C peptide elisa

Human C-peptide levels were measured in mouse serum using ultrasensitive C-peptide ELISA (Mercodia, Uppsala, Sweden) according to the manufacturer’s protocol. Data are represented as average values (±SD) from two donors with 3–5 mice each donor.

For measurement of intracellular of C-peptide content, we harvested two-week old rGBC by rinsing with prewarmed PBS 3 times and lysing in 1X cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) with a complete mini-protease inhibitor cocktail (Roche, Mannheim, Germany). The cells were scraped off the dish, sonicated, and centrifuged at 14,000g at 4^°C, saving the supernatant for C-peptide ELISA (Mercodia, Uppsala, Sweden). The C-peptide secretion assay was done as described previously [17 (link)]. Specifically, 2-week old rGBC in 12-well plates were incubated in fresh prewarmed day 14 reprogramming medium (MM3) for 2 hours at 37^°C, then followed by rinsing three times with plain Krebs-Ringer bicarbonate buffer (KRBB) 20 minutes each at 37^°C. Subsequently, rGBC were incubated with 150 μL KRBB with or without 1mM or 2.8 mM glucose for 2 hours in a humidified 37^°C incubator. Supernatants were collected, centrifuged to remove cell debris, and stored at -80^°C. Into the same wells, we added 150 μL KRBB with 25 mM glucose and incubated for 2 additional hours and harvested the supernatants. Total protein contents were measured using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Glucose-stimulated C-peptide was measured using Ultrasensitive Human C-peptide ELISA kit (Mercodia, Uppsala, Sweden).