Human c peptide elisa kit

At 12 days of differentiation, 2–3 million cells were detached using TrypLE (Invitrogen), pelleted, and mixed with 10–15 μl matrigel (BD Biosciences) before being transplanted into a kidney capsule of a NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mouse (stock number 005557; The Jackson Laboratory), using methods as previously described for the transplantation of islets (19 (link)). Glucose stimulation assay was performed 3 months after transplantation. Mice were deprived of food overnight (12–14 h); water was available ad libitum. Each mouse was injected intraperitoneally with a glucose solution (1 mg/g body weight). Before and half an hour after glucose injection, capillary blood glucose was measured and venous blood samples were collected from the tail and submandibular vein. Human C-peptide concentration in the mouse serum was measured by using an ultrasensitive human C-peptide ELISA kit (Mercodia). Nephrectomy was performed on some mice after human C-peptide was detected in the mouse serum. For ER stress studies, TG was administrated by one dose injection of 1 mg/kg of TG intraperitoneally.

After pre-incubation with Krebs-Ringer solution containing bicarbonate, HEPES and D-glucose (KRBH; 129 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 10 mM HEPES, 2.5 mM D-glucose (all purchased from Wako) and 0.1% (w/v) BSA) at 37°C for 2 h, the samples were incubated with KRBH containing 2.5 mM or 25 mM D-glucose or 30 mM KCl (Wako) or 0.5 μM IBMX (Sigma) at 37°C for 1 h. The C-peptide level in culture supernatants was measured with a human C-PEPTIDE ELISA kit (Mercodia) according to manufacturer's instructions. Cells were lysed in Spheroid Lysis Buffer (Scivax), and dsDNA content was measured by a PicoGreen dsDNA Quantitation Kit (Life Technologies) and Wallac 1420 multilabel/Luminescence counter (PerkinElmer).

Approximately 2 × 10⁶ differentiated cells at the end of stage 6 from 3 different batches were collected and preincubated at 37 °C for 30 min with DMEM (2 mM glucose) containing 10 mM HEPES and 0.1% BSA. Cells were washed twice with DMEM (no glucose) containing 10 mM HEPES+0.1% BSA and then incubated at 37 °C for 1 h in 1 ml DMEM +2.0 mM glucose+10 mM HEPES +0.1% BSA per well. The culture medium was collected and the wells were washed twice with DMEM (no glucose)+10 mM HEPES +0.1% BSA; then, the same cells were further incubated at 37 °C for 1 h in 1 ml DMEM +20 mM glucose +10 mM HEPES +0.1% BSA. The C-peptide concentrations in culture supernatants were determined using a human C-peptide ELISA kit (Mercodia) according to the manufacturer's instructions. Double samples from each supernatant were measured for OD (optical density) by the spectrophotometer (EnVision: Perkin Elmer). After measuring OD, the number of the cells was counted by enzymatic dissociation, and the concentration was converted per 2 × 10⁶ cells for the comparison of each lot of the experiment. We calculated the mean ± SEM for each sample. We compared the concentration of C-peptide between low glucose condition and high glucose condition with the same cells.