Percp cyanine5.5 anti mouse cd45

The spleen, peritoneal macrophages (PMs), and polarized peripheral blood mononuclear cells (pPBMC) were harvested and lysed with red blood cell lysing buffer (420,301; Biolegend). Cells were incubated with an Fc receptor blocker CD16/32 (eBioscience, San Diego, CA, USA) to reduce nonspecific antibody binding prior to adding staining antibodies. For live/dead cell separation 7-AAD (Invitrogen, Carlsbad, CA, USA) staining was used. Cells were labeled with APC anti-mouse F4/80, PerCP-Cyanine5.5 anti-mouse CD45, FITC anti-mouse CD11b, Brilliant Violet 421 anti-mouse CD206, Brilliant Violet 650 anti-mouse CD80 (clone BM8, clone 30-F11, clone M1/70, clone C068C2, and clone 16-10A1 respectively; Biolegend). For intracellular labeling, cells were stimulated with lipopolysaccharide (LPS) (10 μg/mL; Sigma–Aldrich, St. Louis, MO, USA), 3 μg/mL brefeldin A (Biolegend) for 5 h at 37 °C²⁵ (link). The cells were stained with Brilliant Violet 605 anti-mouse IL-10, PE/Dazzle 594 tumor necrosis factor-alpha (TNF-α) (clone JES5-16E3 and clone 506,346, respectively; Biolegend), and PE anti-mouse inducible nitric oxide synthase (INOS) (clone CXNFT, respectively; eBioscience) and then analyzed using an CytoFLEX flow cytometer (Beckman Coulter, Miami, FL, USA). The data were analyzed by using FlowJo software (V.10.4, FlowJo, USA).