Fluorescent eia kit

Venous blood samples were collected after an overnight (≥10 h) fast. Fasting glucose level was measured by glucose oxidase‐peroxidase reagents using a Beckman Glucose Analyzer (Beckman Instruments, Irvine, CA, USA), so was glycated hemoglobin (Hgb_A1C) on Variant analyzer (Variant II TURBO, Bio‐Rad, Hercules, CA, USA). Immunoradiometric assays specific for insulin (Linco, St. Louis, Mo., USA) was used for the measurement of fasting insulin based on an antiserum with <1% cross‐reactivity for pro‐insulin. Triglycerides and total cholesterol were determined by enzymatic methods on the analyzer Beckman Coulter (USA). In order to evaluate the CRP level, the immunoturbidimetric method was applied (analyzer Beckman Coulter, USA). Plasma renin concentration was measured using an immunochemiluminometric assay (Nichols Advantage Direct Renin Assay, Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, USA). Angiotensin concentration was determined through the immuno‐fluorescence assay as described in the instruction manual (Fluorescent EIA Kit, phoenix pharmaceuticals). Serum aldosterone was measured by a sensitive radioimmunoassay (Quest Diagnostics, Cambridge, MA). Enzyme‐linked immunosorbent assay (ELISA) were performed with responding kits (Invitrogen Inc, USA) for IL‐6, TNF‐α, leptin, resistin, adiponectin as manufacturer’s instructions.

These experiments were performed with rat and human islets (10 islets/100 μL medium). The medium contained (in mM): 137 NaCl, 5.4 KCl, 1.3 CaCl₂, 0.81 MgSO₄, 0.34 NaH₂PO₄, 0.44 KH₂HPO₄ + 1 mg/mL BSA, and was at pH 7.4. Islets were maintained for 30 min in a medium containing 25 (rat) or 11.1 mM (human) glucose before being transferred in a medium containing 1 mM glucose and the respective treatments. One hour later, glucagon was determined using a Fluorescent EIA Kit (Phoenix Pharmaceuticals).