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Lysosomes and Lysosomal Diseases - 1st Edition - ISBN: 9780128000793, 9780128002933

Lysosomes and Lysosomal Diseases, Volume 126

1st Edition

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Serial Volume Editors: Frances Platt Nick Platt
Hardcover ISBN: 9780128000793
eBook ISBN: 9780128002933
Imprint: Academic Press
Published Date: 4th February 2015
Page Count: 450
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Table of Contents

  • Series Editors
  • <li>Preface</li> <li>Chapter 1. Methods for monitoring lysosomal morphology<ul><li>Introduction</li><li>1. Lysosomal Form</li><li>2. Lysosomal Function</li><li>3. Lysosomal Failure</li><li>4. Linking Lysosomal Form and Failure</li><li>5. Methods</li><li>6. Discussion</li></ul></li> <li>Chapter 2. A rapid method for the preparation of ultrapure, functional lysosomes using functionalized superparamagnetic iron oxide nanoparticles<ul><li>1. The Endocytic System</li><li>2. The Discovery of Lysosomes and Lysosomal Storage Diseases</li><li>3. Biochemical Features of Lysosomes</li><li>4. Overview of Methods for Purifying Lysosomes</li><li>5. Method for Magnetic Separation of Lysosomes from Whole Cells</li><li>6. Technical Considerations</li><li>7. Techniques for Determining Purity of the Lysosomal Fractions</li><li>8. Discussion</li></ul></li> <li>Chapter 3. TFEB and the CLEAR network<ul><li>Introduction</li><li>1. TFEB Nuclear Translocation Assay</li><li>2. Cell Culture and Treatment</li></ul></li> <li>Chapter 4. Biosynthesis, targeting, and processing of lysosomal proteins: Pulse&#x2013;chase labeling and immune precipitation<ul><li>Introduction</li><li>1. Materials</li><li>2. Methods</li><li>3. Metabolic Labeling and Immune Precipitation of the Lysosomal Protease Cathepsin&#xA0;Z</li><li>4. Discussion</li></ul></li> <li>Chapter 5. Measuring lysosomal pH by fluorescence microscopy<ul><li>Introduction</li><li>1. Materials</li><li>2. Methods</li><li>Conclusion</li></ul></li> <li>Chapter 6. Lysosome fusion in cultured mammalian cells<ul><li>1. Overview of Methods to Study Lysosome Fusion</li><li>2. Choosing an Assay System</li><li>3. Methods</li></ul></li> <li>Chapter 7. RNAi screens of lysosomal trafficking<ul><li>1. Methods of Gene Depletion in Lysosomes</li><li>2. Selection of Screening System</li><li>3. Assay Validation</li><li>4. Validation of Hits</li><li>5. Discussion</li></ul></li> <li>Chapter 8. Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses<ul><li>1. Overview of Wounding Methods and Plasma Membrane Repair Mechanisms</li><li>2. Procedures for Plasma Membrane Wounding</li><li>3. Procedures for Measuring the Extent of Plasma Membrane Repair</li><li>4. Procedures to Measure Exocytosis of Lysosomes</li></ul></li> <li>Chapter 9. Imaging approaches to measuring lysosomal calcium<ul><li>1. Endolysosomal Ca<sup>2+</sup></li><li>2. Assessing Organelle Ca<sup>2+</sup>: General Strategies</li><li>3. Assessing Endolysosomal Ca<sup>2+</sup>: Specific Strategies</li><li>4. Final Remarks</li></ul></li> <li>Chapter 10. Lysosome electrophysiology<ul><li>Introduction</li></ul></li> <li>Chapter 11. Reconstitution of lysosomal ion channels into artificial membranes<ul><li>Introduction</li><li>1. The Bilayer Apparatus</li><li>2. Electrical Equipment Used for Single-Channel Recordings</li><li>3. Painting Bilayers</li><li>4. Ion Channel Incorporation into a Bilayer</li><li>5. Single-Channel Current Amplitude and Conductance Measurements</li><li>6. Choice of Permeant Ion</li><li>7. Measuring the Relative Permeability of Different Ions</li><li>8. Measurements of Liquid Junction Potentials</li><li>9. Single-Channel Gating and Measurements of Open&#xA0;Probability</li><li>10. Noise Analysis</li><li>11. Isolation of Native and Recombinant Purified Lysosomal Ion Channels</li><li>12. Discussion</li></ul></li> <li>Chapter 12. Fluorescence methods for analysis of interactions between Ca<sup>2+</sup> signaling, lysosomes, and endoplasmic reticulum<ul><li>1. ER, Lysosomes, and Ca<sup>2+</sup> Signaling</li><li>2. Pharmacological Tools</li><li>3. Fluorescence Methods</li><li>4. Fluorescence Tools for Analysis of Lysosomes</li><li>5. Ca<sup>2+</sup> Signaling and Lysosomes: Tools and Practical Problems</li><li>6. Single-cell Analyses of Cytosolic Ca<sup>2+</sup> Signals</li><li>7. High-throughput Analyses of Cytosolic Ca<sup>2+</sup> Signals</li><li>8. Tracking Interactions between Lysosomes and ER by Fluorescence Microscopy</li><li>Conclusions</li></ul></li> <li>Chapter 13. Methods for the quantification of lysosomal membrane permeabilization: A hallmark of lysosomal cell death<ul><li>Introduction</li><li>Method 1: Quantification of Cathepsin and &#x3B2;-<i>N</i>-acetyl-glucosaminidase Release into the&#xA0;Cytosol by Enzymatic Activity Measurement</li><li>1. Materials</li><li>2. Protocol</li><li>Method 2: LMP Visualized by Release of Fluorescent Dextran to the Cytosol</li><li>3. Materials</li><li>4. Protocol</li><li>Method 3: LMP Visualized by Cathepsin Immunocytochemistry</li><li>5. Materials</li><li>6. Protocol</li><li>Method 4: Detection of Damaged Lysosomes by Galectin-1 and&#xA0;-3&#xA0;Translocation</li><li>7. Materials</li><li>8. Protocol</li><li>Discussion</li></ul></li> <li>Chapter 14. Measuring the phagocytic activity of cells<ul><li>Introduction</li><li>1. Reasons to Undertake Studies of Phagocytosis</li><li>2. Components of a Phagocytosis Assay</li><li>3. In vitro or In vivo Study?</li><li>4. Methodologies for Analyses of Phagocytosis</li><li>5. Selection of Phagocyte Population</li><li>6. Choice of Target Particle</li><li>7. Targeting Particles to Specific Phagocytic Receptors</li><li>8. Detection of Ingested Particles</li><li>9. Protocol 1. Fc&#x3B3; Receptor-Mediated Phagocytosis of IgG Opsonized Sheep Red&#xA0;Blood&#xA0;Cells by Murine Macrophages</li><li>10. Materials and Reagents</li><li>11. Equipment</li><li>12. Protocol</li><li>13. Protocol 2. FACS Analysis of <i>Mycobacterium bovis</i> Internalization by RAW264.7&#xA0;Cells</li><li>14. Materials</li><li>15. Equipment</li><li>16. Summary</li></ul></li> <li>Chapter 15. Detection and quantification of microbial manipulation of phagosomal function<ul><li>Introduction</li><li>1. Considerations for Choice of Reagents, Cells, and Readouts</li><li>2. Reagents</li><li>3. Methods</li><li>4. Analytical Platforms, Data Collection and Analysis</li><li>5. Discussion</li><li>6. Summary</li></ul></li> <li>Chapter 16. Measuring relative lysosomal volume for monitoring lysosomal storage diseases<ul><li>1. Measuring Relative Lysosomal Volume as an Index of Lysosomal Storage</li><li>2. In Which Circulating Cell Type Should Relative Lysosomal Volume Be Measured?</li><li>3. How to Measure Relative Lysosomal Volume in Blood Cells</li><li>4. Methods</li><li>5. Summary</li></ul></li> <li>Chapter 17. Quantifying storage material accumulation in tissue sections<ul><li>Introduction</li><li>1. Detection of Storage Material</li><li>2. Quantification of Storage Material</li><li>Conclusion</li></ul></li> <li>Chapter 18. Laboratory diagnosis of Niemann&#x2013;Pick disease type C: The filipin staining test<ul><li>Introduction and Rationale</li><li>1. Materials</li><li>2. Methods</li><li>3. Discussion</li><li>Concluding Remarks</li></ul></li> <li>Volumes in Series</li> <li>Index</li>


This new volume of Methods in Cell Biology looks at methods for lysosomes and lysosomal diseases.  Chapters focus upon practical experimental protocols to guide researchers through the analysis of multiple aspects of lysosome biology and function. In addition, it details protocols relevant to clinical monitoring of patients with lysosomal diseases. With cutting-edge material, this comprehensive collection is intended to guide researchers for years to come.

Key Features

  • Covers sections on model systems and functional studies, imaging-based approaches and emerging studies
  • Chapters are written by experts in the field
  • Cutting-edge material


Researchers and students in cell, molecular and developmental biology


No. of pages:
© Academic Press 2015
4th February 2015
Academic Press
Hardcover ISBN:
eBook ISBN:


Praise for the Series:
"The series is invaluable for workers at all levels of cell biology." --Nature

Ratings and Reviews

About the Serial Volume Editors

Frances Platt

Frances Platt

Prof. Frances Platt obtained her PhD from the University of Bath, UK, and was a post-doctoral fellow at Washington University Medical School in St. Louis, USA. She was a Lister Institute Senior Research Fellow and a Reader at the University of Oxford. Prof. Platt’s main research interests include the biology and pathobiology of glycosphingolipids. Her research led to the development of miglustat for the treatment of glycosphingolipid storage diseases. In 1999, Prof. Platt was awarded the Alan Gordon Memorial Award and the Horst Bickel Award for advances in metabolic disease therapy. She was elected a fellow of the Academy of Medical Sciences in 2011.

Affiliations and Expertise

Department of Pharmacology, University of Oxford, Oxford, UK

Nick Platt

Nick Platt

Dr Platt obtained his PhD from the University of Bath, UK and was a post-doctoral fellow at Washington University in St Louis. Since his return to UK he has worked at University of Oxford in the Sir William Dunn School of Pathology and Nuffield Department of Medicine. He has very recently become a senior research fellow in the Department of Pharmacology. His research interests are in innate immunity, particularly the role of specific pattern recognition receptors in immune and inflammatory responses.

Affiliations and Expertise

Department of Pharmacology, University of Oxford, UK