Human insulin elisa kit

Human recombinant insulin (Cell Prime™ r-insulin) was obtained from Millipore Corporation (MA, USA). Zinc acetate (anhydrous) was purchased from Alfa Aesar, (MA, USA). Chitosan (5 kDa, ~10% degree of acetylation) was purchased from Glentham Life Sciences (WIL, UK). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) was procured from Creosalus Inc. (KY, USA). 2,4,6-Trinitrobenzenesulfonic acid solution (TNBSA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (MO, USA). N-hydroxysuccinimide (NHS) was procured from Alfa Aesar (MA, U.S.A.). Oleic acid was purchased from Spectrum Chemical (NJ, USA). Streptozotocin was procured from Enzo Life Sciences (NY, USA). MicroBCA protein assay kit was bought from Pierce Biotechnology Inc. (Rockford, IL, USA). Human insulin ELISA kit was purchased from Mercodia (Uppsala, Sweden). Rat Immunoglobulin G (IgG) was obtained from Alpha Diagnostic International (TX, USA). Human embryonic kidney (HEK 293) cell line, Dulbecco’s modified Eagle’s medium (DMEM) and phosphate buffered saline (PBS) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). All other reagents were purchased in analytical grade and used without any modification.

EndoC-βH1 cells were preincubated with culture medium containing 2.8 mM glucose for 24 hours. Cells were then incubated in Krebs-Ringer buffer (β-KREBS serum-free medium, Univercell-Biosolutions, Toulouse, France) for 1 hour and sequentially stimulated with 0 mM glucose, 20 mM glucose, or 20 mM glucose + 20 μM forskolin for 40 minutes (modified from^{19 (link),20 (link)}). Insulin release and insulin content were measured using the human insulin ELISA kit (Mercodia, Uppsala, Sweden) in cell-free supernatants and acid-ethanol extracted cell lysates, respectively. Results were normalized by total protein content.^{20 (link)}

Perifusion experiments were performed in Krebs buffer containing 125 mM NaCl, 5.9 mM KCl, 1.28 mM CaCl2, 1.2 mM MgCl2, 25 mM HEPES, and 0.1% bovine serum albumin at 37°C using a PERI4–02 machine (Biorep Technologies). Fifty hand-picked human islets were loaded in Perspex microcolumns between two layers of acrylamide-based microbead slurry (Bio-Gel P-4, Bio-Rad Laboratories). Cells were challenged with either low or high glucose (2.8 mM; 16.7 mM) or potassium chloride (KCl = 20 mM) at a rate of 100 μL/min. After 60 min of stabilization in 2.8mM glucose, cells were stimulated with the following sequence: 10 min at 2.8mM glucose, 20 min at 16.7mM glucose, 10 min at 20mM KCl +16.7mM glucose, and 10 min 2.8mM glucose. Samples were collected every minute on a plate kept at <4°C, while the perifusion solutions and islets were maintained at 37°C in a built-in temperature controlled chamber. Insulin concentrations were determined using commercially available ELISA kits (Mercodia). Total insulin release was normalized per total insulin content using a human insulin ELISA kit (Mercodia). See Table S1 for donor information.