Dy737

Prior to being sacrificed, the mice were not allowed access to any food for 8 h. Under anesthesia with isoflurane, blood was withdrawn from the left femoral artery. WBCs, lymphocytes and neutrophils were each measured using routine complete blood counts. Plasma levels of insulin (#630-07289, Shibayagi, Gunma, Japan), TGF-β1 (DY1679-05, R&D Systems, Minneapolis, MN, USA), leptin (MOB00B, R&D Systems, Minneapolis, MN, USA), soluble P-selectin (DY737, R&D Systems, Minneapolis, MN, USA), glutamine (ab197011, Abcam, Cambridge, MA, USA) and HMGB1 (E4864, BioVision, Mountain View, CA, USA) were all measured using Enzyme Immunosorbent Assay (ELISA) kits, following the procedures provided by the respective manufacturers. Plasma levels of circulating citrullinated histone H3 were measured using self-prepared assay plates which had been coated with an antibody against citrullinated histone H3 (NB100-57135, Novus Biologicals, Centennial, CO, USA). The contents of immunocomplexes were quantified using a spectrophotometer at 450 nm. The level of fasting glucose was measured using a hand-held Accucheck glucometer (Roche Diagnostics, Indianapolis, IN, USA). The HOMA-IR index was calculated as [fasting insulin (μU/mL) × fasting glucose (mmol/L)]/22.5, and used as the reported method [32 (link)].

Plasma sCD62P levels were measured by quantitative sandwich enzyme immunoassay (R&D Systems, Inc. DY737, Minneapolis, MN, USA).

Levels of TPO (R&D Systems, catalog MTP00), IL-6 (BD Biosciences, catalog 555240), S100A8/S100A9 heterodimer (R&D Systems, catalog DY8596-05), and P-selectin (R&D Systems, catalog DY737) were measured in the serum of PBS and LCWE-injected mice by ELISA according to the manufacturer’s protocol. IL-1B was quantified in serum using the V-PLEX Mouse IL-1B Assay (Meso Scale Diagnostics, catalog K152QPD-1) per the manufacturer’s instructions using the MSD QuickPlex SQ120 instrumentation and Workbench 4.0 Software (Meso Scale Diagnostics).