Insulin elisa kit

All mouse experiments were approved by the Animal Research and Care Committee at the First Hospital of the Second Hospital of Dalian Medical University. Female C57BL/6 mice (SLAC Laboratory Animal Co. Ltd, Shanghai, China) were used in the experiments at 10 weeks of age. The beta cell toxin STZ (Sigma-Aldrich, St Louis, MO, USA) was injected from the tail vein at 150 mg/kg body weight. The ERK5 inhibitor BIX02189 (Selleck Chemicals, Houston, TX, USA) [11 (link)] was intraperitoneally injected into the mice at a dose of 10 mg/kg body weight in 100 ml saline since the day of STZ injection, at a frequency of twice per week, through the end of experiment at 12 weeks after STZ. The control mice received the same volume of normal saline at the same frequency. Fasting blood glucose, intraperitoneal glucose tolerance test (IPGTT) and serum insulin content were measured as described [15 (link)]. Briefly, the mice were fasted overnight before measurement of blood sugar was performed at 9am via tail tip puncture using a glucometer (Accu-Chek, Roche, Nutley, NJ, USA). To examine the possible glucose tolerance, after a 16 hour fasting, the mice received an intraperitoneal injection of 2 mg/g body weight of dextrose, after which blood samples were obtained by tail clipping at 0, 15, 30, 60 and 120 min. Serum insulin was measured with an insulin ELISA kit (R&D System, Los Angeles, CA, USA).

All rat experiments were approved by the research committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. Sixteen-week old female Wister rats were purchased from Charles River Labs (Beijing, China). Fasting blood glucose and serum insulin were measured as described before [9 (link)]. Streptozotocin (STZ) was intraperitoneally (i.p.) injected at a dose of 150 mg/kg body weight in sodium citrate buffer (pH=4.5) to induce sustained high blood glucose in 18-week-old female rats. Littermate rats that received injection of the same volume of saline were used as controls (saline). Serum insulin was determined with an insulin ELISA kit (R&D System, Los Angeles, CA, USA). Fasting blood glucose measurement and Intraperitoneal glucose tolerance test (IPGTT) were conducted as previously described [9 (link)]. Rapamycin (RAP) and Chloroquine (CQ) were both obtained from Sigma-Aldrich (St. Louis, MO, USA), and were i.p administrated to rats at a dose of 2mg/kg body weight, and 10mg/kg body weight, respectively, once a day for six weeks.

All biochemical parameters were measured using an enzyme-linked immunosorbent assay kit (ELISA). An Alanine Transaminase Colorimetric Activity Assay Kit, Cholesterol Fluorometric Assay Kit, and Glucose Colorimetric Assay Kit were purchased from Cayman Chemical, a TNF-alpha Quantikine ELISA Kit and an Insulin ELISA Kit from R&D Systems, and a Rat Lipopolysaccharides (LPS) ELISA Kit from MyBioSource. Insulin resistance (HOMA-IR) was calculated according to the following formula: fasting serum glucose × fasting serum insulin/22.5.