2.6. LDL-oxidation

Native (n)LDL (Cell sciences, MA, USA) oxidation was performed as described by Galle and Wanner (38) and Steinbrecher (39). nLDL was suspended in endotoxin-free PBS without Ca²⁺, Mg²⁺ (LONZA, Ratingen, Germany) to a final concentration of 2 mg protein/ml, and dialyzed using Vivaspin™ 20—System (Thermo Fisher Scientific GmbH, Schwerte, Germany). The Vivaspin™ 20 centrifugal concentrator was sterilized with 70% EtOH for 10 min at 3,000 × g. Afterward, the Vivaspin™ 20 was washed with aqua dest (endotoxin-free). Then, nLDL suspended in PBS was transferred into the Vivaspin™ 20 and centrifuged for 20 min at 4,500 × g. Two washing steps with PBS were performed to remove ethylene diamine tetraacetic acid (EDTA) from the nLDL. CuSO₄ was added to the EDTA-free nLDL and incubated overnight in the dark by constantly rotating. After 24 h, oxidation was stopped by adding EDTA (50 μM) and set in the dark for 1 h by continually rotating. After that, the oxLDL was washed three times with PBS. Subsequently, the oxLDL/PBS mixture was transferred by filtration through a 0.2 μm syringe filter to an endotoxin-free tube. The protein concentration was measured by Pierce™ BCA (bicinchoninic acid) Protein Assay (Thermo Scientific, Rockford, USA). We used different methods to determine the degree of oxidation: (1) trinitrobenzene sulfonic acid (TNBSA, Thermo Fisher Scientific GmbH), which measures free amino groups (40), (2) relative electrophoretic mobility (REM) by agarose gel electrophoresis and visualization by staining with Coomassie Blue (41), and 3. by spectrophotometric analysis (absorbance spectrum between 400 and 700 nm) (38).