Mouse insulin enzyme linked immunosorbent assay elisa kit

The viability of islets was analysed under each condition by fluorescein diacetate/propidium iodide staining (FDA/PI, Sigma-Aldrich), which was performed by two independent investigators. The ratio of green to red cells determined the percentage of viable cells59 (link). Images were obtained using an Olympus IX51 microscope. The results are expressed as the percentage of viable cells. To determine islet function, 100 pancreatic islets were cultured in RPMI 1640 media with 10% FBS and 5.5–10 mM glucose (Sigma-Aldrich). Each incubation step was performed for 120 min at 37 °C in a humidified 5% CO₂ atmosphere. In some in vitro experiments, islets were cultured in the presence of recombinant cytokines for 24 and 48 h at 37 °C in a humidified 5% CO₂ atmosphere. Supernatants were collected and stored at −80 °C. Insulin levels in culture supernatants were measured using a mouse insulin enzyme-linked immunosorbent assay (ELISA) kit (Mercodia). The results are expressed as ng per ml insulin released per 100 islets.

Bovine α-lactalbumin was provided by Davisco Foods International Inc. (Eden Prairie, MN, USA). Alcalase was purchased from Novozymes Biologicals Inc. (Bagsvaerd, Denmark). Insulin was purchased from Sigma-Aldrich (St. Louis, MO, USA). The mouse Insulin enzyme-linked immunosorbent assay (ELISA) kit was obtained from Mercodia (Uppsala, Sweden). The primary antibody against β-actin (No. ab8227) was purchased from Abcam (Cambridge, UK). The primary antibodies against p-IRS-1 (Ser307) (No. 2381), p-IRS-1 (612) (No. 2386), IRS-1 (No. 3407), p-Akt (Ser473) (No. 4060), Akt (No. 4691), p-IKKα/β (No. 2597), IKKα (No. 2682), IKKβ (No. 2370), p-p38 (Thr180/Tyr182) (No. 4511), p38 (No. 8690), phospho-extracellular signal regulated kinases (p-ERK) (Thr202/Tyr204) (No. 4370), ERK (No. 4696), phospho-c-Jun N-terminal kinases (p-JNK) (Thr183/Tyr185) (No. 4668), JNK (No. 9252), and horseradish peroxidase-conjugated anti-rabbit secondary antibody (No. 7074) were obtained from Cell Signaling Technology (Beverly, MA, USA). All other chemicals used in the study were at least of analytical grade.

The air-dried plant of PDB was provided by Tangxian Dandelion Tea Manufacturing Co., Ltd (Tang country, Hebei Province, China) and was identified and authenticated by the taxonomist of Beijing University of Chinese Medicine. Streptozotocin (STZ) and insulin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The mouse insulin enzyme-linked immunosorbent assay (ELISA) kit was purchased from Mercodia (Uppsala, Sweden). RIPA buffer, phosphatase cocktails and protease for western blot, and BCA protein assay kit were purchased from Beyotime Biotech (Haimen, Jiangsu, China). Primary antibodies against p-GS (Ser641), GS, p-GSK3β (Ser9), GSK3β, p-Akt (Ser473), Akt, p-AMPK (Thr172), and AMPK were purchased from Cell Signaling Technology (Beverly, MA). The primary antibody against β-actin was purchased from Biosynthesis Biotechnology (Beijing, China). The anti-rabbit secondary antibody was purchased from Beyotime Biotech (Haimen, Jiangsu, China). All other chemicals were of analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).