Lysosomal function analysis

Lysotracker red DND-99 (Thermo Fisher Scientific), a fluorescent acid tropic probe for labeling and tracking acidic organelles in live cells, was used to visualize lysosomes⁴⁶. Confluent VSMCs were treated with the calcification culture medium for 7 days, and then added with 1 μM Lysotracker red, followed by culture for 2 h. Moreover, Magic Red Cathepsin B (ImmunoChemistry Technologies) was also used to examine lysosomal function. Confluent VSMCs were treated with the calcification culture medium for 7 days, and then added with cathepsin B substrate reagent, MR-(RR)₂, followed by incubation for 1 h. VSMCs were fixed with 4% PFA and stained with anti-SM22α antibody. Nuclei were stained with DAPI. Cells were visualized and images were captured using with Zeiss LSM 880 Confocal microscope. To quantify the function of lysosomes, the fluorescent intensity of lysosomes in each field was analyzed using Image J software. At least 5 fields per condition were measured.