Chemiluminescent rodent insulin elisa

Blood glucose was measured via tail poke and measured with a OneTouch Ultra Glucometer (Life Scan Inc., Burnaby, Canada). For glucose tolerance tests, mice were fasted for 6 h and subsequently injected intraperitoneally with 2 g/kg (ageing study) or 1.5 g/kg body weight (diet study) glucose, and blood glucose measured at indicated time points as above. Blood was collected from the saphenous vein after a 6-h fast for the measurement of plasma insulin via Stellux Chemiluminescent Rodent Insulin ELISA (#80-INSMR-CH01, Alpco, Salem). For insulin tolerance tests, insulin (Novolin GE Toronto, Novo Nordisk) was injected at the 0.65 U/kg (ageing study) or 0.35 U/kg (diet study) and blood glucose measured at indicated time points.

An Analox GM9 Glucose Analyzer was used to measure glucose levels via the glucose oxidase method. Plasma insulin levels were determined using a Stellux Chemiluminescent Rodent Insulin ELISA (Alpco). Plasma and liver triglycerides were measured using Infinity Triglyceride Reagent (Thermo Fisher TR22421). Liver triglycerides were extracted using a methanol/chloroform-based method^{15 (link)} and the colorimetric Infinity Triglyceride Reagent. Plasma cholesterol levels were measured using Wako Diagnostics Total Cholesterol Kit (999-02601). Plasma fatty acids were measured using Wako Diagnostics NEFA-HR kit (999-34691, 995-34791, 991-34891, 993-35191). Plasma liver enzymes were assessed using Thermo Fisher Infinity alanine aminotransferase and aspartate aminotranferase activity kits (TR71121, TR70121).

Duplicates of 10 size-matched islets per mouse were rested in Krebs-Ringer Bicarbonate buffer (129 mmol/L NaCl, 4.8 mmol/L KCl, 1.2 mmol/L MgSO₄, 1.2 mmol/L KH₂PO₄, 2.5 mmol/L CaCl₂, 5 mmol/L NaHCO₃, 10 mmol/L HEPES, 0.5% bovine serum albumin) containing 2.8 mM glucose for 1 h at 37 °C, and were sequentially incubated for 45 min in KRB containing 2.8 mM (low) glucose, then 16.7 mmol/L (high) glucose, and then 30 mmol/L KCl (103.8 mmol/L NaCl, 30 mmol/L KCl, 1.2 mmol/L MgSO₄, 1.2 mmol/L KH₂PO₄, 2.5 mmol/L CaCl₂, 5 mmol/L NaHCO₃, 10 mmol/L HEPES, 0.5% bovine serum albumin, 2.8 mmol/L glucose). Cell supernatants were collected for measurement of insulin secretion, and islets were lysed in acid:ethanol for insulin content. Insulin was measured via Chemiluminescent Rodent Insulin ELISA (#80INSMR-CH01, Alpco).