Venous blood from normocholesterolemic volunteers was collected into tubes containing ethylenediamine tetraacetic acid (EDTA, 1 mg/mL blood). Pooled plasma was immediately separated by centrifugation, at 2500 rpm for 10 min at 4 °C and 1 mM (PMSF); 2 mM benzamidine, 2 μg/mL aprotinine and 20 mM butylated hydroxytoluene (BHT) were added to prevent protease activity and oxidative reactions. LDL (d ~ 1.019 to 1.063 g/mL) was isolated from plasma by preparative sequential ultracentrifugation, using an Optima XE-90 ultracentrifuge (Beckman Coulter, IN, USA) with a fixed angle rotor (T 70.1). Plasma was centrifuged with saline (density = 1.019 g/mL) at 56,000 G for 7 h at 4 °C and the top fraction was removed. The remaining plasma was adjusted with KBr to a density of 1.063 g/mL and centrifuged with saline (density = 1.063 g/mL). The top LDL fraction was removed and dialyzed against 20 mM Tris-HCl buffer, pH 7.4, 20 mM BHT, and 10 mM EDTA for 4 h, at 4 °C, protected from light, with buffer exchanges each 1 h. The LDL (−) was separated from the native LDL (nLDL) by fast protein liquid chromatography (FPLC, ÄKTA Start, GE Healthcare, Chicago, Illinois, USA) using an ion exchange column (Sepharose UNO Q, Bio-Rad Laboratories Inc, Hercules, CA, USA). Total LDL was injected into the column pre-equilibrated with buffer A (10 mM Tris-HCl, pH 7.4) and eluted with a multi-step gradient of buffer B (10 mM Tris-HCl, 1 M NaCl). The isolated LDL (−) fraction was dialyzed against phosphate buffered saline (PBS) and concentrated using a specific device (Vivaspin 20, 100,000 MWCO, GE Healthcare Life Sciences, Uppsala, Sweden). The presence of LPS in LDL samples isolated from human plasma was determined by the limulus amebocyte lysate (LAL) test (Lonza, Verviers, Belgium). Supplies used for LDL isolation and cell culture were treated with E-Toxa-Clean ®Concentrate (Sigma Aldrich, St. Louis, MO, USA). The endotoxin levels of LDL (−), P1A3, and P2C7 peptides were < 0.1 EU (endotoxin unit) for all samples.