Human insulin elisa kit

For the Glucose-Stimulated Insulin Secretion (GSIS) assay, insulin secretion was measured by incubating 15 IPCs. First, IPCs were washed carefully with PBS and then incubated in Kreps Ringer Bicarbonate Buffer (Table 3; KRB buffer, Sigma-Aldrich, St. Louis, MO, USA), followed by incubation for 1 h in KRB buffer with 2.8 mM glucose (low glucose) or 17.5 mM glucose (high glucose). Insulin levels were measured in the supernatant after 1 h. Insulin secretion was determined using the human insulin ELISA Kit (Cat# ab278123; Abcam, Cambridge, MA, USA).

Mice were fasted for 6 h with free access to water. At the end of the 6 h fasting period, blood samples were collected from the tail vein. Fasting plasma insulin levels were then measured using human insulin ELISA kit following the manufacturer's instructions (Abcam®, Cambridge, UK).

To evaluate insulin degradation in cell lysates human Insulin ELISA Kit (Abcam, Cambridge, MA, USA) was used based on the manufacture’s protocol. First, 300 ng/mL human recombinant insulin (Sigma, St. Louis, MO, USA) was added to cell lysates and incubated for 10 min with gentle shaking at room temperature. Then Ethylenediaminetetraacetic acid (EDTA, 1 mM) which is a metalloproteinase inhibitor was added to solution to inhibit IDE activity. Several experiments were done to adjust concentration of human recombinant insulin and time of incubation. Absorbance was measured at 450 nm and converted to ng/mL using standard curve.

For IPGTT, serum was obtained from tail vein and insulin level was measured using a mouse insulin ELISA kit (Cat#EMINS, Thermo Fisher Scientific); For glucose-stimulated insulin secretion test (GSIS) in vitro, ~20-30 islets of 150–200 μm diameter were kept in 48-well plate and pre-incubated for 1 h at 37 °C in Krebs–Ringer bicarbonate (KRB) buffer (Cat#AAPR108, PYTHONBIO) containing 0.5% bovine serum albumin and 2.8 mM glucose. The buffer was then replaced with KRB buffer supplemented with 2.8 mM or 16.7 mM glucose. After incubation for 1 h at 37 °C, islets were pelleted by a 3 min centrifugation at 800 g, and media were aspirated and frozen for insulin determination. Insulin was also acid-ethanol extracted (1 mol/L HCl/70% ethanol) at −20 °C for 24 h from the islets and cellular debris removed by centrifugation at 15,000 g for 20 min. Insulin levels in the media and islet extract were determined as mentioned above. Total islet insulin content was defined as insulin level in the media plus islet extract. Insulin release was shown as the insulin level in the media as well as the fraction released of the total islet insulin content. Glucose-stimulated insulin release by human islets was determined using a human insulin ELISA kit (Cat#ab100578, Abcam).