Front Cover 1
Methods in Cell Biology 2
Series Editors 3
Methods in Cell BiologyLysosomes and Lysosomal DiseasesVolume 126Edited byFrances PlattDepartment of Pharmacology, Universi ... 4
Copyright 
5 
Contents 6
Contributors 14
Preface 18
1. Methods for monitoring lysosomal morphology 20
Introduction 21
1. Lysosomal Form 21
2. Lysosomal Function 22
3. Lysosomal Failure 24
4. Linking Lysosomal Form and Failure 25
5. Methods 25
5.1 Monitoring Lysosome Morphology in Live Cells 25
5.1.1 LysoTracker® staining 25
5.1.1.1 Materials 26
5.1.1.2 Method 26
5.1.2 Dextran labeling 27
5.1.2.1 Materials 28
5.1.2.2 Method 28
5.2 Monitoring Lysosome Morphology in Fixed Cells 28
5.2.1 Immunofluorescence 28
5.2.1.1 Materials 28
5.2.1.2 Method 29
5.2.2 Electron microscopy 30
5.2.2.1 Conventional EM using chemical fixation 31
5.2.2.2 Pre-embedding labeling 32
5.2.2.2.1 Materials 32
5.2.2.2.2 Method 32
5.2.2.3 Cryo-immuno EM 33
5.2.2.3.1 Materials 33
5.2.2.3.2 Method 33
6. Discussion 34
Acknowledgments 35
References 35
2. A rapid method for the preparation of ultrapure, functional lysosomes using functionalized superparamagnetic iron oxide nan ... 40
1. The Endocytic System 41
2. The Discovery of Lysosomes and Lysosomal Storage Diseases 42
3. Biochemical Features of Lysosomes 43
4. Overview of Methods for Purifying Lysosomes 43
4.1 Density Gradient Centrifugation 43
4.2 Density Gradient Electrophoresis 44
4.3 Magnetic Separation Methods for Purifying Lysosomes 44
4.3.1 Antibody-based purification 44
4.3.2 Purification of lysosomes by intravesicular magnetization, early attempts 44
5. Method for Magnetic Separation of Lysosomes from Whole Cells 47
5.1 Solutions, Reagents, and Equipment 48
5.1.1 Pulse medium 48
5.1.2 Buffer A 48
5.1.3 Buffer B 48
5.1.4 DNase 1 solution 48
5.2 Method 49
5.2.1 Step 1—Incubation of cultured cells with SPIONs (pulse-chase) 49
5.2.2 Step 2—Preparation of the cellular homogenate 49
5.2.3 Step 3—Magnetic separation of lysosomes 49
6. Technical Considerations 50
6.1 Sourcing the Most Suitable SPION 52
6.2 Stability and Potential Toxic Effect of Nanoparticles 53
6.3 Determining the Best Cell Type from which to Purify Lysosomes 55
6.4 Endocytosis of Nanoparticles, Considerations for Lysosomal Disease Cells, or Manipulation of Lysosomal Protein Levels 55
6.5 Choice of Appropriate Buffers 56
6.6 Homogenization Techniques 57
6.7 Consideration of Centrifugation Speeds 57
7. Techniques for Determining Purity of the Lysosomal Fractions 57
7.1 Western Blotting 57
7.2 Enzyme Assays 59
7.3 Electron Microscopy 59
8. Discussion 59
Acknowledgments 60
References 60
3. TFEB and the CLEAR network 64
Introduction 65
1. TFEB Nuclear Translocation Assay 66
2. Cell Culture and Treatment 67
2.1 Starvation 67
2.2 Amino Acid Deprivation 68
2.3 Drug Treatments 68
2.4 RNA Interference 68
2.5 Nuclear/Cytoplasmic TFEB Localization by Western Blot 69
2.6 Nuclear/Cytoplasmic TFEB Localization by Immunofluorescence 70
2.7 Determination of TFEB Phosphorylation Levels 70
2.7.1 Detection of P-S142 TFEB 70
2.7.2 Detection of P-S211 TFEB by using 14-3-3 Motif 71
2.8 Identification of a CLEAR Gene 71
2.9 Cellular Effects of TFEB Activation 73
2.9.1 Lysosomal biogenesis 74
2.9.1.1 Measurement of Lysosomal Volume by FACS 74
2.9.1.2 Immunoblot of LAMP1 74
2.9.1.3 Tracking of Lysosomes by Live Imaging 74
2.9.1.4 Lysosomal Exocytosis Assays 75
2.9.1.5 Immunofluorescence Staining for Surface LAMP1 75
2.9.1.6 Western Blot of PM Proteins 76
2.9.1.7 Tannic Acid Treatment 76
2.9.1.8 Measure of Lysosomal Enzymes in the Media 76
2.10 High Content Imaging to Study TFEB 77
2.10.1 High content nuclear translocation assay 77
2.10.2 Preparation of compound plate 78
2.10.3 Treatment of assay plate with compounds 78
2.10.4 Incubation 78
2.10.5 Fixation and nuclei counterstaining 78
2.10.6 Acquisition of plates and analysis of data using the OPERA system (Perkin Elmer) 78
2.10.7 HCS assay to measure TFEB levels by beta-galactosidase expression 80
References 80
4. Biosynthesis, targeting, and processing of lysosomal proteins: Pulse–chase labeling and immune precipitation 82
Introduction 83
Briefly on Functions and Biogenesis of Lysosomes 83
On Application of Radioactive Isotopes in Studies of Molecular Forms of Lysosomal Proteins 86
1. Materials 88
1.1 Equipment 88
1.2 Materials for Cell Culturing and Metabolic Labeling 88
1.3 Materials for Immune Precipitation and Sample Analysis 89
2. Methods 90
2.1 Cell Culture and Metabolic Labeling 90
2.1.1 Starvation 90
2.1.2 Pulse 91
2.1.3 Chase 93
2.1.4 Cell lysis and preparation of extracts 93
2.2 Immune Precipitation 94
2.3 Sample Preparation and Analysis 95
3. Metabolic Labeling and Immune Precipitation of the Lysosomal Protease Cathepsin Z 96
4. Discussion 99
Acknowledgments 101
References 101
5. Measuring lysosomal pH by fluorescence microscopy 104
Introduction 105
Instrumentation 108
Probe Selection 109
Calibration 111
1. Materials 112
1.1 Cell Lines 112
1.2 Reagents 112
1.3 pH Calibration Buffers 112
2. Methods 113
2.1 Loading Cells with Fluorescein-Dextran 113
2.2 Lysosome pH Measurement 113
2.3 In situ Calibration 114
Conclusion 114
Notes 115
Acknowledgments 115
References 115
6. Lysosome fusion in cultured mammalian cells 120
1. Overview of Methods to Study Lysosome Fusion 121
1.1 Lysosomes and Endolysosomes 121
1.2 Cell-Free Lysosome Fusion Systems 122
1.3 Visualizing Lysosome Fusion in Cultured Cells 123
2. Choosing an Assay System 125
3. Methods 128
3.1 Studying Content Mixing by Transmission Electron Microscopy 128
3.1.1 Materials 128
3.1.2 Protocol 128
3.1.3 Option: perturbation of BSA-15-nm colloidal gold uptake 128
3.1.4 Option: immunogold EM 129
3.2 Visualization of Content Mixing Using Live Cell Microscopy 130
3.2.1 Materials 130
3.2.2 Protocol 130
3.2.3 Option: CLEM 131
3.3 Assaying Content Mixing Using Automated Widefield Microscopy 131
3.3.1 Materials 131
3.3.2 Protocol 132
3.3.3 Option: transfection procedure 132
3.4 Assaying Content Mixing Using Confocal Microscopy 132
3.4.1 Depletion of gene expression using siRNA 132
3.4.1.1 Materials 132
3.4.1.2 Protocol 133
3.4.2 Measurement of delivery of endocytosed fluorescent dextran to lysosomes by confocal fluorescence microscopy 133
3.4.2.1 Materials 133
3.4.2.2 Protocol 134
Acknowledgments 135
References 135
7. RNAi screens of lysosomal trafficking 138
1. Methods of Gene Depletion in Lysosomes 139
2. Selection of Screening System 139
2.1 Choosing an Appropriate Cell Type 139
2.2 siRNA versus shRNA 140
2.3 Pooled versus Arrayed Screens 141
2.4 Assay Development 142
3. Assay Validation 145
3.1 Relationship between Cell Number and Phenotype 145
3.2 Testing Impact of Viral Titer 146
3.3 Testing Run Independence 147
3.4 Assay Controls 148
3.5 Data Analysis 150
4. Validation of Hits 152
4.1 Confirmation of Gene Silencing 152
4.2 Multiple Hairpin Testing and Rescue 153
4.3 Caveats to RNAi Screens 153
5. Discussion 154
Acknowledgments 155
References 155
8. Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses 158
1. Overview of Wounding Methods and Plasma Membrane Repair Mechanisms 159
2. Procedures for Plasma Membrane Wounding 161
2.1 Mechanical Wounding by Three-Dimensional Cellular Contraction 161
2.1.1 Aspects to consider 162
2.2 Mechanical Wounding by Scraping Cells from the Substrate 162
2.2.1 Aspects to consider 163
2.3 Mechanical Wounding Using a Needle/Syringe 163
2.3.1 Aspects to consider 165
2.4 Mechanical Wounding Using Glass Beads 165
2.4.1 Aspects to consider 166
2.5 Wounding Using Pore-Forming Proteins 167
2.5.1 Aspects to consider 168
3. Procedures for Measuring the Extent of Plasma Membrane Repair 168
3.1 PI Influx (Microscopy and Flow Cytometry) 168
3.1.1 Issues to consider 169
3.2 Live Imaging of FM1-43 Dye Influx 169
3.2.1 Aspects to consider 170
4. Procedures to Measure Exocytosis of Lysosomes 171
4.1 Surface Exposure of Lamp1 Luminal Epitopes 172
4.1.1 Aspects to consider 173
4.2 Secretion of Lysosomal Enzymes 173
4.2.1 Aspects to consider 174
Acknowledgments 176
References 176
9. Imaging approaches to measuring lysosomal calcium 178
1. Endolysosomal Ca2+ 179
1.1 Endolysosomal Ca2+: Roles and Regulation 179
1.1.1 Role of Ca2+ 179
1.2 Endolysosomal Ca2+ Homeostasis 180
2. Assessing Organelle Ca2+: General Strategies 180
2.1 Global Cytosolic Ca2+ Measurements 181
2.2 Perivesicular Ca2+ Measurements 182
2.3 Luminal Ca2+ Measurements 184
2.3.2 Free Ca2+ 184
2.3.2.1 Null-point 184
2.3.2.1 Optical recording 185
3. Assessing Endolysosomal Ca2+: Specific Strategies 187
3.1 Indirect Monitoring with Cytosolic Ca2+ Indicators 188
3.1.1 Agents that target acidic Ca2+ stores 189
3.1.2 Ca2+-indicator loading 191
3.1.2.1 Chemical dyes 191
3.1.2.1.1 Reagents 191
3.1.2.1.2 Culture and loading 192
3.1.2.2 Genetically encoded Ca2+ indicators 192
3.1.3 Ca2+ measurements 192
3.1.3.1 Analysis 193
3.1.4 Indirect measurements: pitfalls 194
3.1.5 Conclusion 195
3.2 Direct Luminal Recording 195
3.2.1 Luminal pH 195
3.2.1.1 pH and chromophores 196
3.2.1.2 pH and Ca2+-binding 196
3.2.2 Is pHL always a problem? 196
3.3 Luminal Recording: Practicalities 199
3.3.1 Targeting indicators to acidic vesicles 199
3.3.1.1 Chemical indicators: ester 199
3.3.1.2 Chemical indicators: endocytosis 199
3.3.1.3 Genetic indicators 201
3.3.2 Resting or dynamic [Ca2+] changes? 201
3.3.3 Calibration and correcting for pHL 201
3.3.3.1 pHL correction 201
3.3.3.2 Ratiometric recording 202
3.3.4 Luminal Ca2+ protocol 203
3.3.4.1 Reagents 203
3.3.4.2 In vitro determination of the Kd of the Ca2+ dye 204
3.3.4.2.1 General points 204
3.3.4.2.2 Protocol 205
3.3.4.3 Cell loading with dyes by endocytosis 205
3.3.4.4 Imaging Ca2+ indicator fluorescence—cells 206
3.3.4.5 Imaging Ca2+ indicator fluorescence – calibration 206
3.3.4.5.1 Protocol 207
3.3.4.6 Dynamic luminal Ca2+ changes 207
3.3.5 Conclusions 208
4. Final Remarks 208
References 208
10. Lysosome electrophysiology 216
Introduction 217
1. Lysosome 217
1.1 Lysosome Ion Channels 217
1.2 Methods for Studying Lysosomal Ion Channels 219
1.2.1 Methods to study lysosomal channel localization 219
1.2.2 Methods to study lysosomal Ca2+ channels 220
1.2.3 Studying lysosomal channels in plasma membrane or in artificial membranes using patch clamping 221
1.2.4 Study of lysosomal channels in lysosomes using lysosome patch clamping 221
2. Materials 222
2.1 Cell Culture 222
2.2 Pipettes 222
2.3 Chemicals 223
2.4 Lysosome Patch-Clamp Recording 223
3. Methods 223
3.1 Cell Culture 223
3.2 Pipettes and Solutions 223
3.3 Lysosome Patch-Clamp Recording 225
3.3.1 Isolation of enlarged lysosomes 225
3.3.2 Whole-lysosome patch clamping 225
3.3.3 Other patch configurations 227
4. Discussion 229
5. Summary 230
Acknowledgments 230
References 230
11. Reconstitution of lysosomal ion channels into artificial membranes 236
Introduction 237
1. The Bilayer Apparatus 237
2. Electrical Equipment Used for Single-Channel Recordings 239
3. Painting Bilayers 241
4. Ion Channel Incorporation into a Bilayer 242
4.1 Fusion of Native Vesicles or Purified Proteins with the Bilayer 242
4.2 Ion Channel Orientation 243
5. Single-Channel Current Amplitude and Conductance Measurements 244
6. Choice of Permeant Ion 246
6.1 Native Ion Channels 246
6.2 Recombinantly Expressed and Purified Ion Channels 246
7. Measuring the Relative Permeability of Different Ions 248
8. Measurements of Liquid Junction Potentials 249
9. Single-Channel Gating and Measurements of Open Probability 249
10. Noise Analysis 250
11. Isolation of Native and Recombinant Purified Lysosomal Ion Channels 251
11.1 Native Lysosomal Ion Channels 251
11.2 Purification of Recombinantly Expressed Lysosomal Channels 252
11.2.1 Purification of human TPC1 overexpressed in HEK293 cells 252
12. Discussion 253
References 253
12. Fluorescence methods for analysis of interactions between Ca2+ signaling, lysosomes, and endoplasmic reticulum 256
1. ER, Lysosomes, and Ca2+ Signaling 257
2. Pharmacological Tools 259
3. Fluorescence Methods 260
4. Fluorescence Tools for Analysis of Lysosomes 262
5. Ca2+ Signaling and Lysosomes: Tools and Practical Problems 264
6. Single-cell Analyses of Cytosolic Ca2+ Signals 266
6.1 Materials 266
7. High-throughput Analyses of Cytosolic Ca2+ Signals 269
7.1 Materials 269
8. Tracking Interactions between Lysosomes and ER by Fluorescence Microscopy 270
8.1 Materials 271
Conclusions 273
Acknowledgments 273
References 274
13. Methods for the quantification of lysosomal membrane permeabilization: A hallmark of lysosomal cell death 280
Introduction 282
Method 1: Quantification of Cathepsin and ß-N-acetyl-glucosaminidase Release into the Cytosol by Enzymatic Activity Measurement 285
1. Materials 285
1.1 Reagents 285
1.1.1 ß-N-acetyl-glucosaminidase (NAG) reaction buffer (NAG RB) 285
1.1.2 Caspase reaction buffer (caspase RB) 285
1.1.3 Cathepsin reaction buffer (cathepsin RB) 286
1.1.4 Digitonin extraction buffer (DE buffer) 286
1.2 Equipment 286
1.3 Time Frame 286
2. Protocol 286
2.1 Determination of the Optimal Digitonin Concentration for the Extraction of Lysosome-free Cytosol 286
2.2 Measurement of LMP 288
2.3 Data Analysis 290
2.4 Troubleshooting 290
Method 2: LMP Visualized by Release of Fluorescent Dextran to the Cytosol 290
3. Materials 292
3.1 Reagents 292
3.2 Equipment 292
3.3 Time Frame 292
4. Protocol 292
4.1 Troubleshooting 293
Method 3: LMP Visualized by Cathepsin Immunocytochemistry 293
5. Materials 294
5.1 Reagents 294
5.2 Equipment 294
5.3 Time Frame 294
6. Protocol 294
6.1 Data Analysis 297
6.2 Troubleshooting 297
Method 4: Detection of Damaged Lysosomes by Galectin-1 and -3 Translocation 297
7. Materials 297
7.1 Reagents 297
7.2 Equipment 298
7.3 Time Frame 298
8. Protocol 298
8.1 Data Analysis 299
8.2 Alternative Assay with Fluorescent Constructs 300
8.3 Troubleshooting 300
Discussion 300
Acknowledgments 302
References 302
14. Measuring the phagocytic activity of cells 306
Introduction 307
1. Reasons to Undertake Studies of Phagocytosis 308
2. Components of a Phagocytosis Assay 308
3. In vitro or In vivo Study? 308
4. Methodologies for Analyses of Phagocytosis 309
5. Selection of Phagocyte Population 310
6. Choice of Target Particle 312
7. Targeting Particles to Specific Phagocytic Receptors 313
8. Detection of Ingested Particles 313
9. Protocol 1. Fc. Receptor-Mediated Phagocytosis of IgG Opsonized Sheep Red Blood Cells by Murine Macrophages 314
10. Materials and Reagents 314
11. Equipment 315
12. Protocol 315
13. Protocol 2. FACS Analysis of Mycobacterium bovis Internalization by RAW264.7 Cells 317
14. Materials 318
15. Equipment 319
16. Summary 321
Acknowledgments 321
References 321
15. Detection and quantification of microbial manipulation of phagosomal function 324
Introduction 325
An Overview of the Role of the Macrophage Phagosome in Infection 325
The Role of Lysosomes in Microbial Killing 326
Methods Utilized in the Study of Lysosome Function 327
1. Considerations for Choice of Reagents, Cells, and Readouts 328
1.1 Cell Type 328
1.2 Choice of Phagosomal Reporter Particle 328
1.3 Intraphagosomal Function 330
1.4 Functional Readouts 330
1.5 Analytical Platforms 331
2. Reagents 331
3. Methods 335
3.1 Physical Correlates of Phagosome Maturation: Phagosome Acidification 335
3.1.1 Measurement of phagosomal pH 335
3.1.2 Assessment of the acidification of established microbial vacuoles 335
3.2 Physical Correlates of Phagosome Maturation: Assessing the Extent of Phagosome/Lysosome Fusion 336
3.2.1 Measurement of recruitment of lysosomal markers, such as LAMP-1 and LAMP-2, to phagosomes 336
3.2.2 Measurement of recruitment of lysosomal tracers, such as 10 kDa Alexa Fluor 647-labeled dextran, to phagosomes 336
3.2.3 Utilization of FRET to quantify phagosome/lysosome fusion 336
3.3 Enzymatic Readouts of Phagosomal Function 337
3.3.1 Measurement of the proteolytic capacity of isolated lysosomes 337
3.3.2 Real-time measurement of proteolytic activity in phagolysosomes 338
3.3.3 Real-time measurement of lipase activity in phagosomes 338
3.3.4 Real-time measurement ß-galactosidase activity in phagosomes 338
3.4 Additional Indicators of Phagosomal Function 339
3.4.1 Accumulation of pro-cathepsin D in phagosomes 339
4. Analytical Platforms, Data Collection and Analysis 339
4.1 Fluorescence Plate Reader 339
4.2 Confocal Microscopy 341
4.3 Flow Cytometry 343
4.4 Data Interpretation 344
5. Discussion 345
6. Summary 346
Acknowledgments 346
Supplementary Data 347
References 347
16. Measuring relative lysosomal volume for monitoring lysosomal storage diseases 350
1. Measuring Relative Lysosomal Volume as an Index of Lysosomal Storage 351
2. In Which Circulating Cell Type Should Relative Lysosomal Volume Be Measured? 352
3. How to Measure Relative Lysosomal Volume in Blood Cells 353
4. Methods 355
4.1 Preparation of Human Blood 355
4.1.1 Materials and reagents 355
4.1.2 Protocol 356
4.2 Preparation of Cells from Mouse Spleen or Whole Blood 356
4.2.1 Materials and reagents 356
4.2.2 Protocol 357
4.2.2.1 Isolation of mononuclear cells from mouse spleen 357
4.2.2.2 Isolation of mononuclear cells from mouse blood 357
4.3 B-Lymphocyte Staining 358
4.3.1 Materials and reagents for human cells 358
4.3.2 Protocol for human cells 358
4.3.3 Materials and reagents for mouse cells 358
4.3.4 Protocol for mouse cells 359
4.4 Flow Cytometry, Calibration, Acquisition, and Analysis 359
4.4.1 Materials and reagents 359
4.4.2 Protocol 359
4.5 Isolation of B-Cells for Biochemical Assays or Microscopy 361
4.5.1 Materials and reagents for human cells 361
4.5.2 Protocol for human cells 361
4.5.3 Materials and reagents for mouse cells 361
4.5.4 Protocol for mouse cells 362
4.6 Influence of Patient Blood Shipping Times 362
4.7 Influence of Blood Storage Temperature on LysoTracker Staining 363
4.8 Influence of Delays in Analysis of Samples Post-LysoTracker Staining 364
4.8.1 Some economic considerations relating to B-cell staining 364
5. Summary 365
Acknowledgments 365
References 366
17. Quantifying storage material accumulation in tissue sections 368
Introduction 369
1. Detection of Storage Material 369
1.1 Direct Demonstration of Storage Material 369
1.1.1 Drawbacks of this method 371
1.1.2 Quantification 371
1.2 Immunohistochemical Detection of Storage Material 371
1.2.1 Potential problems 372
1.2.2 Practical issues 372
1.2.3 Alternative approaches 372
1.2.4 Quantification 373
1.3 Histochemical Methods to Detect Storage Material 373
2. Quantification of Storage Material 373
Conclusion 374
References 374
18. Laboratory diagnosis of Niemann–Pick disease type C: The filipin staining test 376
Introduction and Rationale 377
1. Materials 379
2. Methods 380
2.1 General Considerations 380
2.2 Set Up of Experiment and Step for Maximal Expression of LDL-receptors (2–3Days) 381
2.3 Challenge with LDL-enriched Medium (24h) 381
2.4 Fixation Step 381
2.5 Filipin Staining 382
2.6 Fluorescence Microscopic Examination 382
2.7 Enhancing Reliability by Repeating the Test 385
2.8 Reporting Results 385
2.9 Preparation of Bovine LPDS 385
2.10 Preparation of Human LDL 385
2.10.1 Special reagents 385
2.10.2 Procedure 386
3. Discussion 386
3.1 Technical Pitfalls 386
3.1.1 Less than optimal or inappropriate conditions for fluorescence microscopic examination 386
3.1.2 Quality control of reagents and of cell culture 387
3.2 Range of Variability of the Filipin Patterns in NPC 387
3.2.1 Heterogeneity of filipin patterns in NPC fibroblasts: the typical “classic” and “intermediate” and the atypical “variant” f ... 387
3.2.2 Patterns in NPC heterozygotes 388
3.3 Non-NPC Conditions Reported to Result in an Abnormal Filipin Test 388
3.4 The Filipin Test in Clinical Practice 389
Concluding Remarks 392
Acknowledgments 392
References 392
Volumes in Series 396
Index 408