Human ultrasensitive c peptide elisa

Male NMRI-Foxn1^nu/Foxn1^nu and female SCID CB-17/Icr-Prkdc^scid/Rj mice were transplanted with human insulin-producing EndoC-βH1 cells as described^{44 (link),47 (link)}. Male NMRI-Foxn1^nu/Foxn1^nu mice were used for methodological development/validation, but all follow up experiments were performed in female SCID CB-17 mice. At the day of inoculation, 4–6 × 10⁶ EndoC-βH1 cells were seeded on a rubber toric joint (EFJM), supported in Matrigel HC (Corning) supplemented with MmVEGF-164 (1 ng/ml) (BioLegend). The cell-containing or the empty vehicle rubber rings were then inserted under the epimysium in the biceps or quadriceps femoris muscle. The mice were anesthetized with 3 % isoflurane. They received short-term analgesic (Buprenorphine 0.1 mg/kg) and long-term analgesic (3 mg/ml of Acetaminophen-supplemented water for 10 consecutive days) respectively before and after the surgery. Random glycaemia was measured weekly with an ACCU-CHEK Nano glucometer (ROCHE). Once the tumour became palpable, the mice received 20 % glucose-supplemented drinking water to counter the progressive hypoglycemia induced by the EndoC-βH1 cells. Human C-peptide was measured in plasma with a human Ultrasensitive C-peptide ELISA (Mercodia). Kelly tumour-bearing mice were generated by s.c. injections of 5 × 10⁶ cells mixed 1:1 with Matrigel (Corning) in the hind leg of female Crl:NU(NCr)-Foxn1^nu mice.

First, cells of a prefixed number were collected by a cell sorter (differentiated cell clusters: 1 × 10⁶ cells, Human pancreatic islets: 4 × 10⁵ cells). The cells were clustered in a shaking culture and used for the experiment. Human pancreatic islets or differentiated cell clusters from iPS cells were washed twice with Krebs–ringer solution and then pre-incubated for 2 h in low glucose (2.8 mM). The cell clusters were washed twice with glucose-free Krebs–ringer solution, incubated in low glucose Krebs–ringer solution for 1 h, and supernatants were removed. The cells were then incubated in 1 ml of low glucose medium for 45 min, and supernatants were collected, followed by incubated in 1 ml of high glucose (28 mM) Krebs–ringer solution for 45 min and the supernatant was collected. In some cases, cells were again incubated in a low glucose Krebs–ringer solution for 45 min. Insulin in the supernatants were detected using Human Ultrasensitive C-peptide ELISA and Human C-peptide ELISA (MERCODIA).

Cells were washed with Krebs buffer (128 mM NaCl, 5 mM KCl, 2.7 mM CaCl₂, 1.2 mM MgCl₂, 1 mM Na₂HPO₄, 1.2 mM KH₂PO₄, 5 mM NaHCO₃, 10 mM HEPES, 0.1% BSA) and then pre-incubated in 2.8 mM D- (+)-glucose Krebs buffer for 2 h. Cells were then incubated in fresh-low glucose Krebs, followed by 16.7 mM and then in Krebs buffer with 2.8 mM glucose and 30 mM KCl for 30 min at each condition. After each incubation, we collected supernatants. Between incubations, cells were washed two times with Krebs buffer. This procedure was repeated at least three times for different time points and coculture combinations. In the end, cells were dispersed into single cells using TrypLE Express and quantified by Countess (Invitrogen). C-peptide was measured using the Human Ultrasensitive C-peptide ELISA (Mercodia). The C-peptide amount was normalized to the cell number (µIU/3000 cells).