Isolation of Lysosomes from Mammalian Tissues and Cultured Cells.
Lysosomes participate within the cells in the degradation of organelles, macromolecules, and a wide variety of substrates. In any study on specific roles of lysosomes, both under physiological and pathological conditions, it is advisable to include methods that allow their reproducible and reliable isolation. However, purification of lysosomes is a difficult task, particularly in the case of cultured cells. This is mainly because of the heterogeneity of these organelles, along with their low number and high fragility. Also, isolation methods, while disrupting plasma membranes, have to preserve the integrity of lysosomes, as the breakdown of their membranes releases enzymes that could damage all cell organelles, including themselves. The protocols described below have been routinely used in our laboratory for the specific isolation of lysosomes from rat liver, NIH/3T3, and other cultured cells, but can be adapted to other mammalian tissues or cell lines.
Intracellular Protein Degradation and Rare Diseases Laboratory, Centro de Investigación Príncipe Felipe, C./Eduardo Primo Yúfera, 3, Valencia, 46012, Spain. caguado@cipf.es.|Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Valencia, Spain. caguado@cipf.es.
Carmen
C
Aguado
Intracellular Protein Degradation and Rare Diseases Laboratory, Centro de Investigación Príncipe Felipe, C./Eduardo Primo Yúfera, 3, Valencia, 46012, Spain.
Eva
E
Pérez-Jiménez
Intracellular Protein Degradation and Rare Diseases Laboratory, Centro de Investigación Príncipe Felipe, C./Eduardo Primo Yúfera, 3, Valencia, 46012, Spain.
Marcos
M
Lahuerta
Intracellular Protein Degradation and Rare Diseases Laboratory, Centro de Investigación Príncipe Felipe, C./Eduardo Primo Yúfera, 3, Valencia, 46012, Spain.|Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Valencia, Spain.
Erwin
E
Knecht
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002460:Cell Line; D002462:Cell Membrane; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000379:methods; D008099:Liver / Q000166:cytology; D008247:Lysosomes; D051379:Mice; D015388:Organelles / Q000378:metabolism*; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism
Cultured cells; Differential and gradient centrifugation; Liver; Lysosomes; Subcellular fractionation