Rat insulin ria kit

Unless otherwise stated, insulin tolerance tests, glucose tolerance tests and pyruvate tolerance tests were performed on 4 hr, 6 hr and 6 hr fasted conscious mice respectively by injecting human insulin (0.5–0.65 mU insulin/g body weight), D-glucose (2 mg/g body weight), or sodium pyruvate (0.75–1.5 mg/g body weight) into the peritoneal cavity and measuring glucose in tail blood immediately before and at 15, 30, 45, 60, 90 and 120 min after injection using a Accu-Check glucometer (Roche, Germany). Fed and fasted (12 hr fast) plasma insulin levels were determined using a Rat insulin RIA kit (Merck Millipore, CA) or an in-house ELISA (Monash Antibody Technologies Facility) and blood glucose levels were determined using Accu-Check glucometer. The areas under glucose excursion curves were determined and expressed as mmol/l x min.

The metabolic profile in serum blood was determined using commercially available colorimetric tests according to manufacturers’ instructions. The list of kits together with their catalog numbers and the producers are presented here: glucose (catalog number G7519), triglycerides (catalog number T7531), cholesterol (catalog number C7510) (Pointe Scientific, Canton, MI, USA), NEFA (catalog number HR Series NEFA-HR, Wako diagnostics, USA). Insulin and glucagon levels in sera were measured using RIA kits. (Rat insulin RIA kit, catalog number RI-13K; Glucagon RIA kit, catalog number GL-32K, Merck Millipore, USA).

Blood glucose concentration was determined with a glucometer (Accu-Chek Aviva; Roche Diagnostics, Laval, QC, Canada). The plasma insulin and leptin concentrations were determined by RIA (rat insulin RIA kit and rat leptin RIA kit) from Millipore (St. Charles, MO, USA). The homeostasis model assessment (HOMA) index was used to assess insulin resistance by calculating the value of fasting insulin and glucose with the following formula: [insulin (μU·mL⁻¹) × glucose (mmol·L⁻¹)/22.5] (Matthews et al., 1985 (link)).

At 6 weeks after transplantation, the graft-bearing kidneys were removed and homogenized in acid ethanol. After homogenization, the samples were extracted overnight at 4°C. On the following day, they were centrifuged at 2,400 rpm for 30 min and the supernatant was stored at −20°C. The pellet was again homogenized in acid ethanol and insulin was extracted overnight. After centrifugation, this second supernatant was added to the first extraction sample. Insulin was measured by radioimmunoassay with rat insulin RIA kit (Millipore Corporation, Billerica, MA, USA) [27 (link)–29 (link)].

Islet perifusions were performed as described [42] (link). Briefly, batches of 150 islets each were placed in Swinnex chambers (Millipore, Nepean, ON, Canada) and perifused for a total of 80 min with KRB buffer containing 0.1% (wt/vol.) BSA and 2.8 mM glucose. After a 10-min equilibration period with KRB solution with 2.8 mM glucose, islets were perifused for 40 min with 16.7 mM glucose. At 40 min the glucose concentration of the KRB was decreased to 2.8 mM. Intracellular insulin was extracted with acidified ethanol at the end of the perifusion to measure insulin content. For static incubations, batches of ten islets each were starved twice in KRB solution containing 0.1% (wt/vol.) BSA and 2.8 mM glucose for 20 min at 37°C then incubated for 1 h in the presence of 2.8 or 16.7 mM glucose. Each condition was run in triplicate. Intracellular insulin content was measured after acid–ethanol extraction. Insulin was measured by radioimmunoassay using rat insulin RIA kit (Millipore, Billerica, MA, USA).