Mp5 20f3

Mice at 8 weeks of age were injected via tail veins with 50 μg anti-IL-6 antibodies (MP5-20F3; BioLegend) or control IgG antibodies (RTK2071; BioLegend) every other day for 10 days, followed by GTT tests at day 12. In the case of Fig. 8e,f, IL-6 neutralization was conducted in mice at 8 weeks of age.

BM-APCs were plated at 1.5 million cells/ml in Gibco RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS (HyClone, Logan, UT), 1% penicillin/streptomycin (HyClone, Logan, UT), 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, and GM-CSF (20 ng/ml; PeproTech, Rocky Hill, NJ) with 300,000 cells per well in a 96-well plate and treated with PLPs. Brefeldin A was added to cells at 2 hours to enhance intracellular cytokine accumulation, and cells were harvested at 6 hours for intracellular staining. BM-APCs were stained for live/dead discrimination with Zombie Green Fixable Viability Kit (BioLegend, San Diego, CA) and blocked with anti-mouse CD16/CD32 (clone 93) and True-Stain Monocyte Blocker (BioLegend, San Diego, CA). For surface staining, BMDCs were incubated with BUV395 anti-CD11b (M1/70, BD), BV421 anti-CD11c (N418, BioLegend, San Diego, CA), phycoerythrin (PE)–Dazzle 594 anti-Ly6C (HK1.4, BioLegend, San Diego, CA), and allophycocyanin-Cy7 anti-(I-A/I-E) (M5/114.15.2, BioLegend, San Diego, CA). For intracellular staining, cells were fixed with BD Cytofix and permeabilized with Perm/Wash Buffer (BD) and then labeled with allophycocyanin anti–IL-12(p40/p70) (C15.6, BD) or PE anti–IL-6 (MP5-20F3, BioLegend, San Diego, CA) in combination with allophycocyanin anti-TNFα (MP6-XT22, BioLegend, San Diego, CA).

After 2w with regular or high nitrate diet feeding, BMDMs were isolated and cultured as previously described [39] . Endocytic potential of BMDMs was assessed by uptake of fluorescently labeled dextran (ThermoFisher, Sweden). Briefly, BMDMs were cultured in 12-well plate for 24 h then incubated with Alexa Fluor 647-labeled Dextran (1 μg/ml) for 30 min. After washing away the extra dextran with PBS, the cells were detached with 2 mM EDTA. Cells were run in a Gallios flow cytometer (Beckman Coulter, Brea, CA) and analyzed using Kaluza v1.1 software (Beckman Coulter).
The intracellular cytokines of BMDMs was also measured by flow cytometer. Briefly, after 24 h of culture, the cells were first incubated with GolgiPlug (1 µl/ml, BD Biosciences) in complete DMEM for 4 h at 37 °C before incubation with antibodies. Fixation/Permeablization and intracellular staining was conducted using the eBioscience Intracellular Staining Kit and the following antibodies: IL-6 (MP5-20F3, Biolegend), IL-1 beta pro-form (NJTEN3, eBioscience), and TNF alpha (MP6-XT22, Biolegend).