Validation on mouse and rat b-cells

MIN6 mouse ß-cells and INS-1E rat ß-cells were seeded in 24-well plates (400,000 cells/well in 500 µL medium), then cultured for 6 days in complete culture medium at 37°C - 5% CO2. Before stimulation, cells were washed with KRB buffer and a cellular quiescence step was performed in KRB buffer (without glucose) for 2 hours at 37°C. Cells were stimulated with 250 µL of KRB buffer solutions containing increasing concentrations of glucose. After 10 and 30 minutes of secretion for MIN6 cells, or 2 hours of secretion for INS-1E cells, cell supernatants* were collected and insulin was quantified using HTRF detection reagents. Each condition of treatment was measured in triplicate (error bars represent SEM).

Samples were kindly provided by S. Dalle and M. Ravier’s research team: Physiopathology of pancreatic beta-cells, IGF, Montpellier, France.

 

Validation on rat pancreatic islets

Freshly isolated pancreatic islets from a male Wistar rat were incubated at 37°C for 30 min in KRB buffer containing either 2.8 mM glucose (non-stimulant basal condition) or 8.3 mM glucose (stimulant condition) (5 islets/tube;4 tubes/condition). At the end of the incubation period, supernatants* were collected and the insulin concentration determined using the Insulin High Range kit reagents. ​* Samples were generously supplied by the Laboratoire de Pharmacologie du Diabète, Institut des Biomolécules Max Mousseron (IBMM), CNRS UMR-5247 Faculté de Pharmacie, Université de Montpellier, France

 

Validation on an isolated perfused rat pancreas

A pancreas isolated from a male Wistar rat was perfused with KRB buffer containing 1.5 g/L glucose +/- 10 µM Quercetin. The medium was continuously bubbled with 95% O2 ± 5% CO2 and maintained at 37.5°C. A constant pressure was selected to give a flow rate of between 2.5 and 3.3 mL/min. The efferent fluid* was collected over time (13 fractions), and insulin concentration was measured using the kit reagents. Samples were generously supplied by the Laboratoire de Pharmacologie du Diabète, Institut des Biomolécules Max Mousseron (IBMM), CNRS UMR-5247 Faculté de Pharmacie, Université de Montpellier, France

Validation on human pancreatic islets

Pancreatic islets from 3 human pancreases (A, B and C) were placed in 24-well plates (50 islets/well, 3 wells/condition) and pre-incubated for 1h at 37°C in KRBH buffer containing 2.8 mM glucose. The incubation was prolonged for one hour and the supernatant was collected to measure basal insulin secretion. The islets were next stimulated for 1h at 37°C with 16.7 mM glucose before collecting the supernatant to measure glucose-stimulated insulin secretion. The islets were then lysed with a solution of acid-ethanol for 24h and the lysate was collected and centrifuged. The insulin in supernatants* and lysates* was quantified using the Insulin High Range kit reagents. Samples were generated at the Laboratory of Cell Therapy for Diabetes, Institute for Regenerative Medicine and Biotherapy (IRMB), Montpellier University Hospital, France.