Siglecf percp cy5

Cells were prestained with a 1:100 dilution of Zombie Aqua (Fixable Viability Dye; BioLegend, London, UK) for 20 minutes at 4°C in the dark. After 10 minutes, an equal volume of a 1:100 dilution of TruStain FcX PLUS (anti-mouse CD16/32 antibody) and True-Stain Monocyte Blocker (BioLegend) was added. After a washing step, cells were stained with CD3e-PerCP-Cy5.5, CD19-PerCP-Cy5.5 (eBioscience, Thermo Fisher Scientific), NK1.1-PerCP-Cy5.5, CD103-PerCP-Cy5.5, F4/80-FITC, Ly6G-BV785, Ly6C-BV650 (BioLegend), SiglecF-PerCP-Cy5.5, CD45-APC-Cy7, CD11b-PE-Cy7 and Tim4-PE (BD Biosciences, Erembodegem, Belgium) for 20 minutes at 4°C in the dark. Cells were analyzed with a BD FACSAria Fusion flow cytometer (BD Biosciences) and FlowJo software (FlowJo LLC, BD Biosciences), and gated first as live CD45⁺ single cells. Subsequently, CD3e⁺, CD19⁺, NK1.1⁺, CD103⁺ and SiglecF⁺ cells were eliminated, and CD11b⁺Ly6C⁺Ly6G^- monocytes, CD11b⁺Ly6C^-F4/80⁺Tim4⁺ Kupffer cells (KCs) and CD11b⁺Ly6C^-F4/80⁺Tim4^- monocyte-derived macrophages (MoMfs) were gated.

The number of ILCs producing various cytokines in the spleen was measured using flow cytometry (FCM). Analysis was performed on 5–8 animals per group. The spleen was crushed in a petri dish, and the cells were filtered through a mesh and incubated with ACK Lysing Buffer (Thermo Fisher Scientific, Waltham, MA, USA) to lyse the red blood cells. Mononuclear cells were isolated and purified by density gradient centrifugation. The LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) was used to exclude apoptotic and necrotic cells. The cultured mononuclear cells were stained with surface antibodies: CD45R-PerCp-Cy5.5, CD3-PerCp-Cy5.5, CD4-PerCp-Cy5.5, FceRI-PerCp-Cy5.5, CD8a-PerCp-Cy5.5, Gr-1-PerCp-Cy5.5, Siglec-F-PerCp-Cy5.5 (BD Biosciences, Franklin Lakes, NJ, USA) in cell surface staining buffer containing 0.1 M phosphate-buffered saline and 2% FCS (Biowest, Nuaillé, France) [61 (link),62 (link)], and then stained with IFNγ-Brilliant Violet 605, TNFα-APC, IL-4-Brilliant Violet 421, IL-13-FITC, IL-17A-APC-Cy7, and IL-17F-PE antibodies (BD Biosciences). The expression patterns of inflammatory cytokines were analyzed using a BD Lyric flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (v10.9.0) (Tree Star Inc., Ashland, OR, USA).