D glucose 45

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)].

Coring press was obtained from Alabama Research & Development. University of Wisconsin (UW) organ preservation solution was from DuPont Critical Care, Waukegab, IL, USA. Krebs‐Henseleit buffer modified (K3753), NaHCO₃, d‐Glucose (45%), HEPES, 1× HBSS, EDTA Ketanserin (+)‐tartrate salt # S006‐50MG and Endothelin‐1 (ET‐1) # E7764‐1 mg all obtained from Sigma Aldrich. Williams E medium was from Lonza. Glutamax and gentamicin were provided by Gibco. FastPrep System tubes were obtained from MP Biomedicals. ATP Bioluminescence Assay Kit was provided by Roche. Pierce™ protein assay kit was obtained from Thermo Scientific.
RLT buffer was from Qiagen. High capacity cDNA Archive Kit was obtained from Applied Biosystems (#4322169). The quantitative real‐time PCR with gene‐specific primers was from Applied Biosystems. The following primers were used Nfe2 l2 (Rn00582415_m1), Nqo1 (Rn00566528_m1), Hmox1 (Rn00561387_m1) and rRNA Pol2 (Rn01752026_m19).