Measuring Intracellular ROS Production

ROS production was evaluated as previously described [18 (link)]. Briefly, cells were seeded in a 24-well plate at a density of 30,000 cells/well and incubated for 30 min with 5 μM of carboxy-H2DCFDA (Molecular Probes, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), a carboxyl derivative of 2′,7′-dichlorofluorescein, in HEPES buffer. Then, cells were treated with sera or isolated lipoprotein fractions from carriers of LCAT mutations and controls for 1 h. The oxidation of the probe was detected by monitoring fluorescence at 517–527 nm with the Synergy H1 multi-mode reader and the Gen5 software (BioTek, Agilent, Santa Clara, CA, USA). For each sample, fluorescence was normalized by the protein concentration of the total cell lysate, measured by the micro-bicinchoninic acid assay (Thermo Scientific, Waltham, MA, USA).