After isolation, the floating islets were incubated overnight at 37 °C in a humidified atmosphere and 5% CO2 in a RPMI media containing (RPMI medium 1640: Gibco, Invitrogen, France; 10% inactivated fetal calf serum (FCS), 1% HEPES, 11 mmol/L glucose (D-Glucose 45%, Sigma Aldrich, France), 100 IU/mL penicillin, and 100 mg/mL streptomycin).
Next, batches of five size-matched islets were pre-incubated in KRBH-0.1% BSA with 2.8 mm of glucose for 30 min, followed by 60 min incubation at 37 °C in KRBH-0.1% BSA buffer containing 2.8 mm or 16.7 mm of glucose to measure the glucose-induced insulin secretion. The supernatant (500 µL) was immediately collected and stored at −20 °C until being assayed for insulin by ELISA (ultra-sensitive mouse insulin ELISA Kit; Crystal Chem, #90080, the Netherland). The islets were homogenized in protein extraction buffer and stored until insulin content determination by ELISA (ultra-sensitive mouse insulin ELISA Kit; Crystal Chem, #90080, Netherland), as previously described [17 (link)].
Next, batches of five size-matched islets were pre-incubated in KRBH-0.1% BSA with 2.8 mm of glucose for 30 min, followed by 60 min incubation at 37 °C in KRBH-0.1% BSA buffer containing 2.8 mm or 16.7 mm of glucose to measure the glucose-induced insulin secretion. The supernatant (500 µL) was immediately collected and stored at −20 °C until being assayed for insulin by ELISA (ultra-sensitive mouse insulin ELISA Kit; Crystal Chem, #90080, the Netherland). The islets were homogenized in protein extraction buffer and stored until insulin content determination by ELISA (ultra-sensitive mouse insulin ELISA Kit; Crystal Chem, #90080, Netherland), as previously described [17 (link)].
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