Sodium chloride, calcium chloride, sodium hydroxide, ethylenediaminetetraacetic acid (EDTA), Tween 20, and bovine serum albumin (BSA) were from EMD Chemicals (San Diego, CA). Dextrose, RPMI 1640, gentamicin, and fetal bovine serum were from Thermo Fisher Scientific (Waltham, MA). Collagenase P (from Clostrdium histolyticum) was acquired from Roche Diagnostics (Indianapolis, IN). Monoclonal insulin antibody (Ab) was purchased from Meridian Life Science, Inc. (Saco, ME). Fluorescein isothiocyanate labeled insulin (insulin*) and other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless noted otherwise. All solutions were made with Milli-Q (Millipore, Bedford, MA) 18 MΩ·cm ultrapure water and filtered using 0.2 μm nylon syringe filters (Pall Corporation, Port Washington, NY).
Immunoassay reagents (insulin* and Ab) were prepared in TEAT-40 (pH 7.4) composed of 25 mM tricine, 40 mM NaCl, 1 mM EDTA, 0.1% Tween-20 (w/v), and 1 mg mL−1 BSA. 200 nM of the insulin* and Ab were placed in their respective reservoirs for experiments. Islets or insulin standards were placed in a balanced salt solution (BSS) (pH 7.4) that consisted of 125 mM NaCl, 2.4 mM CaCl2, 1.2 mM MgCl2, 5.9 mM KCl, 25 mM tricine, 1 mg mL−1 BSA, and the appropriate glucose concentration as described in the text.
Immunoassay reagents (insulin* and Ab) were prepared in TEAT-40 (pH 7.4) composed of 25 mM tricine, 40 mM NaCl, 1 mM EDTA, 0.1% Tween-20 (w/v), and 1 mg mL−1 BSA. 200 nM of the insulin* and Ab were placed in their respective reservoirs for experiments. Islets or insulin standards were placed in a balanced salt solution (BSS) (pH 7.4) that consisted of 125 mM NaCl, 2.4 mM CaCl2, 1.2 mM MgCl2, 5.9 mM KCl, 25 mM tricine, 1 mg mL−1 BSA, and the appropriate glucose concentration as described in the text.