Lysosomal intracellular activity assay kit

Lysosomal activity was measured using the commercial Lysosomal Intracellular Activity Assay Kit according to the manufacturer’s instructions (Abcam, ab234622). Cells were incubated with a self-quenched substrate, washed with 1 mL of ice-cold 1X assay buffer and immediately imaged. Single plane images were taken using a Leica TCS STED CW SP8 laser scanning microscope using the 63X oil-immersion objective and 1.5X zoom. Images were analyzed with FIJI/ImageJ and mean fluorescence intensity was measured for each individual lysosome. Lysosomal fluorescence intensity values proportionally correlate with the amount of lysosomal activity [86 (link)]. Intensity, lysosomal number and occupied area values were normalized to the cell area. Data are shown normalized to the control of each experiment to reduce interexperimental variability

Seventy-two hours after IP injection of 4 mL of 4% thioglycollate broth (Brewer thioglycollate medium, Fluka, Sigma Aldrich, St. Louis, MO), macrophages were harvested from the peritoneum of euthanized EKO mice with ice-cold PBS, centrifuged, and suspended in DMEM (Dulbecco’s modified Eagle’s medium) medium (Lonza, Basel, Switzerland) supplemented with penicillin/streptomycin (100 U/mL each; Gibco, Rodano, Milan, Italy) and plated. After one hour, plates were washed, and adherent cells were left for 16 hours in DMEM containing 10% fetal calf serum, then treated with DMEM containing 0.2% BSA (Sigma Aldrich, St. Louis, MO) and either Bafilomycin 1×, acetylated LDL 50 μg/mL (ThermoFisher, Milan, Italy; Catalog No. L35354), or acetylated LDL 50 μg/mL supplemented with increasing concentrations of HDL (100, 250, and 500 μg/mL; Merck, Darmstadt, Germany; Catalog No. LP3-5MG), for 48 hours. Lysosomal activity was measured with a self-quenched substrate, with Bafilomycin 1× as control, following manufacturer’s instructions (Lysosomal Intracellular Activity Assay kit, Abcam, Cambridge, United Kingdom; Catalog No. ab234622). Cells were fixed and counterstained with DAPI, then the signal was quantified for each treatment by fluorescence microscopy in 6 randomly chosen 63× fields among biological replicates from 3 independent experiments per group with ImageJ.^{49 (link)}