Bms2038inst

Blood samples were collected after overnight fasting. Fasting glycaemia, HbA1c, total cholesterol, LDL cholesterol, triglycerides, hs-CRP, WBC, troponin, insulin, and uric acid were measured by standard procedures. One EDTA blood tube was centrifuged at 2000g for 15 min at 4 °C to obtain EDTA plasma samples, immediately stored at −80 °C. Plasma EDTA samples were then used to measure IL-6 (BMS213HS, Invitrogen), MPO (BMS2038INST, Invitrogen), and BHB (MAK041, Sigma) with ELISA (IL-6 and MPO) or colorimetric (BHB) kits, according to manufacturer’s instructions.

Dedicated enzyme-linked immunosorbent assay (ELISA) kits were used for quantitative measurement of neutrophil degranulation markers, including polymorphonuclear neutrophil (PMN)-elastase (ab119553; Abcam, Cambrige, UK), neutrophil gelatinase-associated lipocalin (NGAL) (KIT 036RUO; BioPorto Diagnostics A/S, Hellerup, Denmark), and MPO (BMS2038INST; Invitrogen, Waltham, MA, US).
NETs-associated MPO–DNA complexes were measured by adding serum into 96-well plates coated with a monoclonal anti-human MPO antibody (MABX4043-10KC; Millipore, Burlington, MA, US) after saturation of no specific binding site with bovine serum albumin, followed by incubation with peroxidase-labelled anti-DNA monoclonal antibody included in the Cell Death ELISA kit (11774425001; Roche Diagnostics, Mannheim, Germany). The optical absorbance was measured at 405 wavelength by using Berthold Mithras multimode reader (Berthold Technologies GmbH, Bad Wildbad, Germany).