Preparation of Advanced Oxidation Protein Products

The preparation of AOPPs has been described previously [5 (link), 6 (link)]. Briefly, mouse serum albumin (MSA, Sigma, St. Louis, MO, USA) solution (20 mg/ml) was incubated with 40 mM hypochlorous acid (Fluke, Buchs, Switzerland) at room temperature in phosphate-buffered saline (PBS, pH = 7.4) for 30 min. To remove the free hypochlorous acid, the prepared samples were dialyzed against PBS at 4 °C for 12 h. Finally, all samples were passed through a Detoxin-Gel™ Endotoxin Removing Gel (Pierce, Rockford, IL) to remove contaminated endotoxin. The endotoxin levels in AOPPs–MSA and unmodified MSA were measured by Limulus Amoebocyte Lysate kit (Sigma, St Louis, MO) and were found to be below 0.05 ng/mg. The concentration of prepared AOPP was detected as previously described [5 (link), 6 (link)].