Htrf insulin kit

Insulin secretion assays on isolated mouse islets were performed as described previously (18 (link)). In brief, 10 size-matched islets per condition were incubated for 1 h in Krebs-HEPES-bicarbonate (KHB) solution (130 mm NaCl, 3.6 mm KCl, 1.5 mm CaCl₂, 0.5 mm MgSO₄, 0.5 mm KH₂PO₄, 2 mm NaHCO₃, 10 mm HEPES, and 0.1% (w/v) BSA, pH 7.4) containing 3 mm glucose. Subsequently, islets were incubated for 30 min in KHB solution with either 3 mm glucose, 17 mm glucose, or 30 mm KCl. Secreted insulin and total insulin were quantified using an HTRF insulin kit (Cisbio) in a PHERAstar reader (BMG Labtech, UK) following the manufacturer's guidelines.

Homogeneous time resolved fluorescence with the HTRF Insulin Kit (no. 62INSPEB, Cisbio Bioassays, Brisbane, QLD, Australia) was used to measure islet insulin secretion (see ESM Methods for details).

INS-1E cells obtained from Dr. Wollheim (Univ. Geneva, Switzerland), were grown in Roswell Park Memorial Institute medium (RPMI) 1640 (Life technologies) supplemented with 5% of fetal bovine serum (Thermo Fischer Scientific, Inc. Waltham, MA). At 4 or 5 days before the assay, cells were plated in 24-well plates at 2 × 10⁵ cells /well. At the day of the assay, cells were washed twice by Krebs-Ringer bicarbonate (KRB) buffer containing 0.1% BSA (pH7.4) and pre-incubated at 37°C for 30 min. The cells were washed twice again, and the buffer was replaced by KRB buffer containing 0, 1, 3 and 10 μM of compounds and 11.1 mM of glucose. In order to examine whether Zn²⁺ has an enhancing effect on the activity of each compound, 20 μM of Zn²⁺ was added in the incubation buffer as necessary. After cells were stimulated at 37°C for 1 h, the reaction was terminated by placing on ice. The buffer was collected from each well and precipitated (1000 rpm for 2 min at 25°C) in order to be separated from the cells. For each sample, concentration of insulin was measured using HTRF insulin kit (Cisbio).

In brief, ten size-matched islets per condition were incubated for 1 h in Krebs-HEPES-bicarbonate solution. Following incubation for 30 min with either 3 mmol/l glucose (low glucose), 17 mmol/glucose (high glucose) or 30 mmol/KCl, secreted and total insulin were quantified using an HTRF insulin kit (Cisbio, France).

Mice were euthanized by cervical dislocation and pancreatic islets isolated by collagenase digestion as previously described and cultured in RPMI 1640 medium [19 (link)], 11 mM glucose, supplemented with 10% (v/v) FBS plus penicillin (100 units/mL), and streptomycin (0.1 mg/mL) at 37°C in an atmosphere of humidified air (95%) and CO₂ (5%).
Mouse islets (10/well) were incubated in triplicate for each condition and treatment. Islets were preincubated for 1 hour in 3 mM glucose KRH buffer prior to secretion assay (30 min) in 3 mM or 11 mM glucose with vehicle or 100 nM SM102 or Semaglutide. The secretion medium was then collected to measure the insulin and proinsulin concentrations using an insulin HTRF kit (Cisbio Bioassays). INS-1 832/3 cells were treated with the indicated concentrations of SM102 or Semaglutide for 16 h in complete RPMI1640 medium at 11 mM glucose. A sample of supernatant was collected and analyzed for insulin content by HTRF.