eBioscience), MHC class II (M5/114.15.2; Miltenyi Biotec), CD40 (FGK45.5; Miltenyi Biotec), CD80 (16-10A1; Miltenyi Biotec), and CD86 (PO3.3; Miltenyi Biotec) in FACS buffer. CD11c⁺ cells were gated, and MHC class II, CD40, CD80, and CD86 cell-surface expression were measured by flow cytometry (BD FACSVerse; BD Biosciences). Data were analyzed using FlowJo version 10 (BD Biosciences).

Airway contents were recovered by installation and retrieval of 0.7 mL of sterile PBS. The BAL fluid was centrifuged, and the cell pellet was resuspended in PBS. Cells were counted using a hemocytometer under a light microscope. To determine the differential leukocyte count, cells were resuspended in FACS buffer, and Fc receptors were blocked with anti-mouse CD16/32 mAb (93; eBioscience). Cells were stained with a mixture of fluorochrome-conjugated mAbs to CD11c (N418; eBioscience), CD11b (M1/70; eBioscience), Siglec F (E50-2440; BD Biosciences), and Ly-6G (1A8-Ly; eBioscience) in FACS buffer. After lysis of erythrocytes in RBC lysis buffer (eBioscience), cells were resuspended in FACS buffer. Samples were analyzed by flow cytometry (BD FACSVerse; BD Biosciences). Neutrophils were identified as CD11c⁻ Siglec F⁻ CD11b⁺ Ly-6G⁺ cells, eosinophils were identified as CD11c⁻ Siglec F⁺ cells and alveolar macrophages were identified as CD11c⁺ Siglec F⁺ cells. Data were analyzed using FlowJo version 10 (BD Biosciences).