title: Studying Lysosomal Membrane Permeabilization by Analyzing the Release of Preloaded BSA-Gold Particles into the Cytosol.
abstract: In addition to techniques involving assaying the release of endogenous lysosomal molecules into the cytosol, the endocytic system can be preloaded with exogenous fluorescent or electron-dense tracers. These tracers will translocate into the cytosol upon lysosomal membrane permeabilization and have the advantage of being detectable directly without additional labeling. Another benefit is that the tracers can be made more abundant than most endogenous lysosomal molecules, which facilitates their detection. Tracers that can be analyzed with fluorescence microscopy include low-molecular-mass molecules such as sulforhodamine B and also fluorescent polymers of dextran that are available in a wide range of molecular masses. This protocol shows how, for electron-microscopic analysis, cells can be fed with colloidal gold or ferrofluid particles complexed to bovine serum albumin. Although electron microscopy entails a high-resolution analysis, which can be advantageous, we caution how it is important to note that particulate tracers are larger than many endogenous lysosomal molecules and might be released only upon extensive membrane permeabilization.
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: D000818:Animals; D006046:Gold / Q000032:analysis*; D006801:Humans; D007425:Intracellular Membranes / Q000502:physiology*; D008247:Lysosomes / Q000502:physiology*; D008854:Microscopy, Electron / Q000379:methods*; D053758:Nanoparticles / Q000032:analysis*; D010539:Permeability; D012710:Serum Albumin, Bovine / Q000032:analysis*
chemical_list: D012710:Serum Albumin, Bovine; D006046:Gold