Ultrasensitive c peptide elisa

To measure metabolic markers, blood was drawn into EDTA tubes. Plasma was obtained after centrifugation, aliquoted and then stored at −80 °C. Betatrophin concentration was determined using ELISA (Wuhan EIAAB) as reported previously [24 (link), 36 (link), 37 ]. The assay showed linearity at dilutions ranging from 1:10 to 1:40. No significant cross reactivity with other proteins has been observed. Intra-assay coefficients of variation were 1.2–3.8 %, while the inter-assay coefficients of variation were 6.8–10.2 %. C-peptide was measured using Mercodia Ultrasensitive C-peptide ELISA according to the manufacturer’s instructions (Mercodia, Uppsala, Sweden). Inter- and intra-assay coefficient of variation was <5 %.

The pcDNA3.1(+) N-DYK empty vector, pcDNA3.1(+) N-DYK vectors containing cDNA of Got1 or INS1, pcDNA3.1(+) vectors containing cDNA of Slc17a7 or Slc17a6 were purchased from Genscript (Piscataway, NJ, USA). Got1 KO β-cell lines were reverse-transfected with a combination of INS1 (0.4 μg/mL) and either Got1 (0.46 μg/mL) or empty pcDNA3.1(+) N-DYK construct (control, 0.46 μg/mL). VGLUTs (Slc17a7, Slc17a6, and Slc17a8) triple KO β-cell lines were reverse-transfected with either INS1 (0.4 μg/mL, control) or a combination of INS1 (0.4 μg/mL) and Slc17a7 (0.0025 μg/mL) or Slc17a6 (0.0025 μg/mL). For transfection, Lipofectamine 2000 transfection reagent (Life Technologies) was used according to the manufacturer’s instruction. After 48 hours of incubation, human C-peptide secretion experiment was performed in the same manner as in the insulin secretion experiment. Human C-peptide released in the incubation buffer and cellular C-peptide content were measured by Ultrasensitive C-peptide ELISA (Mercodia, Uppsala, Sweden).

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.