Immunosenescence: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Quantification of T-Cell Receptor Excision Circles (TRECs)
	1 Introduction
		1.1 Thymus
		1.2 T-Cell Receptor Excised Circles (TRECs)
	2 Materials
	3 Methods
		3.1 DNA Purification from Whole  Blood
		3.2 DNA Purification from Peripheral Mononuclear Cells
		3.3 Isolation of CD3+ T Cells with Positive Selection
		3.4 Isolation CD4+ and CD8+ T Cells with Negative Selection
		3.5 Analysis of Signal-Joint TREC Concentrations
	4 Notes
	References
Chapter 2: Telomere Length Measurement in Human Tissue Sections by Quantitative Fluorescence In Situ Hybridization (Q-FISH)
	1 Introduction
	2 Materials
		2.1 Materials
		2.2 Equipment
	3 Methods
		3.1 Pretreatment
		3.2 Hybridization
		3.3 Post-hybridization Washes and DAPI Counterstaining
		3.4 Microscopic Observation and Image Analysis
	4 Notes
	References
Chapter 3: Phenotypic Characterization of B-Lymphocyte Subpopulations in Human Peripheral Blood: A Cost-Effective Seven-Color ...
	1 Introduction
		1.1 B Cells
		1.2 Transitional B Cells
		1.3 Naive B Cells
		1.4 Switched Memory B Cells
		1.5 Unswitched Memory B Cells
		1.6 Double-Negative Cells
		1.7 Plasmablasts
		1.8 Protocol Strategy for Identification of B-Lymphocyte Subpopulations in Peripheral Blood Mononuclear Cells (PMBCs)
		1.9 Conclusions
	2 Materials
		2.1 Total Blood Cells
		2.2 Fresh Cells
		2.3 Frozen Cells
	3 Methods
		3.1 Total Blood Cells
		3.2 Fresh Cells
		3.3 Frozen Cells
		3.4 Flow Cytometer Acquisition and Analysis
	4 Notes
	References
Chapter 4: Method to Assess Immunosenescent CD8+ T Cells in Respiratory Viral Infections
	1 Introduction
	2 Materials
	3 Methods
		3.1 Generate Single-Cell Suspension
		3.2 Tetramer Staining
		3.3 Ex Vivo Peptide Stimulation
		3.4 Flow Cytometry Staining for Both Panels
	4 Notes
	References
Chapter 5: γδ T-Cell Immunophenotype for the Study of Human Aging
	1 Introduction
	2 Materials
		2.1 Isolation of Peripheral Blood Mononuclear Cells from Whole Blood Samples
		2.2 Isolation of Tumor-Infiltrating Lymphocytes from Tumor Tissue
		2.3 Cell Count
		2.4 Flow Cytometry
		2.5 Expansion of Vδ2 T Cells from PBMCs or   TILs
		2.6 Stimulating Agents
		2.7 Magnetic Isolation of γδ T Cells
	3 Methods
		3.1 Isolation of Peripheral Blood Mononuclear Cells from Whole Blood Samples
			3.1.1 Preparation of the Sample
			3.1.2 Isolation of PBMCs
		3.2 Isolation of Tumor-Infiltrating Lymphocytes from Tumor Tissue
			3.2.1 Preparation of the Sample
			3.2.2 Isolation of Tumor-Infiltrating Lymphocytes (TILs)
		3.3 Evaluation of Phenotypic Profile of Human γδ T Cells
		3.4 Expansion of Vδ2 T Cells from PBMCs or   TILs
		3.5 Magnetic Isolation of γδ T Cells from Peripheral Blood or Buffy   Coat
			3.5.1 Sample Preparation
			3.5.2 Magnetic Labelling
			3.5.3 Magnetic Separation with MS or LS Columns
		3.6 Assessing Cytokine Production of γδ T Cells After Stimulation with Ionomycin and Phorbol 12-Myristate 13-Acetate
		3.7 Cell Proliferation Assay of γδ T Cells Using Carboxyfluorescein Succinimidyl Ester (CFSE)
		3.8 Evaluation of Cytotoxic Ability of γδ T Cells
	4 Notes
	References
Chapter 6: Investigation of Signaling in Primary Populations of Human Senescent T Cells
	1 Introduction
	2 Materials
		2.1 Human CD27- CD28- CD4+ Tsen-Cell Purification from Peripheral Blood Mononucleate Cells (PBMCs)
		2.2 Western Blotting
		2.3 In Vitro Kinase Assay by ELISA
		2.4 In Vitro sMAC Disruption
		2.5 Sestrin-AMPK Binding Strength Assay
		2.6 Flow Cytometry Analysis
		2.7 sMAC Forming Assay
	3 Methods
		3.1 Human CD27- CD28- CD4+ Tsen-Cell Purification from Peripheral Blood Mononucleate Cells (PBMCs)
			3.1.1 CD4+ T-Cell Isolation
			3.1.2 CD27- CD4+ T-Cell Isolation
			3.1.3 CD28- CD27- CD4+ Tsen-Cell Isolation
		3.2 Western Blotting
		3.3 In Vitro Kinase Assay by ELISA
		3.4 In Vitro sMAC Disruption
		3.5 Sestrin-AMPK Binding Strength Assay
		3.6 Flow Cytometry
		3.7 sMAC Forming Assay
	4 Notes
	References
Chapter 7: Transcription Factor Analysis to Investigate Immunosenescence in Rheumatoid Arthritis Patients
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Reagents
			2.2.1 PBMC Isolation
			2.2.2 Cell Count
			2.2.3 Flow Cytometry Antibody Panel for Aged Th17 and Treg Cells
			2.2.4 Intranuclear Staining Buffers
	3 Methods
		3.1 Isolation of PBMCs
			3.1.1 Cell Count
		3.2 Human Aged Th17 Cell Staining
		3.3 Gating Strategy: Aged Th17 Cells (CD3+CD4+IL17+RORγt+CD28- T Cells) (Fig. 1)
		3.4 Human Aged Treg Cell Staining
		3.5 Gating Strategy: Aged Treg Cells (CD3+CD4+CD25+CD127-FOXP3+CD28-T Cells) (Fig. 2)
	4 Notes
	References
Chapter 8: Evaluation of Gene Expression Profiling by QuantiGene 2.0 RNA Assay
	1 Introduction
	2 Materials
	3 Methods
		3.1 Day 1: Peripheral Blood Mononuclear Cells (PBMCs) Preparation
			3.1.1 PBMCs Isolation
			3.1.2 Preparation of Cell Lysates
		3.2 Day 1: Reagent Preparation
			3.2.1 Day 1: Preparation of Working Bead Mix
		3.3 Day 1: Hybridization with Working Bead Mix
		3.4 Day 2: Reagent Preparation
			3.4.1 Wash Buffer Preparation
			3.4.2 Streptavidin-Conjugated R-Phycoerythrin (SAPE) Preparation
			3.4.3 Pre-amplifier, Amplifier, and Label Probe Preparation
		3.5 Day 2: Luminex Setting
		3.6 Day 2: Pre-Amplifier Hybridization Step
		3.7 Day 2: Amplifier Hybridization Step
		3.8 Day 2: Label Probe Hybridization Step
		3.9 Day 2: SAPE Incubation
		3.10 Day 2: Measurement of the Mean Intensity of Fluorescence (MFI)
	4 Notes
	References
Chapter 9: Application of Multiplex Immunoassay in Aging Research: A Methodological Approach
	1 Introduction
	2 Materials
	3 Methods
		3.1 Sample Preparation
			3.1.1 Plasma and Serum Samples
			3.1.2 CSF Samples
			3.1.3 Preparation of Tissue Culture Supernatant
		3.2 Setting the Experiment
		3.3 Preparation of Assay Reagents
			3.3.1 Reconstitute and Dilute the Standards for Serum and Plasma Samples
			3.3.2 Preparation of Quality Controls
		3.4 Performing the Assay
		3.5 Data Interpretation and Statistical Analysis
			3.5.1 Quality Control
			3.5.2 Data Cleaning and Preprocessing
			3.5.3 Statistical Analysis
	4 Notes
	References
Chapter 10: Analysis of Cytokine-Inducible SH2-Containing Protein (CISH) in the Study of Immunosenescence
	1 Introduction
	2 Materials
	3 Methods
		3.1 Cell Culture
		3.2 RNA Extraction
		3.3 Reverse Transcription (RT)
		3.4 Primer Design
		3.5 qPCR
	4 Notes
	References
Chapter 11: Evaluating the Efficacy of Immune Checkpoint Inhibitors in Elderly Patients: A Systematic Review and Meta-analysis
	1 Introduction
	2 Methods
		2.1 Define a Research Question
		2.2 Register Prospective Meta-analysis on PROSPERO
		2.3 Software Tools for Data Analysis in Meta-analysis
		2.4 Literature Search Within a Systematic Review
			2.4.1 Search Filters Used in PubMed (Sensitivity-Maximizing Version, 2008 Revision)
			2.4.2 Search Filters Used in Embase
		2.5 Evaluating Quality of Evidence
		2.6 Data Collection
		2.7 Evaluation of Publication Bias
		2.8 Meta-analysis
		2.9 Subgroup Analysis
	References
Chapter 12: Computational Analysis of T-Cell Receptor Repertoire Workflow: From T-Cell Isolation to Bioinformatics Analysis
	1 Introduction
	2 Materials
		2.1 Use of Human Biological Samples
		2.2 Peripheral Blood Mononuclear Cell Isolation
		2.3 Freezing and Thawing Cells
		2.4 CD3+ Isolation
		2.5 RNA Isolation and Evaluation
		2.6 Bioinformatics
	3 Methods
		3.1 PBMC Collection
		3.2 CD3+ Cell Isolation
		3.3 RNA Isolation and Quality Assessment
		3.4 Bioinformatics Analysis of the TCR Repertoire
		3.5 Statistics: Insights on Qualitative and Quantitative Parameters
	4 Notes
	References
Chapter 13: In Vitro Interaction Between Yeast Extracellular Vesicles and Human Monocyte-Derived Dendritic Cells
	1 Introduction
	2 Materials
		2.1 Yeast Strains and Growth Conditions
		2.2 Extracellular Vesicle Isolation and Characterization
		2.3 In Vitro Generation of Human Monocyte-Derived Dendritic Cells and Incubation with Yeast  EVs
		2.4 Evaluation of EV Endocytosis by moDCs
		2.5 Analysis of moDC Activation Phenotype After EV Incubation
	3 Methods
		3.1 Yeast Strains and Growth Conditions
		3.2 Extracellular Vesicle Isolation and Characterization
		3.3 In Vitro Generation of Human Monocyte-Derived Dendritic Cells and Incubation with Yeast  EVs
		3.4 Evaluation of EV Endocytosis by moDCs
			3.4.1 EV Staining with  DiI
			3.4.2 Evaluation of Endocytosis
		3.5 Analysis of moDC Activation Phenotype After EV Incubation
	4 Notes
	References
Chapter 14: Recording of Age-Related Changes on Murine and Human Brain Slices
	1 Introduction
	2 Materials
		2.1 Principles and Solutions
		2.2 Recording Solutions and Devices
	3 Methods
	4 Notes
	References
Chapter 15: Immunofluorescence Staining of Murine Spinal Cord Sections to Evaluate Microglial Activation and Astrogliosis
	1 Introduction
	2 Materials
		2.1 Dewaxing of Spinal Cord Sections
		2.2 Antigen Retrieval and Tissue Permeabilization
		2.3 Antibodies and Mounting Medium
		2.4 Sample Observation, Image Acquisition, and Data Analysis
	3 Methods
		3.1 Tissue Slices Dewaxing
		3.2 Antigen Retrieval
		3.3 Permeabilization
		3.4 Slide Staining, Mounting, and Image Acquisition
		3.5 Data Analysis: Astrogliosis and Microglial Activation
	4 Notes
	References
Chapter 16: Characterization of Age-Dependent Changes in Skeletal Muscle Repair and Regeneration Using a Mouse Model of Acute ...
	1 Introduction
	2 Materials
		2.1 Muscle Injury
		2.2 Muscle Processing
		2.3 Cell Staining for Flow Cytometry
	3 Methods
		3.1 Muscle Injury
		3.2 Muscle Harvesting
		3.3 Muscle Dissociation and Cell Isolation
		3.4 Flow Cytometry: Cell Staining, Acquisition, and Analysis
		3.5 Histological Analysis to Assess Myocyte Regeneration
	4 Notes
	References
Chapter 17: Inducing Cellular Senescence in Mouse Embryonic Fibroblasts (MEFs)
	1 Introduction
	2 Materials
		2.1 Routine Reagents
		2.2 Medium
		2.3 Common Equipment
		2.4 Generation of MEFs
		2.5 DNA Damage-Dependent Senescence Induction
			2.5.1 Irradiation-Induced Senescence
			2.5.2 Oncogene-Induced Senescence (OIS)
		2.6 DNA Damage-Independent Senescence Induction: Nutlin-3a and Palbociclib
		2.7 Senescence Detection
			2.7.1 Senescence-Associated (SAβGal) Staining
			2.7.2 Immunofluorescence: γHAX Foci and Lamin B1
	3 Methods
		3.1 Generation of MEFs
		3.2 Senescence Induction Methods Using Primary MEFs
			3.2.1 DNA Damage-Dependent Senescence Induction
				Ionizing Radiation-Induced Senescence (IRIS)
				Oncogene-Induced Senescence (OIS)
			3.2.2 DNA Damage-Independent Senescence Induction by Nutilin-3 or Palbociclib
		3.3 Detecting Senescence Markers
			3.3.1 Senescence-Associated β-Galactosidase (SAβGal) Staining
			3.3.2 Lamin B1 and γHAX Immunofluorescence Staining
	4 Notes
	References
Chapter 18: Polyphenol Treatment of Peripheral Blood Mononuclear Cells from Individuals of Different Ages
	1 Introduction
	2 Materials
		2.1 Polyphenol Solution Preparation
		2.2 Isolation of PBMCs
		2.3 PBMC Count, Viability Check, Freezing, and Thawing
		2.4 PBMC Culture, Treatment with Polyphenols, and Stimulation
		2.5 Collection of Cell Culture Medium and PBMC Harvesting for Analysis
		2.6 PBMC Viability Assessment with Trypan Blue Exclusion Assay
		2.7 PBMC Viability Assessment, Lymphocyte/Monocyte Staining, and ROS Analysis by Flow Cytometry
		2.8 Analysis of Secreted Cytokines
	3 Methods
		3.1 Preparation of the Polyphenol Stock Solution
		3.2 Donor Selection, Database Setup, and Blood Collection
		3.3 PBMC Isolation, Count, Viability Assessment, and Cryopreservation
		3.4 PBMC Culture Setup, Challenge with Polyphenols, and LPS Stimulation
		3.5 Assessment of Cultured PBMC Viability by Trypan Blue Exclusion Assay or Flow Cytometry
		3.6 Assessment of PBMC Pool Composition After Challenge with Polyphenols According to Age
		3.7 Collection and Analysis of PBMC Culture Medium for Quantification of Secreted Cytokines
		3.8 Measurement of ROS Generation
		3.9 Statistical Analysis of Data
	4 Notes
	References
Chapter 19: Antioxidant Features of Humic Products by ABTS Assay
	1 Introduction
	2 Materials
	3 Methods
		3.1 Extraction of Humic Substances
		3.2 Determination of the Antioxidant Properties of Humic Products
	4 Notes
	References
Index