Ultrasensitive mouse insulin elisa kit

Mice were fasted for 16 h, followed by blood sample collection from a facial vein to evaluate serum insulin levels under no glucose stimulation. Mice were then injected intraperitoneally with glucose (2 g/kg body weight) and blood samples were collected from the facial vein 15 min post-glucose injection. Blood samples were allowed to clot in vacutainer tubes (BD Japan) at room temperature followed by centrifugation for 10 min at 4000 rpm and separation of serum. Serum insulin levels were measured using an ultrasensitive mouse insulin ELISA kit (Takara).

Ten islets were pre-cultured in 450 µL Krebs Ringer buffer (KRB) (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH₂PO₄, 2 mM NaHCO₃, 1.5 mM CaCl₂, 0.5 mM MgSO₄, 10 mM HEPES, 0.25% BSA), pH = 7.4, containing 3 mM glucose for 1 h in open Eppendorf tubes. Supernatants were discarded and replaced with 450 µL KRB containing either 3 mM or 17 mM glucose for 1 h. Supernatants were collected after 1 h. Finally, islets were treated with 1 mL 1.5% HCl in ethanol and homogenized by sonication three times (30 s on/off). Acid/ethanol lysates were then centrifuged. Supernatants were collected to measure insulin content and residual islet fragments were used for DNA extraction. Insulin in KRB supernatant (released insulin) and islet lysates (islet insulin content) was measured using an ultrasensitive mouse insulin ELISA kit (Takara). Both released insulin and islets insulin content were normalized to islet DNA content.