Cd40 3 23

Fluorescence-activated cell sorting (FACS) analysis was carried out as described previously30 (link). Briefly, cells in the SVF were suspended in FACS buffer and incubated with anti-mouse CD16/CD32 (93; Biolegend, San Diego, CA) for 15 min. Then, the cells were rinsed and resuspended in FACS buffer and stained with anti-CD11c (HL3; Biolegend) and anti-CD11b (M1/70: Biolegend) antibodies for DC and macrophages, respectively. The expression of co-stimulatory molecules was determined using mAbs to CD80 (16-10A1; BD), CD86 (GL1; BD), CD40 (3/23; BD), and MHC class II (M5/114.15.2; BD). The expression of other surface antigens was analyzed by using mAbs to F4/80 (BM8; Biolegend), CD103 (M290; BD), CD205 (NLDC145; MACS), CD206 (MR5D3; AbD Serotec), mPDCA1 (JF05-1C2.4.1:MACS), and B220 (RA3-6B2; Biolegend). To detect lipid, cells were first stained with 1 μg/ml Nile red (Wako Pure Chemicals, Osaka, Japan) for 15 min, and then analyzed with FACSVerse (BD Biosciences). The expressions of Treg related molecules were determined by using mAbs to CD25 (PC61; Biolegend), FR4 (12A5; Biolegend), CTLA4 (UC10-4B9; Biolegend) and GITR (DTA-1; Biolegend).

Antibodies to the following molecules were purchased from eBioscience unless otherwise stated: MHC class II (I-A^b) (25-9-17), CD11c (N418), CD45.1 (A20), CD80 (16-10A1), CD45R/B220 (RA3-6B2), CD19 (ID3), NK1.1 (PK136), CD90.2 (53-2.1), CD11b (M1/70), CD8α (53-6.7), Streptavidin, CD86 (GL1) (BD Biosciences), CD40 (3/23) (BD Biosciences), CD169 (3D6.112) (Serotec), and 25-D1.16 (grown, purified and labeled in house).

Composition of murine immune cells was analyzed using the following antibodies: CD3 (145-2C11; BioLegend), CD19 (6D5; BioLegend), CD20 (SA275A11; BioLegend), CD11b (M1/70; BioLegend), CD11c (N418; BioLegend), CD45 (30-F11; BioLegend), Ly6C (HK1.4; BioLegend) and Ly6G (1A8: BioLegend). B cell maturation was analyzed using the following antibodies: CD19 (6D5; BioLegend), CD21 (7G6; BD Bioscience), CD23 (B3B4; BD Bioscience), CD93 (AA4.1; BioLegend), CD45R/B220 (RA3-6B2; BioLegend), IgD (11-26c.2a; BioLegend) and IgM (AF6-78; BD Bioscience). Monocyte, macrophage and microglia activation, differentiation and molecules involved in antigen presentation were determined using: CD40 (3/23; BD Bioscience), CD68 (FA-11; BioLegend), CD69 (H1.2F3; BioLegend), CD80 (16-10A1; BioLegend), CD86 (GL-1; BioLegend), MHCII (AF6-120.1; BioLegend) and PD-L1 (MIH5; eBioscience). Fc receptors were blocked using monoclonal antibody specific for CD16/ CD32 (93; BioLegend). Dead cells were stained with a fixable viability kit (BioLegend). Samples were acquired on a BD LSR Fortessa (BD Bioscience). All data evaluation was performed using FlowJo software (FlowJo LLC, Ashland, USA).
Intracellular proteins were analyzed using the BD PhosFlow protocol and analyzed using the following antibodies: BTK (53/BTK; BD Bioscience), pBTK (N35-86, BD Bioscience), iNOS (W16030C, Biolegend) Arg1 (A1exF5, eBioscience).