Nylon syringe filter

Bovine serum albumin (BSA), ethylenediaminetetraacetic acid (EDTA), ammonium hydroxide (NH₄OH), and sodium hydroxide (NaOH) were from EMD Chemicals (San Diego, CA). Dextrose was obtained from Fisher Scientific (Pittsburgh, PA). Sulfuric acid (H₂SO₄), nitric acid (HNO₃), hydrogen peroxide (H₂O₂), and hydrofluoric acid (HF) were from Avantor Performance Materials (Center Valley, PA). Cy5 monofunctional N-hydroxysuccinimide ester was from GE Healthcare Bio-Sciences (Piscataway, NJ). A monoclonal antibody to human insulin C-terminal (Ab) was obtained from Meridian Life Science, Inc. (Saco, ME). All other chemicals were obtained from Sigma-Aldrich (Saint Louis, MO), unless otherwise stated. Labeling of bovine insulin with Cy5 (Insulin*) was performed as previously described.^{29 (link)} All buffers were prepared using ultrapure deionized water (NANOpure Diamond TM deionization system, Barnstead International, Dubuque, IA) and filtered using 0.2 µm nylon syringe filters (Pall Corporation, Port Washington, NY).

Sodium tetraborate, dextrose, and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) were from Fisher Scientific (Pittsburg, PA). Cosmic Calf Serum was from HyClone Laboratories (South Logan, UT). Sodium hydroxide (NaOH) and boric acid were purchased from EMD chemicals (San Diego, CA). RPMI 1640 was from Mediatech (Manassas, Va). Acetonitrile (ACN) was from Avantor Performance Materials (Center Valley, PA). Gentamicin was from Lonza (Walkersville, MD). Collagenase P was purchased from Roche Diagnostics (Indianapolis, IN). All other chemicals were from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. All solutions were made with ultrapure DI water (NANOpure® Diamond system, Barnstead International, Dubuque, IA) and filtered with 0.2 μm nylon syringe filters from Pall Corporation (Port Washington, NY) unless otherwise noted. The pH for all solutions were adjusted using 1 or 5 M NaOH as needed.

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.

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.