Proinsulin elisa

Static insulin secretion assay was performed on days 27–30 of differentiation; 10 islet-like clusters of cells were used per experiment. Islet-like clusters were pre-incubated for 1 h in Kreb’s Ringer Buffer (128 mM NaCl, 5 mM KCl, 2.7 mM CaCl₂, 1.2 mM MgSO₄, 1 mM NaHPO₄, 1.2 mM KH₂PO₄, 5 mM NaHCO₃, 10 mM HEPES, 0.1% Bovine Serum Albumin, pH = 7.4) containing 3.3 mM glucose, washed and incubated for another hour in 3.3 mM glucose and the medium was collected. Subsequently, 200 µl of buffer containing 16.7 mM glucose or 400 µM tolbutamide (abcam, ab120278) or 30.5 mM KCl was used to treat cells for 1 h, after which the medium was collected. Insulin content was measured by acid ethanol extraction; cells were resuspended in 50 µl of water and sonicated for 15 s. The sonicate is mixed with acid ethanol (0.18 M HCl in 96% ethanol (vol/vol)), in a 1:3 ratio of sonicate to acid ethanol. The mixed solution is incubated at 4 °C for 12 h. Human C-peptide, human insulin, and human proinsulin secretion and content were measured using Ultrasensitive C-peptide ELISA (Mercodia, 10-1141-01), Insulin ELISA (Mercodia, 10-1113-01), and ProInsulin ELISA (Mercodia, 10-1118-01) kit according to the manufacturer’s protocol. Mouse insulin was measured using an Ultrasensitive Insulin ELISA (Mercodia, 10-1249-01) kit according to the manufacturer's protocol. All samples were handled the same way.

Proinsulin-related peptides were assessed by a proinsulin ELISA (10-1232-01, Mercodia, Uppsala, Sweden). According to the manufacturer’s instructions, this assay does not cross-react with mouse mature insulin or C-peptide but our data suggest that it also detects split proinsulins. IAPP was measured by ELISA (human mature IAPP, EZHA-52K, Millipore, St. Charles, MO, USA). This assay employs a monoclonal capture antibody (F002; binds all molecular forms of proIAPP) and a monoclonal detection antibody (F025; binds amidated IAPP at its C terminus)^{77 (link)} and is 100% cross-reactive to rodent IAPP. To determine active GLP-1, mice were injected with 25 mg kg⁻¹ sitagliptin (sc-364620, Santa Cruz Biotechnology, Dallas, USA) 30 min prior to oral glucose loading (2 g kg⁻¹ glucose) and blood was collected in tubes containing Diprotin A (H-38250050, Bachem, Bubendorf, Switzerland). Active GLP-1 was assessed by the V-PLEX assay (K1503OD-1, Meso Scale Diagnostics) and serum levels of ghrelin (active), leptin, and PYY (total) were analyzed using the U-PLEX metabolic kit (K152ACL-1, Meso Scale Diagnostics). To determine the ileal content of active GLP-1, 3 cm of the terminal ileum was excised, rinsed with PBS, extracted in 0.18 N HCl in 70% EtOH, and assessed with the kit described above.