Recent Advances in Cytometry, Part B (eBook)

Cover 1
Methods in Cell Biology Volume 103 2
Copyright 5
Contents 6
Contributors 12
Preface To Fifth Edition 16
Section - 1 - New applications in cell biology 20
Chapter - 1 - Recent Advances in Cytometry Applications: Preclinical, Clinical, and•Cell•Biology 22
Abstract 22
Preclinical and Clinical Applications 23
Cell Biology and Cell Transplantation Therapy 30
Acknowledgment 33
References 33
Chapter - 2 - Detection of Hematopoietic Stem Cells by Flow Cytometry 40
Abstract 40
Introduction 41
The side population is highly enriched in hematopoietic stem cells 43
Surface markers for LT-HSC purification 44
References 47
Chapter - 3 - Identification of Very Small Embryonic/Epiblast-Like Stem Cells (VSELs) Circulating in Peripheral Blood During Organ/Tissue Injuries 50
Abstract 51
Introduction 51
Background 54
Very Small Embryonic-Like Stem Cells (VSELs) 54
Developmental Origin of VSELs 55
VSELs and Their Unique Molecular Characteristics 56
Epigenetic Changes of Imprinted Genes that Regulate VSELs Pluripotency 58
Mesenchymal Stem Cells (MSCs) 59
Endothelial Progenitor Cells (EPCs) 59
Materials 59
Preparation of Peripheral Blood (PB) for Analysis 60
Staining of Total PB-Derived Nucleated Cells (TNCs) for Analysis 60
Methods 62
Isolation of Total PB-Derived Nucleated Cells (TNCs) by Lysing Red Blood Cells (RBCs) 62
Staining of PB-Derived TNCs for Flow Cytometric Analysis 62
Setting Up Instrument for FACS Analysis 63
Identification of VSELs in Human PB and UCB 63
Calculation of Absolute Numbers of Target Cells in 1& mu
Sorting of Cells 63
Results 64
Critical Aspects of the Methodology 66
Applications 68
Future Directions 69
Acknowledgments 69
References 70
Chapter - 4 - Apoptosis and Beyond: Cytometry in Studies of Programmed Cell Death 74
Abstract 75
Introduction 75
The Biology of Apoptosis 76
Cytometry in Cell Necrobiology 79
Cytometric Methods to Detect Apoptosis 80
Light Scattering Changes in Apoptotic Cells 80
Dissipation of Mitochondrial Transmembrane Potential (& Delta
Activation of Caspases 86
Fluorochrome-Labeled Inhibitors of Caspases (FLICA) 86
Detection of PARP Cleavage 90
Changes in the Plasma Membrane During Apoptosis 91
Externalization of Phosphatidylserine 91
Changes in Plasma Membrane Permeability 93
Nuclear Hallmarks of Apoptosis 94
Assessment of Fractional DNA Content (Sub-G1 Fraction) 95
Assessment of DNA Strand Breaks (TUNEL Assay) 97
SYTO-Based Detection of Apoptosis 98
Time-Window in Measuring Incidence of Apoptosis 100
Multiparameter Detection of Apoptosis: Choosing the Right Method 101
Beyond Apoptosis & ndash
Autophagy 103
Necrosis 105
Cell Senescence 105
Future Outlook 107
Acknowledgements 109
References 109
Chapter - 5 - Assessment of Oxidative Stress-Induced DNA Damage by Immunoflourescent Analysis of 8-OxodG 118
Abstract 118
Redox Regulation of Cell Fate Signaling 119
8-OxodG as a Marker of Oxidative DNA Damage 120
8-OxodG is a Highly Mutagenic DNA Lesion 121
8-OxodG as a Biomarker of Cellular Oxidative Damage and its Clinical Relevance 121
Detection of Oxidative DNA Damage Involving 8-OxodG 123
Chromatography-Based Direct Assessment of 8-OxodG 124
The Indirect Assessment of OxodG Modification of DNA 125
Indirect Assessment of Oxidative DNA Damage by Immunofluorescence 125
Materials 126
Methodology 126
Results 127
Shortcomings of the Immunofluorescence Assay 128
Concluding Remarks 128
References 128
Chapter - 6 - Analysis of Individual Molecular Events of•DNA Damage Response by Flow- and•Image-Assisted Cytometry 134
Abstract 135
Introduction 135
Events of the DDR 136
Chromatin Decondensation (Relaxation) 136
Activation of Phosphatidyl Inositol 3& prime
Activation of Checkpoint Kinases 139
Histone H2AX Phosphorylation 141
Detection of DDR Events by Cytometry 141
Chromatin Relaxation (Decondensation) 141
Recruitment of Mre11 143
Immunocytochemical Detection of DDR-Associated ATM, DNA-PKcs and Chk2 Activation, Phosphorylation of p53 and Histone H2AX 144
Application of Cytometry to Detect DDR Induced by.Different Genotoxic Agents 145
Assessment of DDR Induced by DNA Topoisomerase Inhibitors 145
Induction of DDR by Ionizing Radiation and UV Light 148
Induction of DDR by Cigarette Smoke (CS) and Other Environmental Mutagens 150
Oxidative DNA Damage 152
Interpretation of Cytometric Data: Role of Image-Assisted Cytometry 154
Role of Microfluidic Lab-on-a-Chip Platforms for DDR Analysis 156
References 158
Chapter - 7 - Fluorescence-Based Detection and Quantification of Features of Cellular Senescence 168
Abstract 169
Introduction 169
Features Associated with Loss of Reproductive Cell Capability 172
Shortened Telomeres in Replicative Senescence 173
Telomere Dysfunction-Induced Foci (TIF) in Replicative Senescence 175
Loss of the Doubling Capacity of Cells 176
Cellular Hypertrophy 176
Changes Associated with Lysosomes 178
Lipofuscin Accumulation 178
Lipofuscin Autofluorescence in Fluorescence Microscopy 179
Flow Cytometric Analysis of Cellular Lipofuscin Levels 180
Increased Lysosome Content 180
Senescence-Associated & beta
In situ Determination of SA & beta
Flow Cytometry for SA & beta
Changes Associated with Mitochondria 183
Increased Mitochondrial Mass 183
Quantification of Changes in Mitochondria Mass 184
Altered Structural Dynamics of Mitochondria 186
Visualization of Altered Mitochondria Structure 187
Decreased Membrane Potential 187
Quantitative Comparison of MMP 188
Decreased Autophagy 190
Determination of Autophagy Activity 190
Changes in the Level of Reactive Oxygen Species (ROS) 192
Changes in Level of Superoxide 193
Changes in Level of Hydroxyl Radicals 194
Changes Associated with Nucleus and Chromosomes 195
Use of Flow Cytometry for Analysis of Cellular Senescence 197
Conclusion 197
Acknowledgments 198
References 198
Chapter - 8 - Measurement of Telomere Length Using•PNA Probe by Cytometry 208
Abstract 208
Introduction to Telomeres 209
Technical Background 210
Methods 212
Our Protocol Versus the Original Flow-FISH Protocol 212
Our Protocol 212
Original Flow-FISH Protocol 213
Results 214
Critical Aspects of the Flow-FISH Methodology 217
Conclusion and Perspectives 219
References 220
Section - 2 - Pre-clinical and clinical applications 222
Chapter - 9 - Cytometry of Intracellular Signaling: From Laboratory Bench to Clinical Application 224
Abstract 224
Introduction 225
Activation States Are Transient 226
Most Signaling Pathways Exist in Their Ground State 226
Constitutive Activation of Signaling Pathways Can Occur in Leukemia 226
Rationale 228
Methods 231
Protocol for Whole Blood Fixation and Red Cell Lysis 231
Samples 231
Sample Suitability 231
Addition of Pathway Activators or Inhibitors 231
Whole Blood Fixation 232
Red Cell Lysis 232
Washing and Alcohol Treatment 232
Antibody Staining 232
Activators and Inhibitors of Signaling Pathways 233
Pathway Activators 233
Pathway Inhibitors 234
Freezing Samples 236
Materials 236
Fixation and Red Cell Lysis 236
Pathway Activators 236
Pathway Inhibitors 237
Antibodies 237
Discussion 237
Summary 238
References 238
Chapter - 10 - Immunophenotypic Pattern of Myeloid Populations by Flow Cytometry Analysis 240
Abstract 240
Introduction 241
Granulocytes 241
Basophils 244
Eosinophils 244
Monocytes 245
Erythroid Precursors 246
Megakaryocytes 247
Acute Myeloid Leukemia (AML) with Minimal Differentiation and AML without Maturation 247
AML with Maturation 247
AML with t(8 21)
Acute Myelomonocytic Leukemia (AML-M4) 258
Acute Monoblastic Leukemia 259
Acute Erythroid Leukemia (FAB: AML-M6) 266
Acute Megakaryoblastic Leukemia (FAB: AML-M7) 267
Blastic Plasmacytoid Dendritic Cell Neoplasm 269
Chronic Myeloid Leukemia (CML, BCR& ndash
Granulocytes/Maturing Myeloid Precursors with Dyspoietic Features Associated with Myelodysplastic Syndrome (MDS) 270
Chronic Myelomonocytic Leukemia (CMML) 281
References 282
Chapter - 11 - Flow Cytometry-Based Pharmacodynamic Monitoring After Organ Transplantation 286
Abstract 286
Introduction 287
T Cells 288
Regulatory T Cells 292
Dendritic Cells 294
Conclusions 298
References 300
Chapter - 12 - Clinical Cytometry and Progress in HLA Antibody Detection 304
Abstract 304
Introduction 305
Cell-Based Assays 306
Lymphocytotoxicity 306
Modified Lymphocytotoxicity Assays 308
Flow Cytometry 310
Antigen-Based Assays 311
Solid-Phase Immunobinding Assays 311
Multiplex Platform for Microparticle Analysis 317
Recombinant HLA Antigens 320
Determining Antibody Specificity 321
Current Frontiers in Transplant-Related Antibody Testing 323
Extended HLA Loci 323
Conundrums in HLA Antibody Assignment 323
Functional Assessments of HLA Alloantibody by Flow Cytometry 324
Summary 324
References 325
Chapter - 13 - Clinical Utility of Flow Cytometry in•the•Study of Erythropoiesis and Nonclonal Red Cell Disorders 330
Abstract 330
Keywords 331
Introduction 331
Normal Erythroid Development 331
Flow Cytometry for Reticulocyte Enumeration 334
Flow Cytometry for the Detection of Fetal& ndash
Flow Cytometry for Quantitation of HbF in Sickle Cell Disease 341
Flow Cytometry in the Evaluation of Hereditary Spherocytosis 342
Measurement of Red Cell Survival and Red Cell Volume 344
Cell Cycle Studies 344
RBC Surface Abnormalities 345
References 345
Chapter - 14 - Immunophenotypic Characterization of Bone Marrow Mast Cells in Mastocytosis and•Other Mast Cell Disorders 352
Abstract 353
Introduction 353
Background 356
Methods 358
Collection of BM Samples 358
Staining of BM Samples 358
Panel of Monoclonal Antibodies 359
Data Acquisition and Analysis 359
Critical Parameters and Troubleshooting 360
Results 362
Identification, Enumeration, and Characterization of BM MC by Flow Cytometry 362
Immunophenotypic Features of Normal BM MC 362
BM MC-Committed Precursors and Normal MC Maturation Patterns 363
Immunophenotype of Mature Resting Normal and Reactive BM MC 363
Activated MC Immunophenotype 366
Immunophenotypic Characteristics of BM MC in Systemic Mastocytosis 367
Good-Prognosis SM: Indolent Systemic Mastocytosis (ISM) with or without Skin Lesions 368
Well-Differentiated Systemic Mastocytosis 368
Poor-Prognosis Systemic Mastocytosis: Aggressive Systemic Mastocytosis and Mast Cell Leukemia 370
Immunophenotypic Analysis of MC from Biological Specimens Other than BM 370
Identification of Circulating MC in Peripheral Blood 370
Identification of MC in Organic Fluids or Tissues 371
Medical Indications 371
Summary 372
Acknowledgments 372
References 373
Index 380
Volumes In Series 390
Color Plate 400