دانلود کتاب Toll-Like Receptors: Methods and Protocols (Methods in Molecular Biology, 2700)

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Preface
Contents
Contributors
Part I: Detection and Analysis of Toll Like Receptors
Chapter 1: Modeling of Transmembrane Domain and Full-Length TLRs in Membrane Models
1 Introduction
1.1 General Introduction to Toll-Like Receptors
1.2 Theory
1.2.1 PDB
1.2.2 Homology Modeling
1.2.3 Protein-Protein Docking
1.2.4 Ab Initio Modeling of Transmembrane α-Helices
1.2.5 All-Atom Molecular Dynamics Simulations
1.2.5.1 Proteins: Amber ff14SB
1.2.5.2 Lipids: Lipid14
1.2.5.3 Carbohydrates: GLYCAM06
1.2.5.4 Small Molecules: GAFF
1.2.6 Molecular Dynamics Simulations Protocols
1.2.6.1 Structure Optimization: Protein Preparation Wizard of the Maestro Package
1.2.6.2 Protein-Membrane System Setup: CHARMM-GUI
1.2.6.3 Protein Protocol
1.2.6.4 Membrane Protocol
1.2.7 Postprocessing of MD Trajectories
1.2.7.1 RMSD
1.2.7.2 Area Per Lipid
1.2.7.3 Free Energy of Binding: The MM/GBSA Method
1.2.8 Visualization of MD Trajectories
2 Materials
2.1 Computer
2.2 Software
2.3 Required Data
3 Methods
3.1 Extracellular Domain of TLR4
3.2 Dimeric TLR4 Transmembrane Domain (TD2 Model)
3.3 Intracellular Domain of TLR4
3.3.1 Monomeric TLR4 Intracellular Domain: Homology Modeling
3.3.2 Dimeric TLR4 Intracellular Domain (Asymmetric Model): Protein-Protein Docking
3.4 Molecular Dynamics Simulations and Analysis of the Full-length (TLR4/MD-2/LPSEc)2
3.4.1 Model Construction
3.4.2 Insertion of the Full-Length TLR4 in a Liquid-ordered Model Membrane
3.4.3 MD Simulation of the Full-Length TLR4 in a Liquid-ordered Model Membrane
3.4.4 Analysis of MD Simulations
4 Notes
References
Chapter 2: Development of Optimal Virtual Screening Strategies to Identify Novel Toll-Like Receptor Ligands Using the DockBox ...
1 Introduction
2 Materials
2.1 Receptor Structures
2.2 Ligand Structures
2.3 VS Chemical Library
2.4 DockBox, CD, and SBCD
3 Methods
3.1 Redocking of Cocrystallized Complexes
3.2 Screening of Active Molecules and Decoys
3.3 VS Campaign
4 Summary
References
Chapter 3: Use of Fluorescent Chemical Probes in the Study of Toll-like Receptors (TLRs) Trafficking
1 Introduction
1.1 Chemical Fluorescent Probes
1.1.1 Receptor Studies Using Chemical Probes
1.2 Toll-Like Receptors, Innate Immunity, and Inflammation
1.3 Toll-Like Receptors Trafficking
1.3.1 TLR4 and Its Trafficking
1.3.2 Endosomal Toll-Like Receptors and Their Trafficking
1.4 Chemical Probes to Study TLR4 Trafficking
2 Materials
3 Methods
3.1 Cell Culture
3.2 Plate Preparation and Cell Differentiation
3.3 Cell Treatment
3.4 Fixation and Immunostaining
3.5 High-Content Imaging Analysis
4 Notes
References
Part II: Toll Like Receptors Function in Immune Responses
Chapter 4: Use of CRISPR/CAS9 Technologies to Study the Role of TLR in Dendritic Cell Subsets
1 Introduction
2 Materials
2.1 Retroviral Particles Production
2.1.1 sgRNA Vector Generation
2.1.2 Cell Isolation and Culture
2.1.3 Cell Transfection
2.2 Isolation of Murine Hematopoietic and Progenitor Stem Cells (HSPCs)
2.2.1 Mice
2.2.2 HSPCs Isolation and Stimulation
2.2.3 Purity of c-Kit+ Cells
2.3 Infection
2.4 Cell Sorting
2.5 Guide Validation
2.5.1 Genomic DNA Extraction
2.5.2 Polymerase Chain Reaction
2.6 TLR Ligands Stimulation
2.7 Cytokine Analysis
2.7.1 Luminex xMAP Technology
2.7.2 Intracellular Staining
3 Methods
3.1 Retroviral Particles Production
3.1.1 sgRNA Vector Generation
3.1.2 Cell Isolation and Culture
3.1.3 Cell Transfection
3.2 Isolation of Murine Hematopoietic and Progenitor Stem Cells (HSPCs)
3.3 Infection
3.4 Cell Sorting
3.5 Guide Validation
3.6 TLR Ligands Stimulation
3.7 Cytokine Analysis
3.7.1 Intracellular Staining
3.7.2 Luminex xMAP Technology
4 Notes
References
Chapter 5: Endotoxin-Tolerance Mimicking to Study TLR in Promotion of Tolerogenic DCs and Tr1 Cells
1 Introduction
1.1 Tolerogenic Dendritic Cells
1.2 An Overview of Different Dendritic Cells Subsets
2 Materials
2.1 Murine Bone Marrow-Derived Dendritic Cells (BMDCs) Differentiation
2.2 cDCs Purification
2.3 cDCs Phenotyping
2.4 In Vitro Induction of Tolerogenic cDCs via TLR4
2.4.1 Extracellular and Intracellular Staining
2.4.2 Cytokines Analysis
2.5 Administration of Tolerogenic cDCs in Endotoxin Mouse Model
2.5.1 Ex Vivo Analysis of Regulatory T Cells
2.5.1.1 Tissue Collection and Processing
2.5.2 Blood Collection
2.5.2.1 Serum Cytokines Analysis
2.5.2.2 Analysis of Regulatory T Cells by Flow Cytometry
3 Methods
3.1 Murine Bone Marrow-Derived DCs (BMDCs) Differentiation
3.2 cDCs Purification
3.3 cDC Phenotyping
3.4 Induction of Tolerogenic cDCs In Vitro via TLR4
3.4.1 Validation of Tolerogenic cDCs Phenotype In Vitro
3.4.1.1 Extracellular Staining: Cytofluorimetric Analysis
3.4.1.2 IDO1 Intracellular Staining
3.4.2 Cytokines Analysis
3.5 Administration of Tolerogenic cDCs in Endotoxin Mouse Model
3.5.1 Serum Cytokines Analysis Over Time
3.5.1.1 Blood Collection
3.5.1.2 Multiplex Analysis
3.6 Ex Vivo Analysis of Regulatory T Cells
3.6.1 Isolation of Murine Splenocytes
3.6.2 Analysis of Regulatory T Cells: Treg and Tr1
4 Notes
References
Chapter 6: Flow Cytometry Analysis of IL-1 Receptors and Toll-Like Receptors on Platelets and Platelet-Derived Extracellular V...
1 Introduction
1.1 Polychromatic Flow Cytometry
1.2 Platelets
1.3 Extracellular Vesicles (EVs)
1.4 EV Validation Through Flow Cytometry
2 Materials
2.1 Isolation of Platelets from Peripheral Blood
2.2 Flow Cytometry Analysis of TLRs and ILRs on Resting Platelets
2.3 Platelet/Neutrophil (Plt/PMN) Hetero Aggregate Evaluation Through Flow Cytometry
2.4 Isolation and Immunophenotyping of Platelet-Derived EVs (PEVs)
2.5 Flow Cytometry Instrument Setting for Platelet and PEV Analysis
3 Methods
3.1 Isolation of Platelets from Peripheral Blood
3.1.1 Collection of Blood
3.1.2 Preparation of Platelet-Rich Plasma (PRP)
3.1.3 Preparation of Washed Platelets
3.2 Flow Cytometry Analysis of TLRs and ILRs on Resting Platelets
3.3 Platelet Neutrophil (Plt/PMN) Hetero Aggregate Evaluation Through Flow Cytometry
3.3.1 Plt/PMN Hetero Aggregate Assessment
3.3.2 Plt/PMN Hetero Aggregate In Vitro Formation Upon Stimulation with LPS, IL1-β, and IL-18
3.4 Isolation and Immunophenotyping of Platelet-Derived EVs (PEVs)
3.4.1 Platelet-Free Plasma (PFP) Preparation
3.4.2 Isolation of Total EVs from PFP
3.4.3 Isolation of PEVs from LPS-Stimulated Washed Platelets
3.4.4 PEV Immunophenotyping Protocol
3.4.5 Absolute Count of EVs
3.5 Flow Cytometry Instrument Setting for Platelet and PEV Analysis
3.5.1 Doublet Discrimination
3.5.2 Setting of Morphological Parameters
3.5.3 Exclusion of Cell Fragments and Apoptotic Bodies During PEV Analysis
3.5.4 Definition of Background/Unspecific Signal
3.5.5 Choice of Fluorochromes
3.5.6 Antibody Titration
3.5.7 Compensation
3.5.8 Gating Strategy for Platelet Analysis
3.5.9 Gating Strategy for Plt/PMN Hetero Aggregate Analysis
3.5.10 Gating Strategy for PEV Analysis
3.5.11 Platelet Acquisition Procedure
3.5.12 Platelet/PMN Hetero Aggregate Acquisition Procedure
3.5.13 PEV Acquisition Procedure
4 Notes
References
Chapter 7: Methods to Study TLRs in Transplantation
1 Introduction
2 Materials
2.1 Donor-Splenocyte Transfer (DST)
2.2 Costimulation Blockade
2.3 TLR Agonist Preparation
2.4 Injection into Mice
2.5 Inactivation of Commensal Microbes by Heat-Killing
3 Methods
3.1 Generation of Donor-Specific Tolerance
3.1.1 DST Preparation
3.1.2 Heart Allograft Tolerance Induction
3.1.3 Skin Allograft Tolerance Induction
3.2 Prevention of Donor-Specific Tolerance Using TLR Agonists
3.2.1 Heart Allograft Prevention of Anti-CD154/DST-Mediated Tolerance
3.2.2 Skin Allograft Prevention of Anti-CD154/DST-Mediated Tolerance
3.3 Reducing Microbiota Variations Among Animals Before Experiments
3.3.1 Randomizing Mice Between Cages by Cohousing Upon Arrival to Investigator´s Animal Facility
3.3.2 Littermate Controls
3.3.3 Breeding In-House
3.3.4 Bedding Transfer
3.4 Inactivation of Commensal Microbes by Heat-Killing
3.5 Use of Transplant Hosts and/or Recipients Genetically Deficient in Individual TLRs or Modules of TLR-Dependent Signaling P...
3.5.1 Use Animals with Targeted Genetic Deficiency of Both Copies of Individual TLR Genes Such as TLR2, TLR4, etc., or C3H/HeJ...
3.5.2 Use Animals with Targeted Genetic Deletion of Both Copies of Adaptor Molecule Genes Required for TLR-Mediated Signaling ...
4 Notes
References
Chapter 8: Determining Endosomal Toll-Like Receptors Gene Expression in NK Cells After Stimulation with Specific Agonists
1 Introduction
2 Materials
2.1 PBMC Isolation
2.2 NK Cells Purification
2.3 NK Cells Stimulation with Endosomal TLR Agonists
2.4 RNA Extraction
2.5 cDNA Synthesis
2.6 Real-Time PCR of Endosomal TLR Genes
3 Methods
3.1 PBMC Isolation
3.2 NK Cells Purification
3.3 NK Cell Stimulation with Endosomal TLRs Agonists
3.4 RNA Extraction
3.5 cDNA Synthesis
3.6 Real-Time PCR of Endosomal TLR Genes
4 Notes
References
Chapter 9: In Vitro Study of TLR4-NLRP3-Inflammasome Activation in Innate Immune Response
1 Introduction
2 Materials
2.1 Immune Cell Stimulation
2.2 NLRP3-Inflammasome and NF-kB Activation Analysis
2.3 NLRP3-Inflammasome Assembly Analysis by Chemical Crosslinking of ASC Oligomers
3 Methods
3.1 Immune Cell Stimulation
3.1.1 THP-1 Differentiation into Macrophages-Like Cells by Phorbol-12-Myristate-13-Acetate (PMA)
3.2 NLRP3-Inflammasome and NF-kB Activation Analysis
3.2.1 Total Protein Extraction
3.2.2 Western Blot Analysis
3.2.3 IL-1β Secretion Analysis
3.3 NLRP3-Inflammasome Assembly Analysis by Chemical Crosslinking of ASC Oligomers
4 Notes
References
Part III: Toll Like Receptors as Novel Therapeutic Targets for Diseases
Chapter 10: Toll-Like Receptor Polymorphisms and the Risk of Cancer: Meta-analysis Study
1 Introduction
2 Materials
2.1 Identification of Study Types
2.2 Identification of Participants
2.3 Keywords
2.4 Searching Strategy
3 Methods
3.1 Quality Assessments
3.2 Data Extraction
3.3 Statistical Analysis
4 Notes
References
Chapter 11: Unrevealing the Role of TLRs in the Pathogenesis of Autoimmune Disease by Using Mouse Model of Diabetes
1 Introduction
2 Materials
2.1 Housing of Animals
2.2 Monitoring of Diabetes Onset
2.3 Monitoring of Serum Cytokines
2.4 Tissue Processing for FFPE
2.5 H&E Staining
2.6 Immunofluorescence for Insulin and Immune Cells Detection
3 Methods
3.1 Housing of Animals
3.2 TLRs Targeting
3.3 In Vivo Analysis
3.3.1 Monitoring of Diabetes Onset
3.3.2 Monitoring of Serum Cytokines
3.4 Ex Vivo Evaluation of Inflammatory Cells Infiltrating Pancreas
3.4.1 Tissue Processing for FFPE
3.4.2 H&E Staining
3.4.3 Immunofluorescence for Insulin and Immune Cells Detection
4 Notes
References
Chapter 12: In Vitro and Ex Vivo Methodologies for T-Cell Trafficking Through Blood-Brain Barrier After TLR Activation
1 Introduction
2 Materials
2.1 Isolation of Murine Naïve and Activated T Cells
2.2 Isolation of Human T Cells
2.3 Murine Cell Cultures
2.4 Primary Human CD4+ T-Cell Cultures
2.5 In Vitro BBB Simulation
3 Methods
3.1 Isolation of Murine T Cells (Fig. 4)
3.2 Isolation of Human T Cells (Fig. 4)
3.3 All Cell Cultures Preparation (Fig. 5)
3.4 Analysis of the Interaction with ECM
3.4.1 Matrigel with Osteopontin
3.4.2 Matrigel with Type I Collagen
3.4.3 In Vitro BBB Simulation
4 Notes
References
Chapter 13: Delineating the Role of Toll-Like Receptors in Inflammatory Bowel Disease
1 Introduction
2 Materials
2.1 TLR Knockout and Wild-Type Mice
2.2 Genotyping (see Note 2)
2.3 DSS-Induced Colitis Model
2.4 Analysis of the Mice
3 Methods
3.1 TLR Wild-Type and Knockout Littermate Mice
3.2 Genotyping
3.3 DSS-Induced Colitis Model
3.4 Analysis of the Mice
4 Notes
References
Chapter 14: Analysis of Differential TLR Activation in a Mouse Model of Multiple Sclerosis
1 Introduction
2 Materials and Reagents
2.1 EAE Induction
2.1.1 Active Immunization Model
2.1.2 Toxin-Induced Demyelination Model
2.2 In Vivo Transcardiac Perfusion and Tissue Dissection
2.3 Histology in Demyelination Model
2.3.1 Demyelination Analysis in Spinal Cord After EAE Induction
2.3.2 Demyelination Assessment in Brain in Cuprizone Model
2.4 TLRs Analysis in EAE Models
2.4.1 Cell Isolation and FACS Analysis of TLRs in APCs
2.4.2 RT-PCR Analysis
2.4.3 Protein Analysis
2.4.4 Immunofluorescence for Detection of TLR2
3 Methods
3.1 EAE Induction
3.1.1 Active Immunization Model
3.1.2 Toxin-Induced Demyelination Model
3.2 In Vivo Transcardiac Perfusion and Tissue Dissection
3.2.1 Perfusion and Fixation
3.2.2 Dissection
3.3 Demyelination Histology Analysis
3.3.1 Demyelination Analysis in Spinal Cord After EAE Induction
3.3.2 Demyelination Assessment in Brain in Cuprizone Model
3.4 TLRs Analysis in EAE Models
3.4.1 Cell Isolation and FACS Analysis of TLRs in APCs
3.4.2 qPCR Analysis
3.4.3 Protein Analysis
3.4.4 Immunofluorescence for Detection of TLR2
4 Notes
References
Chapter 15: Study of Agonists of TLRs as Vaccine Adjuvants
1 Introduction
2 Materials
2.1 TLR-Specific Activation Assay
2.1.1 Freezing, Thawing, and Maintaining HEK293 Transfectant Cells
2.1.2 Activation Assay
2.2 Monocyte Activation Test
2.2.1 Isolation and Freezing of Human Peripheral Blood Mononuclear Cells (PBMC)
2.2.2 Monocyte Activation Test
3 Methods
3.1 TLR-Specific Activation Assay
3.1.1 Thawing of HEK293 Transfectant Cells
3.1.2 Maintaining HEK293 Transfectant Cells
3.1.3 Freezing HEK293 Transfectant Cells
3.1.4 TLR-Specific Activation Assay
3.1.4.1 Day 1-HEK293 Transfectant Cells Plating
3.1.4.2 Day 2-TLR-Specific Assay
3.1.4.3 Calculations
3.2 Monocyte Activation Test
3.2.1 Isolation and Freezing of Human PBMC
3.2.1.1 PBMC Isolation from Buffy Coats and Freezing
3.2.2 Monocyte Activation Test
3.2.2.1 Thawing of PBMC
3.2.2.2 PBMC Stimulation
3.2.2.3 IL-6 Detection in Supernatants by Sandwich ELISA
3.2.2.4 Calculations
4 Notes
References
Chapter 16: Activation of TLRs by Opportunistic Fungi in Lung Organoids
1 Introduction
2 Materials
2.1 Human-Induced Pluripotent Stem Cells (iPSC) Maintaining and Passaging
2.2 Differentiation of Lung Organoids (LOs)
2.3 Freezing of the Organoids
2.4 Thawing of the Organoids
2.5 Quality Check TF-qPCR
2.6 Quality of Differentiation Control Immunofluorescent Staining of Transcription Factors
2.7 Quality Check FACS
2.8 RNA Sequencing
2.9 Stimulation of the Organoids with TLR Ligands
2.10 Stimulation of the Organoids with Whole Fungi
3 Methods
3.1 iPSC Maintaining and Passaging
3.2 Differentiation of LOs
3.3 Freezing of the Organoids
3.4 Thawing of the Organoids
3.5 Quality Check-Expression of Pulmonary Transcription Factors
3.5.1 Isolation of RNA
3.5.2 Reverse Transcription (RT) of cDNA
3.5.3 qPCR Using TaqMan Probes
3.6 Quality Check-Immunofluorescent Staining of Transcription Factors
3.7 Quality Check-Dissociation and FACS
3.8 RNA Sequencing
3.9 Stimulation of the Organoids with TLRs Ligands
3.10 Stimulation of the Organoids with Whole Fungi
4 Notes
References
Index