Phosphatidylcholine assay kit

Serum levels of alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were measured with assay kits for ALT and ALP (Catachem, Bridgeport, CT), respectively. Hepatic contents of triacylglycerol (TG) and non-esterified fatty acids (NEFA) were quantified using Wako TG kit and NEFA kit (Wako Chemicals USA Inc.) as described elsewhere [7 (link)–10 (link)]. Hepatic levels of PC, DG, and malondialdehyde (MDA) were measured by Phosphatidylcholine assay kit, Diacylglycerol assay kit, and OxiSelect™ TBARS assay kit, respectively (Cell Biolabs, Inc., San Diego, CA). Hepatic phospholipase D (PLD) activity was determined by use of a colorimetric assay kit purchased from BioVision (Milpitas, CA). Mitochondial fraction was isolated by differential centrifugation of sucrose and hydrogen peroxide (H₂O₂) and glutathione (GSH) measured using PeroxiDetect kit and glutathione assay kit, respectively (Sigma-Aldrich, Inc., St. Louis, MO) [9 (link)]. Measurement of protein concentrations was carried out using BCA™ protein assay kit (Thermo Scientific, Rockford, IL).

Clear bottom black plates (96-well) were coated with 10 µg/mL fibrinogen (Sigma-Aldrich) followed by blocking with 2% (w/v) bovine serum albumin. Integrin liposomes containing 100 mM glucose were diluted with binding buffer (20 mM Tris-Cl, 100 mM NaCl, 0.5 mM CaCl2, and 0.5 mM MgCl2, pH 7.4) containing 100 mM (303 mOsm/L), 60 mM (263 mOsm/L), or 40 mM (243 mOsm/L) glucose. The diluted liposomes were then added on fibrinogen-coated plate and incubated for 10 min at room temperature in the presence or absence of 10 µM eptifibatide or 1 mM MnCl2. After washing, the amount of fibrinogen-bound liposome was quantified using the Phosphatidylcholine Assay kit (Cell Biolabs, STA-600).