title: The Use of Lysosomotropic Dyes to Exclude Lysosomal Membrane Permeabilization.
abstract: Progressive lowering of pH is characteristic for the endocytic pathway and enables efficient degradation of molecules by hydrolytic enzymes at its distal end. The existence of the proton gradient over the endosomal/lysosomal membranes depends on the action of the vacuolar ATPase (v-ATPase). During lysosomal membrane permeabilization (LMP), protons leak through the destabilized membrane, resulting in loss of the pH gradient. Here, we present a protocol showing how this effect can be detected by staining cells with lysosomotropic dyes, which accumulate in acidic organelles after protonation. During LMP, cells lose the ability to retain these dyes and therefore appear pale. Among the most commonly used lysosomotropic dyes are LysoTracker reagents and acridine orange. Cells can be analyzed with a fluorescence microscope; however, flow-cytometric analysis enables fast, objective, and reliable evaluation of differences between samples. Advantages of the technique include the fact that sample preparation is relatively simple and can be scaled-up to test several different compounds or conditions. However, as we will discuss, cells treated with v-ATPase inhibitors also lose the pH gradient across lysosomal membranes and cannot be stained with lysosomotropic dyes, although this is not accompanied by LMP. Therefore, merely observing loss of staining is not in itself a proof of LMP.
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, J. Stefan Institute, SI-1000 Ljubljana, Slovenia; Department of Biosciences, University of Oslo, NO-0371 Oslo, Norway;
authors.forename: Urška
authors.initials: U
authors.lastname: Repnik
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, J. Stefan Institute, SI-1000 Ljubljana, Slovenia;
authors.forename: Maruša Hafner
authors.initials: MH
authors.lastname: Česen
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, J. Stefan Institute, SI-1000 Ljubljana, Slovenia; Center of Excellence CIPKEBIP, SI-1000 Ljubljana, Slovenia.
authors.forename: Boris
authors.initials: B
authors.lastname: Turk
mesh_terms: D004396:Coloring Agents / Q000378:metabolism*; D003584:Cytological Techniques / Q000379:methods*; D007425:Intracellular Membranes / Q000502:physiology*; D008247:Lysosomes / Q000378:metabolism*; D010539:Permeability; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D004396:Coloring Agents