Insulin Quantification Protocol

Sodium chloride, calcium chloride, sodium hydroxide, ethylenediaminetetraacetic acid (EDTA), Tween 20, bovine serum albumin (BSA), and ammonia were from EMD Chemicals (San Diego, CA). Dextrose, RPMI 1640 containing L-glutamine and 11 mM glucose, gentamicin, and fetal bovine serum (FBS) were from Thermofisher Scientific (Waltham, MA). Collagenase P (from Clostridium histolyticum) was acquired from Roche Diagnostics (Indianapolis, IN). Monoclonal anti-insulin antibody (Ab) was purchased from Meridian Life Science, Inc. (Saco, ME). Cy5-labeled insulin (Ins*) was prepared in-house, as previously described.^{20 (link)} All 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 (Ins* 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. Insulin standards were prepared in balanced salt solution (BSS) (pH 7.4) which consisted of 125 mM NaCl, 2.4 mM CaCl₂, 1.2 mM MgCl₂, 5.9 mM KCl, 25 mM tricine, with pH adjusted to 7.4 with NaOH. An additional 1 mg mL⁻¹ BSA was added to the BSS with different levels of glucose as stated in the text.