Glucagon Secretion Assay in Isolated Islets

Glucagon secretion was performed by static incubation as previously described.⁶⁵ (link) 15–20 size-matched isolated islets were placed into microfuge tubes and pre-incubated for 60 min at 37°C in 150 μL KRB supplemented with 3 mM glucose; pharmacological agents were added as indicated. Media was subsequently replaced with testing KRB buffer supplemented with glucose and/or pharmacological agents, as indicated. After a 60 min incubation at 37°C, the supernatant was removed and stored at −80°C before being assayed using a glucagon enzyme-linked immunosorbent assay (glucagon ELISA, 10-1271-01, Mercodia AB, Uppsala, Sweden). For content measurements, acidic ethanol was added to islets before sonication for content extraction, and storage at −20°C prior to measurement by glucagon ELISA (Mercodia).