LysoTracker leakage assay

RAW264.7 macrophages were seeded in 96‐well CellCarrier Ultra plates (Perkin Elmer) at a density of 6 × 10⁴ cells per well and rested overnight. Cells were loaded with the nuclear dye Nuclear Green LCS1 (Abcam) at a dilution of 1:5,000 and 25 nM LysoTracker DND‐99 (Thermo Fisher Scientific) for 10 min. The cells were washed twice in cDMEM, and the medium was replaced with cDMEM containing 25 nM LysoTracker. The cells were imaged every 3 min at 37°C, 5% CO₂ using an Opera Phenix high‐content screening system (PerkinElmer). First, a baseline was established by imaging four time points, followed by the addition of LLOMe to a final concentration of 1 mM. After 45 min, the cells were washed twice in cDMEM, the medium was replaced with cDMEM containing 25 nM LysoTracker, and lysosomal recovery was followed for 72 min.