Pyroptosis: Methods and Protocols

Preface
References
Contents
Contributors
Chapter 1: Molecular Mechanisms of Pyroptosis
1 Cell Death Nomenclature and the History of ``Pyroptosis´´
2 Inflammasomes and Inflammatory Caspase Activation
3 Cellular Processes Occurring During Pyroptosis
4 Future Directions
References
Chapter 2: Assessment of NLRP3 Inflammasome Activation and NLRP3-NEK7 Complex Assembly
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Assessment of NLRP3 Inflammasome Activation in BMDM
2.3 Assessment of NLRP3-NEK7 Complex Assembly
3 Methods
3.1 Generation of L929 Cells-Conditioned Media
3.2 Generation of Mouse Bone Marrow-Derived Macrophages (BMDM)
3.3 Assessment of NLRP3 Inflammasome Activation in BMDM
3.4 Assessment of NLRP3-NEK7 Complex Assembly in BMDM
4 Notes
References
Chapter 3: Inducing Pyroptosis with FlaTox, RodTox, or NeedleTox
1 Introduction
2 Materials
2.1 In Vitro Stimulation of the NAIP-NLRC4 Inflammasome in Macrophages
2.1.1 Propidium Iodide (PI) Uptake Assay
2.1.2 Lactate Dehydrogenase (LDH) Release Assay
2.2 In Vivo Stimulation of the NAIP-NLRC4 Inflammasome in C57BL/6 Mice
2.2.1 Hematocrit Measurement
3 Methods
3.1 In Vitro Stimulation of the NAIP-NLRC4 Inflammasome in Macrophages
3.1.1 PI Fluorescence
3.1.2 LDH Release Assay
3.2 In Vivo Stimulation of the NAIP-NLRC4 Inflammasome in Mice
3.2.1 Hematocrit
4 Notes
References
Chapter 4: Inducing Pyroptosis Via the Pyrin Inflammasome
1 Introduction
2 Materials
2.1 Pyrin-Expressing U937 Cell Culture
2.2 Bone Marrow-Derived Macrophages (BMDMs)
2.3 Pyrin Inflammasome-Activating Stimuli
2.4 Pyrin Inflammasome Inhibitors
2.5 Real-Time Cell Death Assay
2.6 Lactate Dehydrogenase (LDH) Assay
2.7 Elisa
3 Methods
3.1 Real-Time Cell Death Analysis by Propidium Iodide (PI) Incorporation
3.2 LDH Assay
3.3 Quantification of IL-1β Release by ELISA
4 Notes
References
Chapter 5: Caspase-4 Activation and Recruitment to Intracellular Gram-Negative Bacteria
1 Introduction
1.1 Inflammasomes
1.2 The Genus Burkholderia
2 Materials
2.1 Infection Assays
2.2 Microscopy-Based Analysis
2.3 Western Blot Analysis
2.3.1 SDS-Page
2.3.2 Gel Transfer
2.3.3 Immunoblotting
2.4 PI Uptake Assay
3 Methods
3.1 Microscopy-Based Analysis of Caspase-4 Recruitment on B. Thailandensis
3.2 Detection of Active, Cleaved Caspase-4 by Immunoblot
3.2.1 Sample Preparation for SDS Gel Electrophoresis
3.2.2 SDS Polyacrylamide Gel Preparation
3.2.3 Gel Transfer
3.2.4 Immunoblot
3.3 PI Uptake Assay
4 Notes
References
Chapter 6: CARD8 Inflammasome Activation by HIV-1 Protease
1 Introduction
2 Materials
2.1 Cell Isolation and Culture Conditions
2.2 Virus Preparation and Infection
2.3 Drug Treatments
2.4 HEK293T Cell Transfection
3 Methods
3.1 Isolation of PBMC by Density Centrifugation
3.2 Isolation of CD4+ T Cells from PBMC
3.3 Isolation of Monocytes from PBMC and in Vitro Differentiation
3.4 Virus Preparation and Infection Using HIV-1 Reporter Viruses
3.5 Virus Preparation and Infection Using Clinical Isolates of HIV-1
3.6 HIV-1 Infection with NNRTIs
3.7 HIV-1 Infection with Inhibitors
3.8 HEK293T Cell Transfection
4 Notes
References
Chapter 7: Pyroptosis Markers in Human Primary Specimens: Quantification of Intracellular ASC Specks by Imaging Flow Cytometry...
1 Introduction
2 Materials
2.1 ASC Speck Quantitation in Peripheral Blood Monocytes
2.2 ASC Speck Quantitation in Heterogeneous Peripheral Blood Mononuclear Cells
2.3 Quantification of Oxidized (Mitochondrial) DNA in Plasma
3 Methods
3.1 ASC Speck Quantitation by ImageStream Analysis in Primary Peripheral Blood Monocyte Cell Populations
3.1.1 Cell Staining
3.1.2 Cell Analysis
3.2 ASC Speck Quantitation by ImageStream Analysis in Heterogeneous Peripheral Blood Mononuclear Cell Populations
3.2.1 Cell Staining
3.2.2 Cell Analysis
3.3 Quantification of Oxidized DNA in Primary Patient Plasma
4 Notes
References
Chapter 8: Identifying Inflammasome Activation in Human Tissues
1 Introduction
2 Materials
2.1 Buffers
2.2 Equipment and Materials
2.3 Primary Antibodies
2.4 IF-Specific Materials
2.5 IHC-Specific Materials
3 Methods
3.1 Deparaffinization and Rehydration (a Common Method for IF and IHC)
3.2 Antigen Retrieval (a Common Method for IF and IHC)
3.3 Immunofluorescence on Heart Tissues (ASC/Cardiac Actin or CD31)
3.4 Immunofluorescence on Heart Tissues (ASC/S100A4)
3.5 Immunofluorescence on Lung Tissues (ASC, NLRP3, or Cleaved Caspase-1/Collagen IV)
3.6 Immunohistochemistry on Heart Tissues (NLRP3 and Cleaved-Caspase-1)
4 Notes
References
Chapter 9: Analyzing Caspase-8-Dependent GSDMD Cleavage in Response to Yersinia Infection
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Yersinia Preparation and Infection
2.3 Lactate Dehydrogenase (LDH) Assay
2.4 Cell Extracts and Supernatant Precipitation
2.5 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
2.6 Western Blot
3 Methods
3.1 Cell Culture and Infection Preparation
3.2 Infection
3.3 LDH Assay
3.4 Combining Cell Extracts with Protein Supernatant
3.5 SDS-PAGE and Western Blot
4 Notes
References
Chapter 10: Biochemical Methods for Assessing Gasdermin D Inactivation in Macrophages
1 Introduction
2 Materials
2.1 L929 Cell-Conditioned Medium
2.2 Culture of BMDMs
2.3 Stimulating GSDMD Activation in BMDMs
2.4 Cell Lysis
2.5 SDS/Native PAGE
2.6 Western Blotting
3 Methods
3.1 Preparation of M-CSF L929 Cell-Conditioned Medium
3.2 Isolation and Culture of Bone Marrow-Derived Macrophages
3.3 Treatment of BMDMs
3.4 Lysis and Protein Extraction of BMDMs
3.5 SDS-PAGE/Native PAGE
3.6 Western Blotting for GSDMD Processing and Oligomerization
4 Notes
References
Chapter 11: Detection of Gasdermin C-Mediated Cancer Cell Pyroptosis
1 Introduction
2 Materials
2.1 Transient Transfection
2.2 Western Blotting
2.3 Lactate Dehydrogenase (LDH) Release Assay
2.4 Time-Lapse Imaging
2.5 Confocal Microscopy
3 Methods
3.1 Transient Transfection of Constructs in HeLa Cells
3.2 Detection of GSDMC Cleavage by Western Blotting
3.3 Measurement of GSDMC-Mediated Cytotoxicity by LDH Release Assay
3.4 Monitoring Cell Morphology During GSDMC-Mediated Pyroptosis by Time-Lapse Imaging
3.5 Confocal Microscopy Analysis of GSDMC-N Domain Localization
4 Notes
References
Chapter 12: Inducing Pyroptosis via Gasdermin B and Gasdermin E Cleavage
1 Introduction
2 Materials
2.1 Molecular Cloning
2.2 Cell Culture
2.3 Lentivirus Production and Infection
2.4 Purification of Active Human GZMA from 293F Cells
2.5 Electroporation and FACS Analysis
2.6 Treatment of NCI-H522, CT26, or HeLa Cells with Chemotherapy Drug or TNFα Plus CHX, and FACS Analysis
2.7 Western Blot
3 Methods
3.1 Construction of Expression Plasmids
3.2 Construction of Stable Cell Line
3.3 Purification of Active Human GZMA from 293F Cells
3.3.1 293F Cell Culture
3.3.2 Transfection of 293F Cells with PEI
3.3.3 Purification of GZMA from Supernatant
3.3.4 EK Digestion to Obtain GZMA Protein
3.4 Electroporation of GZAM into GSDMB-Expressing 293T Cells
3.5 Flow Cytometry Analysis of GSDMB Activation-Induced Pyroptosis
3.6 NK-92MI Cell Killing of GSDMB-Expressing 293T Cells
3.7 Measurement of Calcein Release
3.8 TNFα Treatment of HeLa Cells
3.9 Chemotherapy Drug Treatment of HeLa, NCI-H522, and CT26 Cells
3.10 Measurement of LDH Release
3.11 Flow Cytometry Analysis of GSDME Activation-Induced Pyroptosis
3.12 Western Blot
4 Notes
References
Chapter 13: Assessing Interleukin-1β Activation During Pyroptosis
1 Introduction
2 Materials
2.1 Treatment of Cells for Inflammasome Activation
2.2 Collection of Samples for Western Blot
2.3 Immunoblot
2.4 Bioassay
3 Methods
3.1 Treatment of Cells for Inflammasome Activation
3.2 Collection of Samples for Western Blot
3.3 Immunoblot for Mature IL-1β
3.4 Bioassay for Active IL-1β
4 Notes
References
Chapter 14: Monitoring Calcium Fluxes and Lysosome Exocytosis During Pyroptosis
1 Introduction
2 Materials
2.1 Culturing Primary Bone Marrow-Derived Macrophages (BMDMs) (See Note 1)
2.2 Measuring Calcium Influx by Live Cell Microscopy Using Fluo-4
2.3 Surface CD107 (LAMP-1) Staining as a Marker of Lysosomal Exocytosis
3 Methods
3.1 Culturing Primary Bone Marrow-Derived Macrophages (BMDMs)
3.2 Measuring Calcium Influx by Live Cell Microscopy Using Fluo-4
3.3 Surface CD107 (LAMP-1) Staining as a Marker of Lysosomal Exocytosis
4 Notes
References
Chapter 15: Assessing Cytoskeletal Destruction During Pyroptosis
1 Introduction
2 Materials
2.1 Cells
2.2 Cell Culture Medium and Reagents
2.3 Reagents for Immunofluorescent Analysis
2.4 Antibodies and Stains
3 Methods
3.1 Isolation of Bone Marrow for Bone Marrow-Derived Macrophages (BMDMs)
3.2 Propagation and Differentiation of Cells
3.3 Treatment of Cells for Inflammasome Activation
3.4 Fixation and Staining of Cells for Immunofluorescent Analysis
4 Notes
References
Chapter 16: Assessment of HMGB1 Release During Experimentally Induced Pyroptosis
1 Introduction
2 Materials
2.1 Primary Bone Marrow-Derived Macrophage (BMDM) Harvest
2.2 Pyroptosis Activation
2.3 LDH Cytotoxicity Assay
2.4 HMGB1 Elisa
2.5 TCA Precipitation for HMGB1 Immunoblot
2.6 HMGB1 Immunoblot
3 Methods
3.1 Primary Bone Marrow-Derived Macrophage Harvest
3.2 Pyroptosis Activation
3.3 LDH Cytotoxicity Assay
3.4 HMGB1 Elisa
3.5 TCA Precipitation for HMGB1 Immunoblot
3.6 HMGB1 Immunoblot
4 Notes
References
Chapter 17: Time-Lapse Imaging of Inflammasome-Dependent Cell Death and Extrusion in Enteroid-Derived Intestinal Epithelial Mo...
1 Introduction
2 Materials
2.1 Enteroid Culture Maintenance
2.2 Enteroid-Derived 2D Monolayer Establishment
2.3 Murine Enteroid-Derived Monolayer Establishment
2.4 Human Enteroid-Derived Monolayer Establishment
2.5 S.Tm Infection of Enteroid-Derived Monolayers and Cell Death Analysis by Time-Lapse Microscopy
3 Methods
3.1 Murine Enteroid Culture Maintenance
3.2 Human Enteroid Culture Maintenance
3.3 Establishment of Murine Enteroid-Derived Monolayers
3.4 Establishment of Human Enteroid-Derived Monolayers
3.5 Preparation of the S.Tm Inoculum
3.6 Monolayer Infection and Time-Lapse Microscopy Analysis
4 Notes
References
Index