(A) Lysosomal membrane permeabilization (LMP) in cultures of murine J774 macrophages that were treated with azithromycin (AZM) for 45 min or high-molecular-weight desferrioxamine (H-DFO)/ammonium chloride (NH
Cl) for 4 h at the indicated concentrations and then oxidatively stressed. To initiate controlled LMP the lysosomotropic detergent MSDH was used. LMP was assessed by the cytosolic/nuclear green fluorescence due to the acridine orange leakage into the cytosol (arbitrary units; A.U.) in non-oxidatively stressed/non-MSDH treated cells (open symbols) and in oxidatively stressed/MSDH treated cells immediately after a 1-h oxidant exposure (filled symbols). NH4Cl-treated J774 macrophages exhibited pronounced granularity (observed by phase contrast microscopy) that resulted in a reduced emission of green fluorescence. Values are the means ± 1 SD (n = 6; independently performed). Significant differences are indicated as # p < 0.05 (cells either oxidatively stressed or MSDH treated vs. non-oxidatively stressed/non-MSDH treated control cells). * p < 0.05. (B) Detailed micrograph of control cells stained with AO. Lysosomes loaded with AO appear as red-fluorescent dots, while AO at a low concentration (e.g. AO-binding nuclear structures) emits a weak green fluorescence. (C) Detailed micrograph of the same cells following treatment with 200 μM MSDH for 5 min. Note cell shrinkage, an almost complete loss of intact red-fluorescent lysosomes and a massive leakage of AO into the cytosol, the latter resulting in a strong green fluorescence emitted from the nucleus and the surrounding cytoplasm.