Anti f4 80 pe cy5

The SVF cells and splenocytes (2.5 × 10⁵ cells/sample) were incubated with Fc-blocker (anti-CD16/CD32; eBioscience, San Diego, CA) for 20 min and then stained with combinations of anti-CD11b PE-Cy7, anti-Ly-6G PE-Cy5, and anti-F4/80 PE-Cy5, anti-CD11c PE for 20 min. Heparin-treated whole blood cells (50 μL) were stained with anti-CD11b PE-Cy7, anti-Ly-6G PE-Cy5, anti-F4/80 PE-Cy5, or anti-CD11c PE (all from eBioscience). After 20-min incubation in the dark, whole blood cells were incubated with 600 μL Versa Lyse (Beckman Coulter, Tokyo, Japan) at room temperature in the dark. The SVF cells, splenocytes, and blood cells were washed with Staining buffer (BD Pharmingen). Finally, cells were resuspended in Staining buffer and analyzed using fluorescence-activated cell sorting analysis performed with Guava® EasyCyte™ 6HT flow cytometry system (Millipore, Long Beach, CA) and InCyte software (Millipore). A validation of the flow cytometric approach for the identification of total CD11b⁺ Ly-6G⁺ neutrophils is shown in Supplementary Figure S1.

The macrophages isolated via peritoneal lavage (as described above) were fixed with ice-cold formalin. Phenotyping was performed as previously described [23 (link)]. Briefly, ~1.5 × 10⁶ cells fixed-cells were placed into round-bottom polypropylene tubes, blocked with 5% BSA for 30 min on ice. The fixed cells were then mixed with the antibodies anti-F4/80-PE/Cy5 at 0.2 mg/mL (eBioScience), anti-CD11b-PE at 1.6 mg/mL (Abcam), and anti-Ly6c-PE/Cy7 at 0.2 mg/mL (Abcam). The cells were gated for CD11b⁺ F4/80⁺ Ly6c^{[low or high]+}.

Anti-CD11c-PE, anti-CD11b-Biotin (both eBioscience, San Diego, United States), and anti-F4/80 (clone CI:A3-1) and anti-Dectin-1 (clone 2A11), which were affinity-purified from hybridoma cell culture supernatants with protein G-Sepharose (Pharmacia, Uppsala, Sweden), were used for immunofluorescence. For FACS analysis anti-CD11c-Pacific Blue, anti-CD11b-APC, anti-CD11b-PE, anti-CD45-PE-Cy7, anti-F4/80-PE-Cy5 (all eBioscience, San Diego, United States), anti-Dectin-1-FITC (Serotec Oxford, United Kingdom) and unlabeled anti-CX3CR1 (Abcam, Cambridge, United Kingdom) were used. Live/Dead Fixable Near-IR stain fluorescence (Invitrogen, Landsmeer, Netherlands) was used to exclude dead cells.