Ultrasensitive insulin elisa

For each animal (n = 4 per genotype, 8–10 weeks old), quadruplicates of five starved islets were placed in Eppendorf tubes containing 2 ml of Kreb’s buffer containing 2.8 mM glucose, 11 mM glucose, 16.8 mM glucose, or 2.8 mM glucose + 30 mM KCl and incubated for 1 hr, and the supernatant was collected to measure insulin secretion. Quadruplicates of five unstimulated islets were sonicated and extracted by acid-ethanol. Insulin in supernatants and islet lysates was measured by ELISA (ultrasensitive insulin ELISA, ALPCO). Secreted insulin was then normalized with total lysate insulin content and expressed as a percentage of total insulin.
To measure insulin secretion in vivo, 9- to 11-week-old males were fasted for 16 hr, and we collected blood from animals (n = 6 per genotype) before glucose injection and 5, 15, and 30 min after glucose injection. D-glucose solution (15%) was injected intraperitoneally at 3 g/kg body weight. Plasma was separated from blood by centrifugation, and circulating insulin was measured by ELISA (ultrasensitive insulin ELISA, ALPCO).

For GTT and PTT, 12- to 16-week-old male GF and SPF mice were fasted overnight for 14 hours, starting at ZT12. For ITT, mice were fasted for 5 hours, starting at ZT22. Baseline blood glucose was measured via tail snip using a hand-held glucometer. Mice were administered an oral bolus of 20% dextrose (Hospira) in sterile water solution (2 g/kg body weight), an intraperitoneal injection of sterile-filtered sodium pyruvate (Sigma-Aldrich) in PBS (2 g/kg body weight), or an intraperitoneal injection of 0.1 U/mL Humulin R Insulin (Eli Lilly) in PBS (1 U/kg body weight). For GTT and PTT, blood glucose was measured at 1, 30, 60, and 120 minutes after gavage or injection. For ITT, blood glucose was measured at 15, 30, 60, 90, and 120 minutes after injection. During GTT, insulin levels were determined at baseline, 30, 60, and 120 minutes in blood collected in heparin-coated microvette tubes (Sarstedt) using the Ultra-sensitive Insulin ELISA (ALPCO). AUC was calculated using GraphPad Prism v9.