Abstract
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.
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Acknowledgements
Current work in the laboratory is supported by grants from the Spanish Ministry of Economy and Competitivity (BFU2011-22630 and SAF2014-54604-C3-2-R), Generalitat Valenciana (Prometeo 2009/051), and Instituto de Salud Carlos III (ACCI2015 action from CIBER on Rare Diseases). The authors would like to acknowledge networking support by the Proteostasis COST Action (BM1307).
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Aguado, C., Pérez-Jiménez, E., Lahuerta, M., Knecht, E. (2016). Isolation of Lysosomes from Mammalian Tissues and Cultured Cells. In: Matthiesen, R. (eds) Proteostasis. Methods in Molecular Biology, vol 1449. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3756-1_19
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DOI: https://doi.org/10.1007/978-1-4939-3756-1_19
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