2.6. Mitochondrial Superoxide Staining Assay

MitoSOX^TM Red superoxide indicator (Invitrogen) is a fluorogenic dye that is selective for mtO2^•− in live cells. It localizes into cellular mitochondria and is readily oxidized by superoxide, but not other sources of ROS or nitrogen species. The oxidation of the probe is prevented by superoxide dismutase and exhibits a bright red fluorescence upon binding to nucleic acids (excitation=emission maxima ¼ 510 = 580 nm). After Lp(a) exposure, ventricular cardiomyocyte AC16 cells were incubated with MitoSOX^TM Red (3 μM) for 10 min at 37 °C as indicated by the manufacturer’s instructions. The AC16 cells were collected by trypsinization and washed in phosphate-buffered saline (PBS) supplemented with 2% fetal bovine serum (FBS). AC16 cells were fixed in 2% paraformaldehyde and suspended in PBS. Measurements were performed in duplicates using the BD flow cytometer (BD Biosciences). MitoSOX^TM Red was excited at 488 nm, and the data were collected by a 575 = 26 nm (FL2) channel. The data were presented by histograms in terms of the mean intensity of MitoSOX^TM fluorescence normalized to those of the garcinol controls.