Chemokine-Glycosaminoglycan Interactions: Methods and Protocols

Preface
References
Contents
Contributors
Chapter 1: A Surgical Approach to Hindlimb Suspension: A Mouse Model of Disuse-Induced Atrophy
1 Introduction
2 Materials
2.1 Surgical Hanger Implantation
2.2 Hindlimb Suspension Setup
2.3 Cage Setup
3 Methods
3.1 Surgical Hanger Implantation
3.2 Hindlimb Suspension Setup: Attaching Mouse to Hanger
3.3 Hindlimb Suspension Setup: Attaching Mouse to Hanger
3.4 Cage Setup and Monitoring
3.5 Recovery and Equipment Maintenance
4 Notes
References
Chapter 2: Remote Ischemic Pre-Conditioning (RIPC)
1 Introduction
2 Materials
2.1 RIPC Surgery
2.2 Confirmation of MG53 Secretion After RIPC
3 Methods
3.1 Surgery
3.2 Confirmation of MG53 Secretion After RIPC
4 Notes
References
Chapter 3: Mouse Corneal Transplantation
1 Introduction
2 Materials
3 Methods
3.1 Anesthesia
3.2 Corneal Grafting
3.2.1 Obtaining the Donor Cornea
3.2.2 Preparing the Graft Bed in the Recipient Mouse
3.2.3 Suturing the Graft
3.2.4 Suture Removal
3.3 Clinical Evaluation
4 Notes
References
Chapter 4: Analysis of Chemokine-to-GAG Interactions in Model of Donor Renal Allograft Transplant
1 Introduction
2 Materials
2.1 Rationale
2.2 Materials
3 Methods
3.1 Mouse Kidney Transplant and Usual Procedure Plan Time Course
3.2 Kidney Transplant: Microsurgical Procedure
3.3 Treatment with M-T7 or Saline Control
3.4 Postoperative Care
3.5 Follow-Up Analysis for Histological Microscopic Analysis
3.6 Follow-Up Analysis for HS and CS Disaccharide Content
4 Notes
References
Chapter 5: Mouse Models of Renal Allograft Transplant Rejection: Methods to Investigate Chemokine-GAG Interaction and Therapeu...
1 Introduction
2 Materials
2.1 Mice
2.2 Surgery (See Note 3)
2.3 All Medications Used for Surgical Procedures
2.4 Expression and Purification of M-T7 Protein
2.5 Tissue Histology, Processing, Embedding, and Staining
3 Methods
3.1 Subcapsular Renal Allograft Surgery Procedure
3.1.1 Preparation of Mice for Surgery
3.1.2 Surgical Procedure
3.2 Subcutaneous Renal Allograft Surgery Procedure
3.2.1 Preparation of Mice for Surgery
3.2.2 Surgical Procedure (See Fig. 2)
3.3 Pretreatment of Subcapsular and Subcutaneous Renal Allograft Models with M-T7, the Virus-Derived Chemokine -Modulating Pro...
3.3.1 Pretreatment of Donor Mouse
3.3.2 Pretreatment of Donor Organ Immediately Before Implant by Soaking
3.3.3 Expression and Purification of M-T7 from CHO Cells
3.4 Collection of Tissue for Histological Analysis
3.4.1 Subcapsular Transplants
3.4.2 Subcutaneous Transplants
3.4.3 Subcapsular and Subcutaneous Transplant Models
3.5 Tissue Processing for Paraffin Sections (See Fig. 3 and See Note 11)
3.6 Histology and Immunohistology Staining Protocols
3.7 Immunohistochemical (IHC) Staining (See Note 15)
3.8 Analysis of Immune Responses in Transplanted Allografts: Histological Analysis
4 Notes
References
Chapter 6: Ex Vivo Perfusion System to Analyze Chemokine-Driven Leukocyte Adhesion
1 Introduction
2 Materials
2.1 Isolation of Carotid Arteries
2.2 Cell Suspension of Bone Marrow-Derived Leukocytes
2.3 Mounting of Artery
2.4 Flow Assay
3 Methods
3.1 Isolation of Carotid Arteries
3.2 Cell Suspension of Bone Marrow-Derived Leukocytes
3.3 Mounting of Artery
3.4 Flow Assay
4 Notes
References
Chapter 7: In Vitro (Trans)Migration Experiment Using Chemokines as Stimulatory Factor
1 Introduction
2 Materials
2.1 Spleen Isolation
2.2 Cell Dissociation of the Spleen
2.3 CD4+ T Cell Isolation
2.4 Transmigration Assay
2.5 Quantification of Migrated CD4+ T Cells
3 Methods
3.1 Isolation of the Spleen
3.2 Cell Dissociation of the Spleen
3.3 CD4+ T Cell Isolation
3.3.1 Magnetic Labeling
3.3.2 Magnetic Separation (See Notes 13 and 14)
3.4 Transmigration Assay (See Note 17)
3.5 Quantification of Migrated CD4+ T Cells
4 Notes
References
Chapter 8: The Use of Campenot Trichambers for the Study of Peripheral Neuronal Growth and Survival in Presence of Thrombotic ...
1 Introduction
2 Materials (See Note 1)
3 Methods
3.1 Preparation of 35-mm Dishes
3.2 Assembling Campenot Trichambers
3.3 Dissociation of Peripheral Ganglia
3.4 Maintaining Neuron Cultures
3.5 Experiments
4 Notes
References
Chapter 9: Flow Cytometry Analysis of Immune Cell Responses
1 Introduction
2 Materials
2.1 Murine Organ Isolation and Organ Processing
2.2 Murine Submandibular Vein Blood Collection and Processing
2.3 Antibody Staining
2.4 Flow Cytometry
3 Methods
3.1 Organ Isolation
3.2 Organ Processing and Cell Counting
3.3 Submandibular Blood Collection and Processing
3.4 Antibody Staining for CD4+, CD8+, CCR7+ T Cells
3.5 Flow Cytometry of Antibody Stained Cells
3.6 Analysis of Flow Cytometry Data
4 Notes
References
Chapter 10: Detection of Chemokine Binding Proteins Association to Cell Surface Glycosaminoglycans by Flow Cell Cytometry and ...
1 Introduction
2 Materials
3 Methods
3.1 CHO Cells Subculturing Procedure
3.2 Detection of vCKBPs Binding to Cell Surface by Flow Cytometry
3.3 Detection of vCKBPs Binding to Cell Surface by Indirect Immunofluorescence Microscopy
4 Notes
References
Chapter 11: Protein Microarrays and their Fabrication
1 Introduction
2 Materials
2.1 Preparation of Plasmid DNA
2.2 Preparing the Slide and Master Mix
3 Methods
3.1 Preparation of Plasmid DNA
3.2 Slide Coating and Master Mix Preparation
3.3 Pin Cleaning and Microarray Printing
3.4 Detection of GAG-Chemokine Interactions Using Microarray
4 Notes
References
Chapter 12: NMR Methods for Characterization of Glycosaminoglycan-Chemokine Interactions
1 Introduction
2 Materials
2.1 Glycosaminoglycan
2.2 Chemokine
2.3 NMR
3 Methods
3.1 Production of Defined GAG Oligosaccharides
3.2 Production of 15N-Labeled Chemokines
3.3 NMR Methods Applied to GAG-Chemokine Studies
3.3.1 NMR Titrations
3.3.2 Saturation Transfer Difference (STD)
3.3.3 Paramagnetic Relaxation Enhancement (PRE)
4 Notes
References
Chapter 13: Glycosaminoglycan Analysis: Purification, Structural Profiling, and GAG-Protein Interactions
1 Introduction
2 Materials
2.1 Biotinylation of GAG-Binding Proteins on Heparin-Sepharose
2.2 Cell Surface Binding Assays and Flow Cytometry Analysis
2.3 Purification of Heparan Sulfate and Chondroitin/Dermatan Sulfate from Cells
2.4 Compositional Analysis of Purified HS and CS/DS: Disaccharide Profiling
2.5 [35S] Metabolic Labeling of Cellular GAGs
2.6 GAG Chain Sizing by Gel Filtration Chromatography
2.7 Filter Binding Assays
3 Methods
3.1 Biotinylation of GAG-Binding Proteins on Heparin-Sepharose
3.2 Cell Surface Binding Assays and Flow Cytometry Analysis
3.3 Purification of Heparan Sulfate and Chondroitin/Dermatan Sulfate from Cells
3.4 Compositional Analysis of Purified HS and CS/DS: Disaccharide Profiling
3.5 [35S] Metabolic Labeling of Cellular GAGs
3.6 GAG Chain Sizing by Gel Filtration Chromatography
3.7 Filter Binding Assay
4 Notes
References
Chapter 14: Isolation and Compositional Analysis of Glycosaminoglycans
1 Introduction
2 Materials
3 Methods
3.1 Homogenization, and Protein/Nucleic Acid Digestion
3.2 Anion Exchange Chromatography
3.3 β-Elimination
3.4 Desalting
3.5 Compositional Analysis of Heparan Sulfate by GAG Lyase Digestion and SAX-HPLC (UV-Detection)
4 Notes
References
Chapter 15: How to Design Peptides
1 Introduction
2 Materials
2.1 PDB Preparation
2.2 RosettaScripts Syntax
2.3 Other Programs to Use
3 Methods
3.1 Get Started
3.2 Define the Scoring Function(s)
3.3 Outline the Manipulations to Perform on Your Peptide Sequence
3.4 Define the Residues to Be Manipulated
3.5 Define the PackerPalette
3.6 Define the TaskOperations
3.7 Define the Filters
3.8 Define the Movers
3.9 Call the Functions
3.10 Execute the Protocol
3.11 Evaluate the Protocol
3.12 Debugged Protocol Run
4 Notes
References
Chapter 16: Methods to Assess Chemokine Binding and Anti-chemotactic Activity of Virus Proteins
1 Introduction
2 Materials
2.1 Materials for Inhibition ELISA
2.2 Solutions for Inhibition ELISA
2.3 Materials for Chemotaxis Inhibition Assay
2.4 Solutions for Chemotaxis Inhibition Assay
3 Methods
3.1 Determination of Optimal Chemokine Concentration for Inhibition ELISA
3.2 Inhibition ELISA
3.3 Determination of Optimal Chemokine Concentration for Chemotaxis Inhibition Assay
3.4 Chemotaxis Inhibition Assay
4 Notes
References
Chapter 17: Producing Biologics with Defined N-Glycosylation in Plants
1 Introduction
2 Materials
2.1 Plant Growth
2.2 Bacterial Culture (See Note 1)
2.3 Syringe Infiltration
2.4 Harvesting
2.5 Protein a Affinity Chromatography
2.6 N-Glycan Analysis
3 Methods
3.1 Plant Growth
3.2 A. Tumefaciens Culturing and Preparation for Syringe Infiltration
3.3 Syringe Agroinfiltration
3.4 Harvesting, Isolation, and Characterization of Recombinant Anti-WNV and Anti-HIV IgG 1 mAbs with Defined Mammalian Glycosy...
3.5 N-Glycan Analysis of mAbs by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS)
4 Notes
References
Chapter 18: Native Deglycosylation and Size Exclusion Chromatography of Viral Chemokine Binding Proteins for Structural Discov...
1 Introduction
2 Materials
2.1 Deglycosylation Optimization
2.2 Size Exclusion Chromatography
3 Methods
3.1 Sequence Analysis
3.2 Deglycosylation Optimization
3.3 Size Exclusion Chromatography (SEC)
4 Notes
References
Index