Ultra sensitive mouse insulin elisa kit

Fed blood was collected from a tail nick in heparinised capillary collection tubes (Sarstedt; 20.1309, Germany) coated tubes between 9 and 10 p.m. Food was then removed overnight, and fasted bloods were collected at 9–10 a.m. the following morning. Blood samples were centrifuged at room temperature, 2000 × g and plasma was frozen for later analysis. Insulin concentrations were assayed using Crystal Chem Ultra-Sensitive Mouse Insulin ELISA Kit (Crystal Chem Inc., 90080, USA) according to manufacturers’ instructions except that test samples and standards were incubated overnight at 4 °C.
Terminal blood draws were collected under isoflurane anaesthesia via retroorbital bleed in ETDA-coated capillary tubes (Sarstedt; 15.1671.100, Germany). Hematology and clinical biochemistry panels were performed by Laverty Pathology Vetnostics (West Ryde, NSW, Australia). Haematological analysis was performed using a Sysmex XN-V Multisciences hematology analyser (Sysmex, USA). Plasma was then extracted by centrifugation and clinical biochemistry panel was performed using a Cobas 8000 modular analyser (Roche, USA).

Alzet Micro-osmotic pumps, model 1002 with pumping rate 0.25μl/hr (DURECT Corporation) were filled with Humulin (Eli Lilly) diluted in 1X sterile PBS to allow Insulin release of 0.2 or 0.3U/day. Pumps were implanted subcutaneously under isoflurane anesthesia. The mice were allowed to recover and their plasma glucose was monitored using glucose meter (ACCU-CHEK Aviva, Roche). Plasma insulin levels was measured by ELISA (Crystal Chem, Ultrasensitive mouse insulin ELISA kit, Catalogue #90080, Lot # 16SEUMI411 that detects both human and mouse insulin)

Plasma fatty acid, triglyceride, cholesterol, alanine transaminase and aspartate transaminase were analysed with the serum analyser (Beckman Coulter, AU480). Insulin levels were determined by using the Ultra-Sensitive Mouse Insulin ELISA Kit (CrystalChem, Zaandam, Netherlands) according to manufacturer’s instructions. Ketone body measurements were analysed in tail vein blood (Wellion, Marz, Austria). Plasma TNFα levels were analysed by using the TNF alpha Mouse ProQuantum Immunoassay Kit (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s instructions. Murine plasma proteome was analysed by using the Proteome Profiler Mouse Adipokine Array Kit (R&D systems, Minneapolis, Canada) according to the manuals.

Whole blood glucose levels were measured from the tail veins using a Contour Plus glucometer (Ascensia Diabetes Care, Basel, Switzerland). Blood serum was isolated using BD Microtainer tube with serum separator (365967, Becton Dickinson, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Serum insulin was determined by Ultra Sensitive Mouse Insulin ELISA kit (90080, Crystal Chem, Downers Grove, IL, USA). Homeostatis model assessment of insulin resistance (HOMA-IR) was calculated as fasting blood glucose (mmol/L) × fasting insulin (mU/L)/22.5 [15 (link)].

Oral Glucose Tolerance Tests (OGTTs) were performed 1 p.m. after a 4 h fast by oral administration of 2 g glucose per kg body weight. Blood (12 µl) was sampled from the tail vein at 0, 15, 30, 60 and 120 min for determination of glucose (2 µl, Accu-Chek, Roche Diagnostics, Mannheim, Germany) and insulin (2×5 µl, Ultra-sensitive mouse insulin ELISA kit, Crystal Chem. Inc., Downers Grove, IL) levels.