UID:
almafu_9958130722802883
Format:
1 online resource (451 p.)
Edition:
First edition.
ISBN:
0-12-800293-X
Series Statement:
Methods in Cell Biology, Volume 126
Content:
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.Covers sections on model systems and functional studies, imaging-based approaches and emerging studiesChapters are written by experts in the
Note:
Description based upon print version of record.
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Front Cover; Methods in Cell Biology; Series Editors; Methods in Cell BiologyLysosomes and Lysosomal DiseasesVolume 126Edited byFrances PlattDepartment of Pharmacology, Universi ...; Copyright; Contents; Contributors; Preface; 1. Methods for monitoring lysosomal morphology; Introduction; 1. Lysosomal Form; 2. Lysosomal Function; 3. Lysosomal Failure; 4. Linking Lysosomal Form and Failure; 5. Methods; 5.1 Monitoring Lysosome Morphology in Live Cells; 5.1.1 LysoTracker® staining; 5.1.1.1 Materials; 5.1.1.2 Method; 5.1.2 Dextran labeling; 5.1.2.1 Materials; 5.1.2.2 Method
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5.2 Monitoring Lysosome Morphology in Fixed Cells5.2.1 Immunofluorescence; 5.2.1.1 Materials; 5.2.1.2 Method; 5.2.2 Electron microscopy; 5.2.2.1 Conventional EM using chemical fixation; 5.2.2.2 Pre-embedding labeling; 5.2.2.2.1 Materials; 5.2.2.2.2 Method; 5.2.2.3 Cryo-immuno EM; 5.2.2.3.1 Materials; 5.2.2.3.2 Method; 6. Discussion; Acknowledgments; References; 2. A rapid method for the preparation of ultrapure, functional lysosomes using functionalized superparamagnetic iron oxide nan ...; 1. The Endocytic System; 2. The Discovery of Lysosomes and Lysosomal Storage Diseases
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3. Biochemical Features of Lysosomes4. Overview of Methods for Purifying Lysosomes; 4.1 Density Gradient Centrifugation; 4.2 Density Gradient Electrophoresis; 4.3 Magnetic Separation Methods for Purifying Lysosomes; 4.3.1 Antibody-based purification; 4.3.2 Purification of lysosomes by intravesicular magnetization, early attempts; 5. Method for Magnetic Separation of Lysosomes from Whole Cells; 5.1 Solutions, Reagents, and Equipment; 5.1.1 Pulse medium; 5.1.2 Buffer A; 5.1.3 Buffer B; 5.1.4 DNase 1 solution; 5.2 Method; 5.2.1 Step 1-Incubation of cultured cells with SPIONs (pulse-chase)
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5.2.2 Step 2-Preparation of the cellular homogenate5.2.3 Step 3-Magnetic separation of lysosomes; 6. Technical Considerations; 6.1 Sourcing the Most Suitable SPION; 6.2 Stability and Potential Toxic Effect of Nanoparticles; 6.3 Determining the Best Cell Type from which to Purify Lysosomes; 6.4 Endocytosis of Nanoparticles, Considerations for Lysosomal Disease Cells, or Manipulation of Lysosomal Protein Levels; 6.5 Choice of Appropriate Buffers; 6.6 Homogenization Techniques; 6.7 Consideration of Centrifugation Speeds; 7. Techniques for Determining Purity of the Lysosomal Fractions
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7.1 Western Blotting7.2 Enzyme Assays; 7.3 Electron Microscopy; 8. Discussion; Acknowledgments; References; 3. TFEB and the CLEAR network; Introduction; 1. TFEB Nuclear Translocation Assay; 2. Cell Culture and Treatment; 2.1 Starvation; 2.2 Amino Acid Deprivation; 2.3 Drug Treatments; 2.4 RNA Interference; 2.5 Nuclear/Cytoplasmic TFEB Localization by Western Blot; 2.6 Nuclear/Cytoplasmic TFEB Localization by Immunofluorescence; 2.7 Determination of TFEB Phosphorylation Levels; 2.7.1 Detection of P-S142 TFEB; 2.7.2 Detection of P-S211 TFEB by using 14-3-3 Motif
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2.8 Identification of a CLEAR Gene
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English
Additional Edition:
ISBN 1-322-88938-4
Additional Edition:
ISBN 0-12-800079-1
Language:
English
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