NLR Proteins: Methods and Protocols (Methods in Molecular Biology, 2696)

✍ Scribed by Pablo Pelegrín (editor), Francesco Di Virgilio (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 301
• Edition: 2nd ed. 2023
• Category: Library

✦ Synopsis

This second edition provides a sound basis for the molecular investigation of NLR function in health and disease. Chapters focus on NLR as innate immune receptors, “atypical” inflammasomes, biochemical and novel bioluminescence techniques for the measurement of IL-1b as readout of NLR-based inflammasomes, bioluminescent probe, biochemical and microscopy techniques, techniques to measure caspase-1 activation, cell free systems for the study of inflammasome function, and inflammasome activation. 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 key tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, NLR Proteins: Methods and Protocols, Second Edition aims to be a useful and practical guide to new researcher sand experts looking to expand their knowledge.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Canonical Inflammasomes
1 Introduction
2 The NLRP1 Inflammasome
3 The NLRP3 Inflammasome
4 The NLRC4 Inflammasome
5 The AIM2 Inflammasome
6 The Pyrin Inflammasome
References
Chapter 2: Inflammasome-Independent Roles of NLR and ALR Family Members
1 Introduction
2 NLRC4
3 NLRP12
4 AIM2
5 Concluding Remarks
References
Chapter 3: Measuring IL-1β Processing by Bioluminescence Sensors: Using a Bioluminescence Resonance Energy Transfer Biosensor
1 Introduction
2 Materials
2.1 Expression of the Bioluminescence Sensor in Macrophage Cell Lines
2.2 BRET Recording Using a Plate Reader
2.3 BRET Recording by Microscopy and Data Analysis
3 Methods
3.1 Macrophage Transfection and Priming
3.2 Measuring IL-1β Processing in Real Time Using a Plate Reader
3.3 Monitoring Real-Time IL-1β Processing in Individual Macrophages
4 Notes
References
Chapter 4: Methods to Measure NLR Oligomerization I: Size Exclusion Chromatography, Co-immunoprecipitation, and Cross-Linking
1 Introduction
2 Materials
2.1 Size Exclusion Chromatography (SEC)
2.2 Co-immunoprecipitation (Co-IP)
2.3 Caspase-1 Activity Assay
2.3.1 Fluorometric Caspase-1 Activation Assay
2.3.2 In Vitro Caspase-1 Activation Assay
2.4 Native Gel Electrophoresis
2.5 Cross-Linking
3 Methods
3.1 Size Exclusion Chromatography (SEC)
3.2 Co-immunoprecipitation (Co-IP)
3.3 Fluorometric Caspase-1 Activity Assay
3.4 In Vitro Caspase-1 Activation Assay
3.5 Native Gel Electrophoresis
3.6 Cross-Linking
4 Notes
References
Chapter 5: Measuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immunofluorescence
1 Introduction
2 Materials
2.1 Cells and Tissue Culture Buffers
2.2 Stimulation and Cell Staining
2.3 Microscopy
3 Methods
3.1 Live-Cell Imaging of ASC Speck Formation
3.1.1 Cell Culture and Seeding
3.1.2 Microscope Preparation and Live-Cell Imaging
3.2 Immunofluorescence Staining of ASC Specks in Primary Cells
3.2.1 Human Macrophage Preparation, Seeding, and Stimulation
3.2.2 Primary Cell Fixation, Permeabilization, and Immunostaining
3.3 Detection of ASC Specks by Imaging Flow Cytometry
3.3.1 Sample Preparation
3.3.2 Imaging Flow Cytometry Analysis
4 Notes
References
Chapter 6: Measuring NLR Oligomerization III: Detection of NLRP3 and NLRC4 Complex by Bioluminescence Resonance Energy Transfer
1 Introduction
2 Materials
2.1 Reagents and Solutions
2.2 Materials and Equipment
3 Methods
3.1 Expression of NLRP3 BRET Sensors in HEK293 Cells
3.2 Determination of Specific Intermolecular (Model a) or Intramolecular (Model B) BRET Signal
3.3 Detection of NLRP3 BRET Signal Variation during Activation
3.4 Detection of NLRC4 BRET Signal
4 Notes
References
Chapter 7: Method to Measure Ubiquitination of NLRs
1 Introduction
2 Materials
2.1 Cell Lysis
2.2 Transfection
2.3 Immunoprecipitation
2.4 Western Blot
3 Methods
3.1 Transfection
3.2 Cell Lysis for Detection of NLRP3-Flag Tagged with Ubiquitin Antibody
3.3 Cell Lysis for Detection of NLRP3-Flag Tagged with TUBE-Biotin
3.4 Cell Lysis in NP40 Buffer for Detection of Endogenous NLRP3 with Ubiquitin Antibody
3.5 Immunoprecipitation of Flag-Tagged NLRP3
3.6 Immunoprecipitation of Endogenous NLRP3
3.7 Western Blot Analysis
3.8 Detection of Poly-ubiquitination by Using Ubiquitin Antibodies
3.9 Detection of Poly-ubiquitination by Using TUBE-Biotin
4 Notes
References
Chapter 8: Methods to Study NLR in Human Blood Cells
1 Introduction
2 Materials
2.1 Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
2.2 PBMC Characterization
2.3 PBMC Culture
2.4 PBMC Agonists and Inhibitors
2.5 Transfection, RNA Isolation, and Amplification
2.6 Measurement of Interleukin-1β (IL-1β) Release
2.7 Measurement of Intracellular ROS
2.8 Measurement of Cysteine Release
2.9 Measurement of ATP Release
3 Methods
3.1 Isolation of Primary Monocytes from Patients with Autoinflammatory Diseases
3.2 Culture Conditions and Stimulation of Enriched Monocyte Preparations from Patients with Autoinflammatory Diseases (AID)
3.3 Analysis of pro- and Mature IL-1β Production and Secretion
3.4 NLRP3 mRNA Silencing in Primary Cells
3.5 Evaluation of Intracellular Reactive Oxygen Species in Primary Cells from AID Patients
3.6 Determination of Cysteine in Cellular Culture Media
3.7 Determination of ATP Secretion from Freshly Isolated Monocytes
3.8 Evaluation of the Expression and Modulation of Redox Gene and Cytokine Gene in Adherent Cells by AID Patients by Real-Time...
4 Notes
References
Chapter 9: Noncanonical NLRP3 Inflammasome Activation: Standard Protocols
1 Introduction
2 Materials
2.1 Reagents for Mice Stimulation: Macrophage Culture
2.2 Reagents for Monocyte Culture and Stimulation
2.3 Reagents for PCR
2.4 Reagents for Western Blot or Immunocytochemistry
3 Methods
3.1 Macrophage Differentiation and Sample Preparation
3.2 In Vivo Stimulation and Peritoneal Macrophages Isolation
3.3 Monocyte Isolation and Stimulation
3.4 Real-Time Quantitative PCR
3.5 Western Blot
3.6 Immunocytochemistry
4 Notes
References
Chapter 10: Pyroptosis Induction and Visualization at the Single-Cell Level Using Optogenetics
1 Introduction
2 Materials
2.1 Plasmid
2.2 Cell Culture
2.3 Microscope
2.4 Data Analysis
3 Methods
3.1 Cell Seeding
3.2 Cell Transfection
3.3 Microscopy Preparations for the Confocal Optogenetics Experiments
3.4 Microscopy: Optogenetics Experiments for Inducing Pyroptosis
3.5 Data Analysis
3.6 Statistics
4 Notes
References
Chapter 11: Determination of Gasdermin Pores
1 Introduction
2 Materials
2.1 Molecular Cloning
2.2 Protein Purification from E. coli
2.3 Liposome Preparation
2.4 Liposome Binding Assay
2.5 Coomassie Blue Staining
2.6 Liposome Leakage Assay
2.7 Negative-Stain Electron Microscopy Imaging
2.8 Activation of Inflammatory Caspases to Cleave GSDMD in Macrophages
2.9 Western Blot
2.10 Immunocytochemistry and Immunohistochemistry
3 Methods
3.1 Construction of Expression Plasmids
3.2 Protein Purification from E. coli
3.3 Liposome Preparation
3.4 Liposome Binding of Gasdermin N-Domain
3.5 Coomassie Blue Staining Analysis
3.6 Liposome Leakage by Gasdermin-N Pores
3.7 Imaging Gasdermin-N Pores by Negative-Stain EM
3.8 Activation of Inflammatory Caspases to Cleave GSDMD and Induce Macrophage Pyroptosis
3.9 Western Blot to Detect Caspase-Cleaved GSDMD
3.10 Immunocytochemistry to Detect Caspase-Cleaved GSDMD
4 Notes
References
Chapter 12: Methods to Activate the NLRP3 Inflammasome
1 Introduction
2 Materials
2.1 Cells and Media
2.2 Cell Culture Equipment and Reagents
2.3 Standard Laboratory Equipment and Consumables
2.4 Reagents for Experimental Treatment
2.5 Canonical NLRP3 Inflammasome Activators
2.6 Measurements
2.6.1 ELISA
2.6.2 Western Blotting
2.6.3 Fixed-Cell Imaging
2.6.4 Live Cell Imaging
2.6.5 LDH Release Assay
3 Methods
3.1 Cells
3.2 Priming
3.3 NLRP3 Inflammasome Inhibition
3.4 Canonical NLRP3 Inflammasome Activation
3.5 Read-Outs for Inflammasome Activation
4 Notes
References
Chapter 13: Quantifying Cell Death Induced by the NLRC4 Inflammasome
1 Introduction
2 Materials
2.1 Human NLRC4 Activation in THP-1 Cells
2.2 Murine NLRC4 Activation in Bone Marrow Macrophages
2.3 Quantification of NLRC4-Induced Cell Death by SYTOX
2.4 Quantification of NLRC4-Induced Cell Death by Lactate Dehydrogenase (LDH) Assay
3 Methods
3.1 Human NLRC4 Activation in THP-1 Cells
3.2 Murine NLRC4 Activation in Bone Marrow-Derived Macrophages (BMMs)
3.3 Quantification of NLRC4-Induced Cell Death by Plate-Based SYTOX Green Assay (see Notes 11 and 12)
3.4 Quantification of NLRC4-Induced Cell Death by Microscopy for SYTOX Positive Cells (see Notes 11 and 12)
3.5 Quantification of NLRC4-Induced Cell Death by LDH Assay (see Notes 11 and 12)
4 Notes
References
Chapter 14: Methods to Activate the NLRP1 Inflammasome
1 Introduction
2 Materials
2.1 Bacillus Anthracis Lethal Factor (LF) Preparation
2.2 Bacillus Anthracis Protective Antigen (PA) Preparation
2.3 LDH Cytotoxicity Assays
2.4 Western Blotting
2.5 RFP-ASC Speck Formation Assays
3 Methods
3.1 Purification of Bacillus anthracis Lethal Factor (LF)
3.2 Purification of Bacillus anthracis Protective Antigen (PA)
3.3 Measuring NLRP1B Inflammasome Activation-Induced Pyroptosis by LDH Assay
3.4 Measuring NLRP1B Inflammasome Activation by Western Blot
3.5 Analyzing NLRP1B Inflammasome Activation by ASC Speck Formation
3.6 Analyzing VbP-Induced NLRP1 Activation by ASC Speck Formation in Reconstituted Cells
3.7 Analyzing TEV Protease-Induced NLRP1 Activation by ASC Speck Formation in Reconstituted Cells
4 Notes
References
Chapter 15: Methods to Study Inflammasome Activation in the Central Nervous System: Immunoblotting and Immunohistochemistry
1 Introduction
2 Materials
2.1 Protein Extraction
2.2 Immunoblotting
2.3 Immunofluorescence on Free-Floating Sections and Paraffin-Embedded Sections
3 Methods
3.1 Protein Extraction
3.2 Immunoblotting
3.3 Immunofluorescence on Free-Floating Sections
3.4 Immunofluorescence on Paraffin-Embedded Sections
4 Notes
References
Chapter 16: Inflammasome Activation in Human Macrophages: IL-1β Cleavage Detection by Fully Automated Capillary-Based Immunoas...
1 Introduction
2 Materials
2.1 Isolation and Differentiation of hMoMacs
2.2 Treatment and Stimulation of hMoMacs
2.3 Protein Separation and Detection
3 Methods
3.1 Isolation and Differentiation of hMoMacs
3.2 Treatment and Stimulation of hMoMacs
3.3 Protein Sample Preparation
3.4 Preparation of Antibodies and Chemiluminescence
3.5 Fill the Microplate
3.6 Setting Up the Wes Instrument for Fully Automated Capillary-Based Immunoassays
3.7 Analysis and Interpretation of Results
4 Notes
References
Chapter 17: Assessing the ATP Binding Ability of NLRP3 from Cell Lysates by a Pull-down Assay
1 Introduction
2 Materials
2.1 Cell Culture, Priming, and Transfection of Cell Lines
2.2 Lysis
2.3 Determination of Total Protein Concentration
2.4 Hydration and Preparation of ATP Agarose
2.5 Incubation of Samples with ATP-Conjugated Beads
2.6 Western Blot for Detection of ATP-Bound NLRP3
3 Methods
3.1 Transfection
3.2 Lysis
3.3 Determination of Total Protein Concentration
3.4 Incubation of Samples with ATP-Conjugated Beads
3.5 Western Blot for Detection of ATP-Bound NLRP3
3.6 Interpretation of the Experimental Results
4 Notes
References
Chapter 18: Theoretical 3D Modeling of NLRP3 Inflammasome Complex
1 Introduction
2 Materials
3 Methods
3.1 Sequence Alignment and Secondary Structure Prediction
3.2 Three-Dimensional (3D) Model of the NLRP3 Protein
3.2.1 Modelling of NLRP3 Monomer in Open Conformation
3.2.2 Modeling of NLRP3 Inflammasome
4 Notes
References
Chapter 19: A Knock-In Mouse Model of Cryopyrin-Associated Periodic Syndromes
1 Introduction
2 Materials
2.1 Mice Generation
2.2 Mice Genotype
2.3 Cultures of Bone Marrow-Derived Dendritic Cells and Macrophages
2.4 Reagents for Cell Cultures
2.5 Measurement of Cytokine Release
2.6 Cytofluorimetric Analysis
3 Methods
3.1 Mice Generation
3.2 Animal Husbandry
3.3 DNA Extraction
3.4 Identification of NLRP3 Gene Mutation
3.5 Identification of Cre Gene
3.6 Clinical Phenotype
3.7 Bone Marrow Precursor Isolation
3.8 Culture Conditions of Bone Marrow-Derived Dendritic Cells
3.9 Culture Conditions of Bone Marrow-Derived Macrophages
3.10 Bone Marrow-Derived Cell Stimulation
3.11 Peripheral Blood Isolation
3.12 Spleen Cell Isolation
3.13 Cell Surface Staining
4 Notes
References
Index