Apoptosis and Cancer: Methods and Protocols

Description

This volume discusses methods used in the fields of molecular and cellular biology for detecting and studying cell death, especially in cancer and cancer therapy. Chapters in this book cover topics such as non-destructive, real-time Annexin V apoptosis assay; assessment of the immune response to tumor cell apoptosis and efferocytosis; mass cytometry assessment of cell phenotypes and signaling states in human whole blood; cell cycle analysis of ER stress and autophagy; and simultaneous detection of inflammasome activation and membrane damage during pyroptosis. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and practical, Apoptosis and Cancer: Methods and Protocols is a valuable resource and practical guide for both novice and expert researchers looking into the “meaning of death.” Preface Contents Contributors Chapter 1: A Nondestructive, Real-Time Annexin V Apoptosis Assay 1 Introduction 2 Materials 2.1 Equipment 2.2 Reagents 3 Methods 3.1 Kinetic Characterization of an Apoptotic Induction Time Course 4 Notes References Chapter 2: Cell Death Analysis in Cancer Spheroids from a Microfluidic Device 1 Introduction 2 Materials 2.1 Microfluidic Device Generation 2.2 Cell Culture and Cell Treatment 2.3 Clonogenic Assay 2.4 Flow Cytometry 3 Methods 3.1 Microfluidic Fabrication 3.2 Microfluidic Preparation 3.3 Cells Seeding 3.4 Spheroids Treatment Within the Microfluidic Device 3.5 Cell Death Measurement 3.5.1 Spheroid Size Evaluation 3.5.2 Cells Collection and Analysis Clonogenic Assay Flow Cytometry 4 Notes References Chapter 3: Apoptosis Detection by Quantification of Cell Debris in Bright-Field Microscopy Images 1 Introduction 2 Materials 2.1 Equipment 2.2 Reagents 3 Methods 3.1 Sample Collection and Imaging 3.2 Image Processing and Analysis 4 Notes References Chapter 4: Accurate Enumeration of Apoptotic Cancer Cells Using Flow Cytometry 1 Introduction 1.1 Apoptosis Measurement 1.2 Discrimination Between Whole Cells and Cell Fragments by Flow Cytometry 1.3 Apoptosis Quantification 2 Materials 2.1 Equipment 2.2 Reagents 3 Methods 3.1 Preparation of the Microbeads 3.2 Preparation of the Cell Suspension 3.3 Preparation of the Culture 3.4 Preparation of the Basal Condition 3.5 Acquisition of Cells After Culture 3.6 Calculation of the Apoptotic Rate 4 Notes References Chapter 5: Assessment of the Immune Response to Tumor Cell Apoptosis and Efferocytosis 1 Introduction 2 Materials 2.1 Establish Orthotopic Mammary Tumors in Immune-Competent Mice 2.2 Induce Apoptosis and Efferocytosis In Vivo 2.3 Analyze Immune Response to Efferocytosis in the Tumor Microenvironment 3 Methods 3.1 Establish Orthotopic Mammary Tumors in Immune-Competent Mice 3.2 Induce Apoptosis and Efferocytosis In Vivo 3.3 Analyze Cytokines by RT-qPCR 3.4 Analyze Leukocytes by Flow Cytometry 3.5 Histologic Analysis of Tumor Microenvironment 4 Notes References Chapter 6: Quantitative Analysis of Apoptosis and Necrosis in Live Cells Using Flow Cytometry 1 Introduction 2 Materials 2.1 Cell Lines 2.2 Plasmids 2.3 Cell Culture Reagents 2.3.1 Media 2.3.2 Other Cell Culture Reagents and Kits 2.4 Instruments and Other Essential Requirements 3 Methods 3.1 Generation of Cells Expressing FRET-Based Cellular Sensor for Detection of Apoptosis 3.1.1 Transfection of Cells with pcDNA3 ECFP-DEVD-EYFP Plasmid 3.1.2 Antibiotic Selection for the Generation of Stably Transfected Cells 3.1.3 Fluorescent-Associated Cell Sorting (FACS) 3.1.4 Clonal Selection and Expansion 3.2 Generation of Cells Expressing Fluorescent Probe for Discriminating Apoptosis and Necrosis 3.2.1 Transfection of Cells with pDsRed2-Mito Plasmid 3.3 Flow Cytometric Analysis of Apoptosis and Necrosis 3.3.1 Quantification of Necrosis in a Scatter Plot of ECFP/EYFP vs PE 3.3.2 Quantification of Apoptotic Fraction in the Sample 4 Notes References Chapter 7: A Fluorescence-Microscopic System for Monitoring the Turnover of the Autophagic Substrate p62/SQSTM1 1 Introduction 2 Material 2.1 Disposables 2.2 Equipment 2.3 Reagents 3 Methods 3.1 Cell Culture Routine 3.2 Plasmid Transfection 3.3 Sample Preparation for Microscopic Analysis 3.4 Cellular Imaging and Image Analysis 3.5 Sample Preparation and Immunoblot Detection 4 Notes 5 Concluding Remarks References Chapter 8: Single-Cell Profiling of the Intrinsic Apoptotic Pathway by Mass Cytometry (CyTOF) 1 Introduction 2 Materials 2.1 Reagents 2.2 Solution Preparation (See Note 3) 3 Methods 3.1 Single-Cell Sample Preparation for CyTOF 3.2 Barcoding for CyTOF 3.3 Cell Staining 3.4 Sample Acquisition 3.5 Data Analysis 4 Notes References Chapter 9: High-Throughput, Parallel Flow Cytometry Screening of Hundreds of Cell Surface Antigens Using Fluorescent Barcoding 1 Introduction 2 Material and Equipment 2.1 Single-Cell Preparation for FCB 2.2 Fluorescent Barcoding and Viability Staining 2.3 FCB Quality Control and Cell Surface Immunophenotyping 2.4 Data Acquisition and Analysis 3 Methods 3.1 Single-Cell Preparation for FCB 3.2 Fluorescent Barcoding and Viability Staining 3.3 FCB Quality Control 3.4 Plate Preparation 3.5 Cell Surface Immunophenotyping 3.6 Data Acquisition 3.7 Data Analysis 4 Notes References Chapter 10: Mass Cytometry Assessment of Cell Phenotypes and Signaling States in Human Whole Blood 1 Introduction 2 Materials 3 Methods 3.1 Whole Blood Sample Stimulation and Storage 3.2 Thawing and Lysing Erythrocytes 3.3 Sample Barcoding 3.4 Surface Staining 3.5 Additional Fix with 4% PFA 3.6 Methanol Permeabilization 3.7 Intracellular Staining 3.8 Ir Staining and CYTOF Acquisition 3.9 Analysis 4 Notes References Chapter 11: Quantification of Neutrophils Undergoing NET Formation and Distinguishing Mechanisms of Neutrophil Cell Death by U... 1 Introduction 2 Materials 2.1 Isolation of Neutrophils 2.2 Staining of Neutrophils 2.3 Assessment of NET Formation and of Other Forms of Cell Death 3 Methods 3.1 Neutrophil Isolation 3.2 Staining of the Neutrophils 3.3 Visualization of NETting Neutrophils by IncuCyte S3 3.4 Quantification of Neutrophils Undergoing NET Formation 4 Notes References Chapter 12: Profiling of Protein Carbonylations in Ferroptosis by Chemical Proteomics 1 Introduction 2 Materials 3 Methods 3.1 Identifying Carbonylated Proteins in Ferroptosis 3.1.1 Cell Treatment 3.1.2 Cell Lysis 3.1.3 m-APA Labeling 3.1.4 Conjugation by Click Chemistry 3.1.5 Streptavidin Enrichment 3.1.6 Reduction and Alkylation 3.1.7 Trypsin Digestion 3.1.8 Reductive Dimethylation 3.1.9 Pre-Fractionation of the Tryptic Peptides 3.1.10 LC-MS/MS Analysis 3.1.11 MS Data Analysis 3.2 Identifying Carbonylated Sites in Ferroptosis 3.2.1 Cell Treatment 3.2.2 Cell Lysis 3.2.3 m-APA Labeling 3.2.4 Conjugation by Click Chemistry 3.2.5 Streptavidin Enrichment 3.2.6 Reduction and Alkylation 3.2.7 Trypsin Digestion 3.2.8 Release of Carbonylated Peptides 3.2.9 LC-MS/MS Analysis 3.2.10 MS Data Analysis 4 Notes References Chapter 13: Cell Cycle Analysis of ER Stress and Autophagy 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Chemicals 2.3 ER Mass and Cell Cycle Flow Cytometric Analysis 2.4 Autophagy and ER Stress 2.5 Cellular Protein Aggregate Determinations 3 Methods 3.1 Induction of Autophagy and ER Stress 3.2 Cell Cycle, ER Tracker, Apoptosis, and Cell Viability Labeling 3.3 Data Analysis of Cell Cycle, ER Tracker, Apoptosis, and Cell Viability 3.4 Indirect Immunofluorescence LC3B and Direct PERK Labeling with Cell Cycle Analysis 3.5 Data Analysis of LC3B (Autophagy) and ER Stress (PERK) 3.6 Live, Apoptotic, and Necrotic Cell Analysis with Misfolded Protein Labeling 3.7 Data Analysis of Misfolded Proteins 4 Notes References Chapter 14: Proximity Ligation in Situ Assay to Monitor Autophagy-Related Protein Interactions and Autophagy in Cancer Cells 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Immunofluorescence/PLISA 2.3 Analysis 3 Methods 3.1 Cell Culture 3.2 Proximity Ligation In Situ Assay (PLISA) 3.3 Analysis 3.4 Quantification of Autophagy 3.5 Antibody Validation for PLISA 4 Notes References Chapter 15: Simultaneous Detection of Inflammasome Activation and Membrane Damage During Pyroptosis 1 Introduction 2 Materials 2.1 Harvest of Murine Bone Marrow 2.2 Generation of L929-Conditioned Medium 2.3 Generation, Collection, and Seeding of Murine Bone Marrow-Derived Macrophages 2.4 Activation and Detection of Inflammasomes 3 Methods 3.1 Collection of Bone Marrow Cells 3.2 Generation of L929-Conditioned Medium 3.3 Generation, Collection, and Seeding of Murine Bone Marrow-Derived Macrophages 3.4 Induction of Caspase-1 Activation Using Salmonella Typhimurium 4 Notes References Chapter 16: Cancer and Apoptosis 1 Cancer 2 Link Between Extracellular Tumor Matrix and Cancer Progression 3 Programmed Cell Death/Apoptosis in Cancer 4 Apoptotic Dysregulation in Cancer 5 Targeting Apoptosis Regulators and Limitations of Potential Anticancer Drugs References Index