C peptide elisa kit

Mice were made to fast for 6 h during glucose and insulin tolerance tests as well as glucose-stimulated insulin secretion tests. After that, they were injected with D-glucose solution (2 g/kg) via intraperitoneal injection (IP). In addition, the mouse blood glucose level was recorded at selected intervals after the IP. Blood samples were kept on ice during collection and centrifuged at 450× g for 10 min at 4 °C, and the obtained plasma was stored at −20 °C. Plasma samples were analyzed using an ultrasensitive C-peptide ELISA kit (Mercodia, Uppsala, Sweden). Measurements were performed on a spark plate reader (TECAN Group Ltd., Männedorf, Switzerland) and analyzed using Prism8 software (GraphPad Software Inc., San Diego, CA, USA.)

Basal c-peptide levels in culture supernatants of EndoC-βH1 cells upon miR-204 up- and down-regulation were measured using the Mercodia c-peptide ELISA kit (Mercodia AB, Uppsala, Sweden) following the manufacturer’s instructions. Differences in c-peptide levels across treatments were compared by calculating the percent change relative to the appropriate control reference.

Both ECs and ECCs were preincubated in Krebs-Ringer bicarbonate with HEPES buffer (KRBH; 115 mM NaCl, 24 mM NaHCO_3, 5 mM KCl, 2.5 mM CaCl_2, 1 mM MgCl₂, 25 mM HEPES) supplemented with 2% BSA (Sigma) containing 2.5 mM glucose (Invitrogen) at 37 °C for 2 h for equilibration. After an additional incubation in fresh buffer for 1 h, cells were incubated in KRBH containing 27.5 mM glucose (Invitrogen) or 2.5 mM glucose (Invitrogen) with 30 mM KCl (Sigma), 100 μM tolbutamide (Sigma) at 37 °C for 1 h. Secreted insulin was measured from the supernatant. To measure total insulin, the cells were lysed in 0.5 ml of acid-ethanol by sonication on a Vibra-Cell sonicator™ (Sonics & Materials, Inc., Newtown, CT, USA) for 40 sec; the cells were then neutralized with the same volume of 1 M Tris-HCl (pH 7.5). Secreted insulin and total insulin were measured using an Ultrasensitive Insulin ELISA Kit (Alpco, Salem, USA) according to the manufacturer’s instructions. A Mercodia C-peptide ELISA Kit (Mercodia, Sylveniusgatan 8A, Sweden) was used to measure secreted human c-peptide after the same procedure described above.

All participants underwent structured assessment at baseline including demographic status, medical history, clinical measurements and laboratory tests after an 8 h overnight fast. All participants eligible for analysis had measurements of FPG, HbA1c, CP, lipid profile (fasting total cholesterol [TC], triglycerides [TG], HDL‐C and calculated LDL‐C), estimated glomerular filtration rate (eGFR calculated by the Chronic Kidney Disease Epidemiology Collaboration equation)²⁵ and random spot urinary albumin to creatinine ratio. All biochemical assays were performed by the Department of Chemical Pathology at the PWH with external accreditation. Plasma CP levels in the HKDR cohort were measured by Mercodia@ C‐peptide ELISA kit with lower detection limit of 25 pmol/L, within‐assay variation of <4.8% and total assay variation of <6.8%. Plasma CP levels in the BHBHK and HKFDS cohorts were measured by radioimmunoassay (Novo Nordisk, Copenhagen, Denmark) with lower detection limit of 0.1 nmol/L, an intra‐assay variation of 3.4% and an inter‐assay variation of 9.6%.²⁶