Mono 125i tyr a14 labeled human insulin

Tissue and cell culture medium RPMI 1640 was obtained from GIBCO BRL (Paisley, UK). Guinea pig anti-porcine insulin antibody, mono-¹²⁵I-(Tyr A14)-labeled human insulin, and porcine insulin were from Novo Nordisk (Bagsvaerd, Denmark). Collagenase P was obtained from Boehringer Mannheim GmbH (Mannheim, Germany) and Hanks’ balanced salt solution (HBSS), bovine serum albumin (BSA), and other chemicals were obtained from Sigma Chemical (St. Louis, MO, USA). ISV was purchased from Wako Pure Chemical Industries (Tokyo, Japan) and was added to the medium from a stock solution (10⁻² M) prepared in 99% ethanol.
50 mM palmitic acid: palmitic acid (Sigma) was prepared by dissolving and heating equal molar amounts of NaOH, supplemented with distilled water, to obtain a concentration of 100 mM. It was further diluted with 10% BSA (fatty acid free) to 50 mM fatty acid, with 5% BSA. The stock solution was frozen at −20 °C until usage.
Modified Krebs-Ringer Buffer (M-KRB): 125 mM NaCl, 1.2 mM MgCl₂, 5.9 mM KCl, 1.28 mM CaCl₂, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 5.0 mM NaHCO₃ (pH 7.4; All Sigma). SYTO 24 solution: 5 mM SYTO 24 green fluorescent nucleic acid stain (molecular probes, Invitrogen, Eugene, OR, USA) in dimethyl sulfoxide was diluted to a final concentration of 0.01 mM.

Insulin was analyzed by radioimmunoassay using guinea pig anti-porcine insulin antibody (Novo Nordisk, Bagsvaerd, Denmark). Mono-¹²⁵I-(Tyr A14)-labeled human insulin (Novo Nordisk) was used as tracer and rat insulin (Novo Nordisk) was used as a standard. Ethanol was added to separate bound and free radioactivity. The inter- and intra-assay variation coefficients were both less than 5%.