Ultra sensitive rat insulin elisa kit

Animals were euthanized using an overdose of sodium pentobarbital (200–300 mg/kg, Vortech; Dearborn, MI) administered by intraperitoneal injection, followed by exsanguination. Weight, nose-to-tail length, and abdominal girth were measured, and body mass index (BMI) was calculated. Blood was collected from the vena cava in EDTA-treated collection tubes and cells were analyzed using a ProCyte Dx Hematology Analyzer (IDEXX Laboratories, Inc.; Norcross, GA). Blood used for serum samples was collected in BD Vacutainer™ Serum Separation Tubes, left at room temperature for 1.5 h, and centrifuged at 25,000 g for 20 min. Fresh serum was used for measurements of high-density lipoprotein (HDL) (#ab65390, Abcam, Cambridge, MA) and blood chemistry (Catalyst Dx Chemistry Analyzer, IDEXX Laboratories, Inc). Epididymal fat pads were removed and weighed. Serum samples were stored at -80 °C until used for ELISA and cytokine analysis. ELISAs were conducted following the manufacturers’ protocols: leptin (#MOB00B, R&D Systems, Minneapolis, MN) and adiponectin (#Acrp30, R&D Systems). Insulin was measured using the Ultra-Sensitive Rat Insulin Elisa Kit (#90060, Crystal Chem; Elk Grove Village, IL). Serum cytokines were measured using the MSD V-PLEX Proinflammatory Panel 2 (rat) kit and MESO QuickPlex SQ 120 (Meso Scale Diagnostics; Rockville, MD).

This test was carried out using a Crystal ChemInc ultrasensitive rat insulin ELISA kit (Illinois, USA). The detection range is 0.1 to 64 ng/ml. Insulin antibody had a 100 percentage cross-reactivity with rat insulin. There was a 10.0 percent intra-assay
coefficient of variance and the results are given in nanograms per millilitre.

The concentration of insulin in the serum was determined in triplicates using an ultra-sensitive rat insulin ELISA kit (Crystal Chem Inc, Elk Grove Village, IL, USA). Total cholesterol, triglycerides, and HDL cholesterol were determined using an automated Siemens ADVIA 2400 Chemistry Analyzer (Erlangen, Germany). Total cholesterol and triglycerides were measured based on the enzyme-coupled reaction, whereas HDL cholesterol was determined using a direct HDL-cholesterol assay. LDL cholesterol was calculated using the Friedewald equation. The body weights of the rats were measured on day 0 and day 12 by using an electronic balance (Navigator^TM, Ohaus Corporation, Nanikon, Switzerland).

Glucose tolerance was evaluated by means of oral glucose tolerance test (OGTT) three times during the study; at 5, 11 and 16 weeks of age. Mice were fasted for four hours prior to testing and given a glucose solution (2 g/kg, Fresenius Kabi, Copenhagen, Denmark, concentration 500 g/l) by oral gavage. Blood samples were collected at t = 0, 30, 60, 90, 120 and 180 minutes post gavage. Glucose levels were measured by transferring 5 μl whole-blood to 500 μl EBIO buffer and analysing glucose levels by a Biosen glucose auto analyser (Eppendorf, Hamburg, Germany).
Insulin levels were measured just prior to glucose dosing at all three time points and again at 30 minutes post dosing in the 16 weeks old animals. Plasma samples were analysed for insulin using the "Ultra-sensitive rat insulin ELISA kit" (Crystal Chem, Downers Grove, IL, USA) with the modifications that in-house rat insulin standards, prepared using heat-treated rat plasma, were used.