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Kacal M, Vakifahmetoglu-Norberg H. Isolation of Autophagy Competent Lysosomes from Cancer Cells by Differential Large-Scale Multilayered Density Gradient Centrifugations. Methods Mol Biol. 2022;2445:27-38doi: 10.1007/978-1-0716-2071-7_2.
Kacal, M., & Vakifahmetoglu-Norberg, H. (2022). Isolation of Autophagy Competent Lysosomes from Cancer Cells by Differential Large-Scale Multilayered Density Gradient Centrifugations. Methods in molecular biology (Clifton, N.J.), 244527-38. https://doi.org/10.1007/978-1-0716-2071-7_2
Kacal, Merve, and Vakifahmetoglu-Norberg, Helin. "Isolation of Autophagy Competent Lysosomes from Cancer Cells by Differential Large-Scale Multilayered Density Gradient Centrifugations." Methods in molecular biology (Clifton, N.J.) vol. 2445 (2022): 27-38. doi: https://doi.org/10.1007/978-1-0716-2071-7_2
Kacal M, Vakifahmetoglu-Norberg H. Isolation of Autophagy Competent Lysosomes from Cancer Cells by Differential Large-Scale Multilayered Density Gradient Centrifugations. Methods Mol Biol. 2022;2445:27-38. doi: 10.1007/978-1-0716-2071-7_2. PMID: 34972983.
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Methods Mol Biol. 2022;2445:27-38. doi: 10.1007/978-1-0716-2071-7_2.

Isolation of Autophagy Competent Lysosomes from Cancer Cells by Differential Large-Scale Multilayered Density Gradient Centrifugations.

Methods in molecular biology (Clifton, N.J.)

Merve Kacal, Helin Vakifahmetoglu-Norberg

Affiliations

  1. Department of Physiology and Pharmacology, Solnavägen 9, Biomedicum, Karolinska Institutet, Stockholm, Sweden.
  2. Department of Physiology and Pharmacology, Solnavägen 9, Biomedicum, Karolinska Institutet, Stockholm, Sweden. [email protected].

PMID: 34972983 DOI: 10.1007/978-1-0716-2071-7_2

Abstract

Accurate isolation of functional and intact lysosomes enables the quantification and analyses of abundances, dynamic changes and enrichment levels of lysosomal content, allowing specific lysosomal investigations induced by autophagy. In this protocol chapter, we describe detailed practical instructions and advices for an efficacious lysosomal enrichment and isolation procedure by differential multilayered density gradient centrifugations using human cancer cell lines. By this method, intact and autophagy competent lysosomes can be isolated from cancer cells based on their distinct density and obtained fractions can further be analyzed for functional lysosomal assays, as well as for protein or metabolic loads to identify select spatiotemporal changes by comparative quantitative measurement. This method has been used to enrich lysosomes from a variety of cancer cells with activated chaperone-mediated autophagy, but can be optimized for other cell lines and tissues for multiple autophagy-induced conditions.

© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Keywords: Autophagy; Cancer; Chaperone-mediated autophagy; LAMP-2A; Lysosomes

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