Cd45 apc

Liver leukocytes were incubated with Fc-blocker (BD Biosciences, Franklin Lakes, NJ, USA) to prevent non-specific binding and labeled with APC-CD45 (Thermo Fisher, Waltham, MA USA) and PerCP-eFluor710-Ly-6G (Thermo Fisher). To exclude non-immune cells and neutrophils, CD45⁺Ly6G⁻ mononuclear cells (MNCs) were gated. FITC-F4/80 and PE-CD11b (Thermo Fisher) were used to define each macrophage population. For the analysis of PE-Ly6c, PE-CD36 Ab (Thermo Fisher), and PE-MerTK (R&D Systems, Minneapolis, MN, USA), PerCP-Cy5.5-CD11b was selected to identify each cell population. The cells with the highest expression of CD11b in addition to high FS and SS were defined as neutrophils and were excluded from the analysis. In the case of CLEC4F (C-type lectin domain family 4 F) staining, cells with or without permeabilization of cell membranes were labeled with a polyclonal goat anti-mouse CLEC4F antibody (R&D Systems) and an FITC-donkey polyclonal anti-goat IgG antibody (R&D Systems). PE-F4/80 (Thermo Fisher) was used for macrophage detection. Similarly, after permeabilization of the cell membrane (BD Biosciences), intracellular TNF-α was labeled with FITC-anti-TNF (Thermo Fisher). The cells labeled with fluorescent antibodies were analyzed using a Novocyte flow cytometer (Agilent, Santa Clara, CA, USA).

2x10⁶ lung or spleen cells were resuspended in PBS and stained with Fixable Viability Dye eFluor 450 (eBiosciences) for 20 minutes, then washed and resuspended in PBS containing 1% heat-inactivated FBS, 1 mM EDTA, 10 μg/mL CD16/32 and 0.1% sodium azide. Cells were stained for 30 minutes with appropriate antibodies, fixed in BD Stabilizing Fix, and stored at 4°C until analysis on an LSR II (Becton Dickenson). Antibodies used to identify monocytes, macrophages, neutrophils, and DCs were as follows: neutrophils (CD11c^-CD11b⁺SiglecF^loLy6G⁺), monocytes (CD11c^-CD11b⁺MHCII^-CD64⁺), CD103⁺ cDCs (MHCII⁺CD11c⁺CD11b^-CD24⁺CD103⁺), CD11b⁺ cDCs (MHCII⁺CD11c⁺CD11b⁺CD103^-), and pDCs (CD11c^+/-CD11b^-CD103^-B220⁺). APC-CD45, PE-Cy7-B220, and APC.780-MHCII (eBioscience), Alexa700-CD11c and PE-Cy7-SiglecF (Miltenyi Biotec), BV510-CD103, BV510-Ly6G, BV605-CD11b and PE-CD24 (Becton Dickenson), and BV711-CD64 and BV421-CD19 (BioLegend). The antibodies used to identify T cells and NK cells were as follows: APC-CD4, PerCp-Cy5.5-CD8, PE-Cy7-CD69, and PE-CD69 (Becton Dickenson) and PerCp-Cy5.5-NK1.1, FITC-CD3ε, and APC-gamma delta T cell receptor (γδ TCR) (eBiosciences). Flow cytometry data was analyzed using FlowJo version 7.6.5.