Mouse glucagon elisa kit

A total of 30 islets/well were seeded in a 96-well plate and transfected with either the circGlis3 overexpression plasmid or circGlis3 knockdown plasmid and cultured for 48 h. Then, the islets were preincubated for 30 min at 37 °C in KRBH balanced buffer and supplemented with 0.1% bovine serum albumin and 2.5 mM glucose. Subsequently, the buffer was changed, and the islets were incubated with 2.5 mM or 16.7 mM glucose for 2 h. Immediately after incubation, aliquots of the medium were removed for glucagon and somatostatin assays. The islets were then lysed via ultrasonication to extract the total cellular glucagon content. Secreted glucagon and somatostatin in the supernatants or lysates were quantified using either a mouse glucagon ELISA kit or somatostatin ELISA kit (both from Mercodia).

Plasma insulin from the neonates was measured on post-natal day 4 using Ultrasensitive mouse insulin ELISA kit (Catalog no. 10-1249-01). In adult mice, plasma insulin measurements were conducted in mice fasted for ~4 hours followed by intraperitoneal injection of 2mg dextrose/g body weight at 0-, 15-, and 30-min post injection. Tail blood samples were collected in Microvette^® CB 300 K2 EDTA tubes (Catalog no. 16.444.100, Sarstedt) at the indicated time points and plasma insulin were measured using mouse ultrasensitive insulin ELISA kit (Mercodia Inc., Sweden). For in vitro insulin and glucagon secretion assays, islets were isolated from mice fed a standard chow diet and were incubated over-night in RPMI supplemented with 0.5mg/mL BSA. On the following day, islets were equilibrated in DMEM containing 0.5 mg/mL BSA, 0.5 mM CaCl₂ and 10.0 mM HEPES (DMEM*) supplemented with 10% FBS and 5.5 mM glucose for 1 h at 37°C, 5% CO₂. 20 islets/well were picked into 400 uL DMEM* without FBS at glucose concentrations specified in the figures in 24-well plate(s) and insulin or glucagon secretion was measured over 1 h at 37°C and stored at −20°C until analysis. Insulin secretion was measured using mouse insulin ELISA kit (Catalog no. 10-1247-01, Mercodia Inc, Sweden) and glucagon secretion was measured using the mouse glucagon ELISA kit (and 10-1281-01, Mercodia Inc, Sweden).