Tissue-Resident Macrophages: Methods and Protocols (Methods in Molecular Biology, 2713)

دانلود کتاب Tissue-Resident Macrophages: Methods and Protocols (Methods in Molecular Biology, 2713)

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کتاب ماکروفاژهای مقیم بافت: روش‌ها و پروتکل‌ها (روش‌ها در بیولوژی مولکولی، 2713) نسخه زبان اصلی

دانلود کتاب ماکروفاژهای مقیم بافت: روش‌ها و پروتکل‌ها (روش‌ها در بیولوژی مولکولی، 2713) بعد از پرداخت مقدور خواهد بود
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توضیحاتی در مورد کتاب Tissue-Resident Macrophages: Methods and Protocols (Methods in Molecular Biology, 2713)

نام کتاب : Tissue-Resident Macrophages: Methods and Protocols (Methods in Molecular Biology, 2713)
ویرایش : 1st ed. 2024
عنوان ترجمه شده به فارسی : ماکروفاژهای مقیم بافت: روش‌ها و پروتکل‌ها (روش‌ها در بیولوژی مولکولی، 2713)
سری :
نویسندگان :
ناشر : Humana
سال نشر : 2023
تعداد صفحات : 572
ISBN (شابک) : 1071634364 , 9781071634363
زبان کتاب : English
فرمت کتاب : pdf
حجم کتاب : 28 مگابایت



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فهرست مطالب :


Preface
Contents
Contributors
Chapter 1: Macrophage Development and Function
1 The History of Macrophages
2 Macrophage Development
3 Macrophage Core Functions: From Homeostasis to Pathogenesis
References
Chapter 2: Fate-Mapping Macrophages: From Ontogeny to Functions
1 Introduction
2 Developmental Origins and the Microenvironment: Does Ontogeny Matter?
2.1 Current Understanding of Macrophage Ontogeny
2.2 Macrophage Specification: Origin, Environment, or Both?
2.3 Stage- and Niche-Specific Functions for Fetal-Derived Macrophages?
2.4 Macrophage Ontogeny: Key to Nonhomeostatic Conditions?
2.5 Ontogeny: More than Just Origins
3 How to Determine the Origins of Macrophages: The Case for Genetic Fate Mapping
3.1 Macrophage Fate-Mapping Models
3.1.1 Fate-Mapping Fetal-Derived Macrophages
3.1.2 Fate-Mapping BM Monocyte-Derived Macrophages
3.1.3 Monocyte Input in a Specified Window (Pulse-Chase)
4 Practical Considerations
4.1 Recombination Efficacy, Cre Toxicity, and Zygosity
4.2 Reporter Choice
4.3 Limitations of Cre(ER) Systems: Specificity, Precision of Labeling, and Overlap of Hematopoietic Waves
4.4 Caveats of Tamoxifen Treatment
5 Concluding Remarks: Future Approaches to Current Limitations
References
Chapter 3: Studying Autophagy in Microglia: Overcoming the Obstacles
1 Microglia and Autophagy: The Generalities
2 Autophagy: Regulation of Microglial Cell Biology
3 Technical Aspects: What You Need to Know for Correctly Analyzing Autophagy in Microglia
4 Experimental Techniques Used to Analyze Autophagy Flux in Microglia
4.1 Western Blotting of Autophagy-Related Proteins
4.1.1 In Vitro Analysis
Advantages
Limitations
4.1.2 In Vivo Analysis
4.2 Fluorescence and Confocal Microscopy-Based Methods
4.2.1 In Vitro Analysis
Advantages
Limitations
4.2.2 In Vivo Analysis
4.3 Transmission Electron Microscopy-Based Methods
4.3.1 In Vitro Analysis
Advantages
Limitations
4.3.2 In Vivo Analysis
4.4 Concluding Remarks
References
Chapter 4: Hemocyte Nuclei Isolation from Adult Drosophila melanogaster for snRNA-seq
1 Introduction
2 Materials
2.1 Transgenic Drosophila melanogaster Lines
2.2 Plastic Ware
2.3 Buffers and Solutions
2.4 Equipment
3 Methods
3.1 Fly Dissection and Nuclei Isolation
3.2 Hemocyte Nuclei Sorting
4 Notes
References
Chapter 5: Isolation of Tissue Macrophages in Adult Zebrafish
1 Introduction
2 Materials
2.1 Zebrafish Lines (See Notes 1 and 2)
2.2 Plasticware and Equipment
2.3 Media and Reagents
3 Methods
3.1 Tubes to Prepare Before to Start
3.2 Dissection of Adult Zebrafish Organs or Tissues
3.3 Processing the Organs
3.3.1 WKM
3.3.2 Brain and Liver
3.3.3 Skin
3.4 Pelleting the Cells
3.5 Filtering and Staining for Dead Cell Discrimination
3.6 Data Collection
3.7 Expected Outcomes for Each Tissue
4 Notes
References
Chapter 6: Genetic and Immunohistochemistry Tools to Visualize Rat Macrophages In Situ
1 Introduction
2 Materials
2.1 Equipment
2.2 Solutions and Reagents
2.3 Consumables
3 Methods
3.1 Preanalytical Phase: Tissue Processing and Section Preparation
3.1.1 PFA Fixation
3.1.2 Embedding and Sectioning
3.2 Analytical Phase: Antigen Detection
3.2.1 Dewax Slides (See Note 7)
3.2.2 Rehydrate Tissue
3.2.3 Block Endogenous Peroxidase Activity (See Note 10)
3.2.4 Antigen Retrieval
3.2.5 Block Nonspecific Binding
3.2.6 Primary Antibody Reaction
3.2.7 Detection and Colour Reaction
3.2.8 Dehydrate and Clear the Tissue (See Note 26)
3.3 Postanalytical Phase: Visualization
4 Notes
References
Chapter 7: Phenotyping of Macrophages in Human Immune System Mice
1 Introduction
2 Materials
2.1 Mice
2.2 HSC Purification
2.3 HSC Transplantation
2.4 Verification of Humanization by Flow Cytometry Analysis
2.5 Phenotyping of Human Macrophages by Immunofluorescence Microscopy
3 Methods
3.1 PBMC Isolation
3.2 HSC Isolation Using Miltenyi CD34 Microbead Kit
3.3 Analysis of HSC Purity by Flow Cytometry
3.4 Generation of Humanized Mice
3.5 Verification of Humanization
3.6 Phenotyping of Human Macrophages by Immunofluorescence Microscopy
4 Notes
References
Chapter 8: Fate-Mapping of Yolk Sac-Derived Macrophages
1 Introduction
2 Materials
2.1 Mouse Model
2.2 Consumables
2.3 Buffers and Solutions
2.4 Equipment
3 Methods
3.1 In Utero Pulse Labeling by OH-TAM
3.2 Preparation of Cell Suspension from Embryonic Tissue for Flow Cytometry Analysis
3.2.1 Embryo and Organ Isolation
3.2.2 Cell Suspension Preparation
3.2.3 Staining of Cell Suspension with Antibodies
3.2.4 Flow Cytometry Analysis
4 Notes
References
Chapter 9: Fate-Mapping of Hematopoietic Stem Cell-Derived Macrophages
1 Introduction
2 Materials
2.1 Mouse Model
2.2 Consumables
2.3 Buffers and Solutions
2.4 Equipment
3 Methods
3.1 Tamoxifen Administration and Labeling Efficiency Control
3.2 Preparation of Cell Suspension from Adult Mouse Spleen for Flow Cytometry Analysis
3.2.1 Isolation of Mouse Spleen for Flow-Cytometry Analysis
3.2.2 Cell Suspension Preparation
3.3 Staining of Cell Suspension with Antibodies Conjugated with Fluorochromes
3.4 Flow Cytometry
3.5 Data Analysis
4 Notes
References
Chapter 10: Isolation and Flow Cytometry Analysis of Macrophages from White Adipose Tissue
1 Introduction
2 Materials
2.1 Consumables
2.2 Buffers and solutions
2.3 Antibodies/Dyes
2.4 Equipment
3 Methods
3.1 Harvesting of WAT
3.2 Tissue Digestion and Preparation of Single-Cell Suspension from WAT
3.3 Antibody Staining of Cell Surface Markers
4 Notes
References
Chapter 11: Isolation and Flow Cytometry Analysis of Macrophages from the Dermis
1 Introduction
2 Materials
2.1 Required Reagents
2.2 Equipment
3 Methods
3.1 Preparation
3.2 Sample Processing and Enzymatic Digestion
3.3 Staining for Flow Cytometry
3.4 Flow Cytometry and Gating Strategies
4 Notes
References
Chapter 12: Isolation and Flow Cytometry Analysis of Macrophages from the Kidney
1 Introduction
2 Materials
2.1 Chemical Reagents
2.2 Equipment
2.3 Antibodies and Live/Dead Stain
3 Method
3.1 Isolation and Flow Cytometry Analysis of Kidney Macrophages
3.1.1 Day 1: Prep
3.1.2 Day 2: Harvest
3.1.3 Day 3: Flow Analysis
3.2 Expected Outcomes
3.3 Quantification and Statistical Analysis
4 Notes
References
Chapter 13: Isolation and Flow Cytometry Analysis of Intestinal Macrophages
1 Introduction
1.1 Intestinal Macrophages: From Monocytes to Macrophages
1.2 Heterogeneity of Intestinal Macrophages
2 Materials
2.1 Equipment
2.2 Buffers
2.2.1 Common Buffers
2.2.2 Muscularis Externa
2.2.3 Lamina Propria/Whole Tissue (See Note 1)
3 Methods
3.1 Sample Preparation
3.2 Tissue Digestion
3.2.1 Muscularis Externa
3.2.2 Lamina Propria/Whole Tissue
3.3 Antibody Staining
3.4 Identification of Intestinal Macrophages via Flow Cytometry
3.4.1 Muscularis Externa
3.4.2 Lamina Propria
4 Notes
References
Chapter 14: Isolation and Characterization of Testis Macrophages Using Flow Cytometry
1 Introduction
2 Materials
2.1 Equipment
2.2 Consumables
2.3 Workflow Solutions
2.3.1 Stock Solutions to Be Prepared in Advance
2.3.2 Freshly Prepared Solutions
3 Methods
3.1 Organ Harvesting
3.2 Organ Digestion
3.3 FACS Staining
3.4 Samples Fixation (Optional)
3.5 Sample Acquisition and Analysis
4 Notes
References
Chapter 15: Studying Macrophages in the Murine Steatotic Liver Using Flow Cytometry and Confocal Microscopy
1 Introduction
1.1 Macrophage Functions in MASLD
1.2 Different Diets to Study MASLD and MASH
1.3 Isolating Macrophages from the Steatotic Liver
1.4 Macrophage Heterogeneity in the Healthy and Steatotic Liver
1.5 Autofluorescence Problems in the Steatotic Liver
2 Materials
2.1 Diets to Induce MASLD/MASH
2.1.1 Western Diet
2.1.2 CDA-HF Diet
2.2 Isolating Macrophages from the Murine Steatotic Liver
2.2.1 Ex Vivo Digestion
2.2.2 In Vivo Digestion
2.3 Flow Cytometry for Macrophages in the Steatotic Liver
2.4 Confocal Microscopy for Macrophages in the Steatotic Liver
2.4.1 Antigen-Fix Perfusion
2.4.2 Cutting Sections, Staining, and Imaging
3 Methods
3.1 Western Diet vs Choline-Deficient L-Amino Acid Defined High-Fat Diet to Induce MASLD/MASH
3.2 Isolating Macrophages from the Steatotic Liver
3.2.1 Ex Vivo Digestion
3.2.2 In Vivo Digestion
3.3 Preparing Cells for Flow Cytometry
3.4 Removal of Autofluorescence Using Spectral Flow Cytometry
3.5 Phenotypic Identification of Macrophages in the Steatotic Liver
3.5.1 Identifying Macrophages in the Steatotic Liver Using Conventional Low Cytometry
3.5.2 Identifying Macrophages in the Steatotic Liver Using Spectral Flow Cytometry
3.5.3 Comparing Results Obtained with the Different Digestion Protocols across Diets
3.6 Preparing Liver Tissue for Confocal Microscopy
3.7 Staining Steatotic Liver Tissue for Confocal Microscopy
4 Notes
References
Chapter 16: Isolation, Ex Vivo Expansion, and Lentiviral Transduction of Alveolar Macrophages
1 Introduction
2 Materials
2.1 Conditioned Medium Containing Murine GM-CSF
2.2 AM Harvest
2.3 AM Culture
2.4 AM Cryopreservation
2.5 Lentiviral Production and Transduction
2.6 Common Solutions and Equipment
3 Methods
3.1 Preparation of Conditioned Medium Containing Murine GM-CSF
3.2 Isolation of BAL AM
3.3 Establishing Culture
3.4 Long-Term AM Culture and Cryopreservation of exAM
3.4.1 Cryopreservation of AM
3.4.2 Thawing of exAM
3.5 Lentiviral-Vector-Mediated Gene Transfer in AM
3.5.1 Lentivirus Production and Harvesting
3.5.2 Concentration of Lentiviral Vectors by Ultracentrifugation
3.5.3 Lentivirus Titration by Flow Cytometry
3.5.4 Lentivirus Transduction of exAM
4 Notes
References
Chapter 17: Translatome Profiling of Tissue-Resident Macrophages Using the RiboTag Approach
1 Introduction
1.1 The RiboTag Approach
1.2 Strengths of the RiboTag Approach
1.2.1 Avoidance of Cell Isolation and Associated Artifacts
1.2.2 Exclusion of Sequestered RNAs and RNA Associated with Phagocytosed Transcripts
1.2.3 Fate Mapping Feature
1.3 Additional Critical Features of the RiboTag Approach
1.3.1 Reliance on mRNA Enrichment
1.3.2 Dependence on Cre Recombinase
1.3.3 Dependence on Rpl22 Expression for HA Tagging
2 Materials
2.1 Mice
2.2 General Tools and Machines
2.3 General Reagents
3 Methods
3.1 Brain Isolation and Homogenization (seeNote 1)
3.2 Antibody and Beads Incubation
3.3 Washing
3.4 RNA Purification with Dynabeads mRNA DIRECT Purification Kit (seeNote 8)
3.5 Quality Control
3.6 Analysis of RiboTag Data
4 Notes
References
Chapter 18: Spectral Flow Cytometry Analysis of Resident Tissue Macrophages
1 Introduction
2 Materials
2.1 Buffers
2.2 Equipment and Disposables
3 Methods
3.1 Step-by-Step Sample Preparation for Mouse Tissue (Spleen, White Adipose Tissue, Heart, and Lung) Macrophages
3.2 Step-by-Step Sample Preparation for Mouse Tissue (Skin) Macrophages
3.3 Panel Design and Staining Protocol
3.4 Step-by-Step Operational Use of Cytek Aurora
3.5 Data Analysis
4 Notes
References
Chapter 19: Unveiling Macrophage Heterogeneity and Their Spatial Distribution Using Multiplexed Tissue Imaging
1 Introduction
2 Materials
2.1 Consumables, Chemicals, and Equipment Needed for the Staining and Acquisition
2.2 Solutions Required for Staining Frozen Sections
2.3 Solutions Required for Antibody Detection in Multicycle Acquisition Experiment
2.4 Antibody Panel
3 Methods
3.1 Tissue Preparation (Intestine, Lung, and Liver)
3.2 Pre-staining
3.3 Photobleaching
3.4 Staining
3.5 Preparation of Reporter Plate for Imaging
3.6 Imaging and Data Export
3.7 Image Processing
3.8 Image Analysis
4 Notes
References
Chapter 20: Three-Dimensional Imaging of Macrophages in Complete Organs
1 Introduction
2 Materials
3 Methods
3.1 Adapted iDisco Method
3.2 Benzyl Benzoate-Based Mild Method
4 Notes
References
Chapter 21: Whole-Mount Imaging of Adipose Tissue Macrophages
1 Introduction
2 Materials
2.1 Equipment
2.2 Buffers and Solutions
2.3 Antibodies
2.4 Image Acquisition and Analysis
3 Methods
3.1 Preparation for Tissue Harvesting
3.2 Harvesting the Adipose Tissue
3.3 Fixation
3.4 Blocking and Permeabilization
3.5 Adding the Antibodies
3.6 Mounting
3.7 Visualization of Adipose Tissue Macrophages and Structures
4 Notes
References
Chapter 22: Functional In Vivo Imaging of Macrophages
1 Introduction
2 Materials
2.1 Materials for Surgery
2.2 Image-Processing Resources
2.3 Imaging Setup
3 Methods
3.1 Preparation of the Animal
3.2 Midline Laparotomy
3.3 Preparation of the Imaging Situs
3.4 Data Analysis and Applications of Two-Photon Imaging
4 Notes
References
Chapter 23: Elucidating Immune Monitoring of Tissue-Resident Macrophages by Intravital Microscopy
1 Introduction
2 Materials
2.1 Reagents
2.1.1 General Reagents
2.1.2 Reagents for In Vivo Injection
2.2 Equipment
2.3 Software
3 Methods
3.1 Microscope Setup
3.2 Mouse Handling and Preparation
3.3 Surgery
3.4 Intravital Microscopy
3.5 Post-acquisition Processing of Intravital Imaging Datasets
4 Notes
References
Chapter 24: Combined Host-Pathogen Fate Mapping to Investigate Lung Macrophages in Viral Infection
1 Introduction
2 Materials
2.1 Required Reagents
2.2 Equipment
3 Methods
3.1 Respiratory Tract Infection with MCMV
3.1.1 Intranasal Infection
3.2 Preparation of Lung Cell Suspension for Flow Cytometry
3.2.1 Preparation
3.2.2 Cell Extraction
3.2.3 Staining for Flow Cytometry
3.3 Flow Cytometry (Gating Strategies)
3.3.1 Myeloid Cell Populations in the Lung
3.3.2 Host-Pathogen Fate Mapping Enables Identification of Infected Cells
4 Notes
References
Chapter 25: Measuring the Metabolic State of Tissue-Resident Macrophages via SCENITH
1 Introduction
2 Materials
2.1 Preparation of Buffers and Cells
2.2 SCENITH Kit (GammaOmics, Cat. Number GO-0002PE-50; GO-0002AF488-50; GO-0002AF647-50) (see Note 1)
2.3 Viability/Fc-Blocking and Surface Staining
2.4 Intracellular Staining
2.5 Equipment and Machines
3 Methods
3.1 Preparation of Single-Cell Suspensions
3.2 Inhibitor and Puromycin Treatment
3.3 Red Blood Cell Lysis (Optional)
3.4 Fc-Block/Viability and Surface Staining
3.5 Intracellular Staining
3.6 Analysis
4 Notes
References
Chapter 26: Analyzing Fcγ-Receptor Interactions on Monocytes with the Proximity Ligation Assay (PLA)
1 Introduction
2 Materials
2.1 General Materials and Equipment
2.2 Buffers and Solutions
2.3 For Direct PLA
2.3.1 Duolink In Situ Probemaker PLUS or MINUS Kit (See Note 4)
2.3.2 Duolink In Situ Detection Reagents FarRed (See Note 5)
3 Methods
3.1 Proximity Ligation Assay (PLA)
3.2 Direct PLA of FcγRIIb on Monocytes
3.2.1 Conjugation of Primary Antibodies to PLA Probes PLUS or MINUS
3.2.2 Cell Preparation and Coating on Eight-Well Glass Slides
3.2.3 Fixing and Blocking of Cells
3.2.4 Addition of Primary Antibodies Coupled to Duolink Probemakers
3.2.5 Ligation: Generating the Rolling Circle Template
3.2.6 Amplification with Incorporation of Fluorescent Oligonucleotides
3.3 Mounting of Cells and Preparation for Imaging Preparation
3.4 Image Analysis
4 Notes
References
Chapter 27: Studying Efferocytosis Dynamics in Tissue-Resident Macrophages Ex Vivo
1 Introduction
2 Materials
2.1 Buffers and Reagents
2.2 Consumables
2.3 Equipment
2.4 Analysis of IFC Data
3 Methods
3.1 Isolation and Culture of Primary Peritoneal Macrophages
3.2 Isolation of Thymus Tissue
3.3 Preparation of Apoptotic Thymocytes
3.4 Labeling of Apoptotic Cells (ACs)
3.5 Phagocytosis Assay
3.6 Detachment of Macrophages
3.7 Antibody Labeling of Macrophages for Imaging Flow Cytometry
3.8 Sample Acquisition
3.9 Data Analysis
3.9.1 Generation of the Compensation Matrix
3.9.2 Custom Mask Generation
3.9.3 Generation of Analysis Features
3.9.4 Gating Strategy
4 Notes
References
Untitled
Chapter 28: Monitoring of Inflammasome Activation of Macrophages and Microglia In Vitro, Part 1: Cell Preparation and Inflamma...
1 Introduction
2 Materials
2.1 Common Consumables and Equipment
2.2 Common Buffers, Solutions, and Reagents
2.3 Consumables for the Isolation of Different Myeloid Cell Types
2.3.1 Human PBMC Isolation and Differentiation
2.3.2 Preparation of Murine Bone Marrow-Derived Macrophages (BMDMs)
2.3.3 Preparation of Murine Microglia
2.4 Buffers, Solutions, and Reagents for the Isolation of Different Myeloid Cell Types
2.4.1 Human PBMC Isolation and Differentiation
2.4.2 Preparation of Murine Bone Marrow-Derived Macrophages (BMDMs)
2.4.3 Preparation of Murine Microglia
2.5 Consumables for Activation of Inflammasomes
2.5.1 Non-Canonical Inflammasome Activation Using LPS Transfection
2.5.2 Activation of NLRC4 and Non-Canonical Inflammasome Using Salmonella Infection
2.6 Buffers, Solutions, and Reagents for Activation of Inflammasomes
2.6.1 AIM2Inflammasome
2.6.2 NLRC4Inflammasome
2.6.3 NLRP1 and CARD8
2.6.4 NLRP3Inflammasome
2.6.5 Pyrin Inflammasome
2.6.6 Non-Canonical Inflammasome Activation Using LPS Transfection
2.6.7 Activation of NLRC4 and Non-Canonical Inflammasome Using Salmonella Infection
3 Methods
3.1 Protocols for the Isolation of Different Myeloid Cell Types
3.1.1 Human PBMC Isolation and Differentiation
Preparation of the MACS Buffer
Isolation of PBMCs Using Density Gradient
Monocyte Isolation Using CD14 Beads
Differentiation of Monocytes into M-CSF or GM-CSF Macrophages
Harvesting of M-CSF or GM-CSF Macrophages
3.1.2 Preparation of Murine Bone Marrow-Derived Macrophages (BMDMs)
Bone Marrow Flushing and Cultivation
Harvesting and Plating the BMDMs
3.1.3 Preparation of Murine Microglia
Coating of the T75 Flasks
Brain Dissection
Tissue Dissociation and Mixed Glia Culture
Microglia Isolation
3.2 Activation of Inflammasomes
3.2.1 AIM2Inflammasome
3.2.2 NLRC4Inflammasome
3.2.3 NLRP1 and CARD8
3.2.4 NLRP3Inflammasome
3.2.5 Pyrin Inflammasome
3.2.6 Non-Canonical Inflammasome Activation Using LPS Transfection
3.2.7 Activation of NLRC4 and Non-Canonical Inflammasome Using Salmonella Infection
NLRC4 Activation
Non-Canonical Inflammasome Activation
4 Notes
References
Chapter 29: Monitoring of Inflammasome Activation of Macrophages and Microglia In Vitro, Part 2: Assessing Inflammasome Activa...
1 Introduction
2 Materials
2.1 Common Consumables and Equipment (SeeNotes 1 and 2)
2.2 Consumables and Buffers for the ASC Speck Staining
2.3 Reagents and Consumables for Pyroptotic Cell Detection
2.4 Reagents for Pyroptosis Detection Using Propidium Iodide Incorporation
2.5 Reagents and Equipment for Western Blot Analysis and ASC Cross-Linking
2.6 Reagents for IL-1β Detection Using ELISA
2.7 Reagents for LDH Assay
2.8 Homogeneous Time-Resolved Fluorescence (HTRF) Assay
2.9 Reagents for the Detection of IL-1β and IL-18 Processing Using WES
3 Methods
3.1 Quantification of ASC Specks Using ASCAntibody Staining and Microscopy
3.2 Visualization of the Pyroptotic Cell Death Using Microscopy
3.3 Measuring Pyroptosis Using Propidium Iodide (PI) Incorporation
3.4 Immunoblot for Cleaved IL-1β, Cleaved Caspase 1, and Inflammasome Components
3.4.1 Lysis of Stimulated Cells and Sample Preparation (6 Wells)
3.4.2 Supernatant Harvesting and Precipitation
3.4.3 Gel Electrophoresis
3.4.4 Protein Transfer to PVDF Membrane
3.4.5 Developing the Immunoblot (Fig. 4)
3.5 ASC Cross-Linking and Detection Using Western Blot
3.6 Detection Inflammasome Activation and IL-1β Release Using WES Capillary Protein Electrophoresis
3.7 Detection of IL-1β Release Using ELISA
3.8 Measuring Pyroptosis Using Lactate Dehydrogenase Activity Assay (LDH)
3.9 Homogeneous Time-Resolved Fluorescence (HTRF) Assay
4 Notes
References
Chapter 30: Detection of G-Quadruplex DNA Structures in Macrophages
1 Introduction
2 Materials
2.1 General Equipment
2.2 Reagents
3 Methods
3.1 G4 Staining
3.2 Quantification
3.3 Alternative Methods to Detect G4 Signal
4 Notes
References
Chapter 31: Adaptation of Human iPSC-Derived Macrophages Toward an Alveolar Macrophage-Like Phenotype Post-Intra-Pulmonary Tra...
1 Introduction
2 Materials
2.1 Instruments and Equipment
2.2 Reagents, Media, and Buffers
3 Methods
3.1 Generation and Characterization of Human iPSC-Derived Macrophages
3.2 Intratracheal Application of iPSC-Derived Macrophages
3.3 Alveolar Macrophage Recovery and Further Analysis
4 Notes
References
Chapter 32: Tackling Tissue Macrophage Heterogeneity by SplitCre Transgenesis
1 Introduction
2 Materials
2.1 Mice
2.2 General Tools and Machines
2.3 Reagents
3 Methods
3.1 Design Strategy-Bioinformatic
3.1.1 Strain Considerations
3.1.2 Genomic Considerations
3.1.3 Guide Design
3.1.4 Choosing Guides
3.1.5 Repair Template
3.2 Gene-Targeted ``Knock-In´´ of splitCre Insertions Using CRISPR/Cas9
3.2.1 Annealing of crRNA with gRNA (SeeNote 8)
3.2.2 Microinjection of Guides and HDR Templates
3.2.3 Preparation of RNP Complexes for Microinjection
3.2.4 Embryo Microinjection
3.3 Genotyping and Validation
3.4 Primary Breeding
3.5 Establishment of splitCre Mouse Lines
3.5.1 Considerations for Choice of the Promoter-Driving NCre Expression
3.5.2 In Vivo Validation-Breeding to a Reporter Strain
4 Notes
References
Chapter 33: Automated Cell Counting of Macrophages In Situ
1 Introduction
2 Materials
3 Methods
3.1 Creating the Pipeline for Automated Macrophage Quantification
3.2 Importing Images
3.3 Identification of Macrophage Soma and Nuclear Diameters
3.4 Identification of Macrophages Using DAPI Masks
3.5 Saving Output Images and Data Tables
4 Notes
References
Chapter 34: Morphometric Analyses of Macrophages
1 Introduction
2 Materials
2.1 Installing FIJI and the MotiQ Plugins to Your Computer
2.2 Preparation of Input Images
2.3 Validate Input Image Files
2.4 Re-order Hyperstack in .tif Files (If Applicable)
2.5 Inspect the Images to Find Good Settings
3 Methods
3.1 Cropping of Individual Cells
3.2 Segmentation of the Images
3.3 Validating the Segmented Images
3.4 Analysis of the Images
3.5 Validating Results
3.6 Convolving Analysis Results
3.7 Data Interpretation
3.8 Reduce the File Size of a Finished MotiQ Analysis for Long-Term Storage
4 Notes
References
Chapter 35: Combined Analysis of mRNA Expression and Open Chromatin in Microglia
1 Introduction
2 Materials
2.1 SHARE-seq Preparations
2.2 Microglia Nuclei Isolation and Sorting
2.3 SHARE-seq on Microglial Nuclei-Transposition
2.4 SHARE-seq on Microglial Nuclei-Reverse Transcription
2.5 SHARE-seq on Microglial Nuclei-Nuclei Barcoding
2.6 SHARE-seq on Microglial Nuclei-Reverse Crosslinking and Library Splitting
2.7 SHARE-seq on Microglial Nuclei-Template Switch
2.8 SHARE-seq on Microglial Nuclei-Tagmented DNA Cleanup
2.9 SHARE-seq on Microglial Nuclei-cDNA Amplification
2.10 SHARE-seq on Microglial Nuclei-Tagmentation and RNA Library Preparation
2.11 SHARE-seq on Microglial Nuclei-ATAC Library Preparation
2.12 Sequencing
2.13 Preprocessing
3 Methods
3.1 SHARE-seq Preparations
3.2 Microglia Nuclei Isolation and Sorting
3.3 SHARE-seq on Microglial Nuclei-Transposition
3.4 SHARE-seq on Microglial Nuclei-Reverse Transcription
3.5 SHARE-seq on Microglial Nuclei-Nuclei Barcoding
3.6 SHARE-seq on Microglial Nuclei-Reverse Crosslinking and Library Splitting
3.7 SHARE-seq on Microglial Nuclei-Template Switch
3.8 SHARE-seq on Microglial Nuclei-Tagmented DNA Cleanup
3.9 SHARE-seq on Microglial Nuclei-cDNA Amplification
3.10 SHARE-seq on Microglial Nuclei-Tagmentation and RNA Library Preparation
3.11 SHARE-seq on Microglial Nuclei-ATAC Library Preparation
3.12 Sequencing
3.13 Preprocessing
4 Notes
References
Index




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