Gap Junction Protocols (Methods in Molecular Biology, 1437)

Annotation Presenting state-of-the-art protocols to study gap junctions, this detailed book first focuses on the use of methods and tools to investigate the different aspects of connexin expression and gap junction regulation. The second part of the volume describes several methods to probe gap junction functionality as such. 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 and readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Gap Junction Protocols is intended for basic and applied researchers in the area of biomedical and life sciences, both in academic and industrial settings Preface References Contents Contributors Chapter 1: Analysis of Liver Connexin Expression Using Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction 1 Introduction 2 Materials 2.1 RNA Extraction 2.2 RNA-DNA Quantification and Purity Control 2.3 cDNA Synthesis 2.4 cDNA Purification 2.5 Real-Time qPCR 2.6 Data Processing 3 Methods 3.1 Maintenance of a Contamination-Free Workplace 3.2 RNA Extraction 3.2.1 Cultured Hepatic Cells 3.2.2 Liver Tissue 3.3 RNA-DNA Quantification and Purity Control 3.4 cDNA Synthesis 3.5 cDNA Purification 3.6 Real-Time qPCR 3.7 Data Processing 4 Notes References Chapter 2: DNA Methylation Analysis of Human Tissue-Specific Connexin Genes 1 Introduction 2 Materials 2.1 Genomic DNA Extraction from Tissues 2.2 Sodium Bisulfite Conversion of Unmethylated Cytosines in DNA 2.3 PCR Reagents 2.4 Methylation- 2.5 Bisulfite-Specific PCR Sequencing 2.6 MassArray Assay 3 Methods 3.1 Genomic DNA Extraction from Human Gastric Tissues 3.2 Sodium Bisulfite Conversion of Unmethylated Cytosine 3.2.1 Bisulfite DNA Conversion 3.2.2 Clean-Up of Bisulfite-­Converted DNA 3.3 Primer Design and Methylation Positive Control Preparation 3.4 Connexin DNA Methylation Analysis 3.4.1 Methylation- 3.4.2 Bisulfite-Specific PCR Sequencing 3.4.3 MassArray Analysis 4 Notes References Chapter 3: Detection of Connexins in Liver Cells Using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Immunoblot Analysis 1 Introduction 2 Materials 2.1 Protein Extraction 2.2 Protein Quantification Assay (See Note 3) 2.3 Preparation of Gels (Table 2) 2.4 SDS-PAGE 2.5 Protein Transfer 2.6 Immunoblotting and Protein Detection 2.7 Stripping of the Immunoblot 3 Methods 3.1 Protein Extraction 3.2 Protein Quantification Assay 3.3 Preparation of Gels (See Note 14) 3.4 SDS-PAGE 3.5 Protein Transfer (See Note 21) 3.6 Immunoblotting and Protein Detection 3.7 Stripping of the Immunoblot (See Note 31) 3.8 Processing of Results 4 Notes References Chapter 4: Immunohisto- and Cytochemistry Analysis of Connexins 1 Introduction 2 Materials 2.1 Frozen Tissue Sections 2.2 Paraffin- 2.3 In Vitro Cultures 2.4 General Procedure for Fluorescent IHC 2.5 Tyramide Signal Amplification (TSA) 3 Methods 3.1 Fluorescent IHC in Frozen Tissue Sections 3.2 Fluorescent IHC in Paraffin-­Embedded Tissue Sections 3.3 Fluorescent Immunocytochemistry 3.4 Tyramide Signal Amplification (TSA) Method 4 Notes References Chapter 5: Small Interfering RNA-Mediated Connexin Gene Knockdown in Vascular Endothelial and Smooth Muscle Cells 1 Introduction 2 Materials 2.1 Cell Culture of Primary Vascular Endothelial Cells 2.2 Transfection of Endothelial Cells 2.3 Dissection and Transfection of Mouse Thoracodorsal Arteries (TDAs) 3 Methods 3.1 Setting Up Plates of Vascular Cells for Experiments 3.2 Transfection of HUVEC Cells 3.3 Dissection of Mouse TDA 3.4 Ex Vivo Endothelial or Smooth Muscle Cells Transfection 4 Notes References Chapter 6: Generation and Use of Trophoblast Stem Cells and Uterine Myocytes to Study the Role of Connexins for Pregnancy and Labor 1 Introduction 2 Materials 2.1 Culture and Differentiation of Mouse Trophoblast Stem Cells 2.2 Colorimetric TSC Proliferation Assay 2.3 Derivation and Culture of Primary Rodent Uterine Myocytes 2.4 In Vitro Stretch of Primary Uterine Myocyte Cultures 3 Methods 3.1 Comparative Analysis of Connexin-­Deficient TSC Differentiation 3.2 MTT-­Proliferation Assay for TSC 3.3 Derivation and Culture of Primary Rodent Uterine Myocytes 3.4 Application of Stretch to Rat Smooth Muscle Cells 4 Notes References Chapter 7: Identification of Connexin43 Phosphorylation and S-Nitrosylation in Cultured Primary Vascular Cells 1 Introduction 2 Materials 2.1 Cell Culture of Primary Vascular Human Cells 2.2 Membrane Protein Isolation 2.3 Biotin Switch Analysis of Nitrosylated Proteins 2.4 Western Blotting 2.5 Immunoblotting and Protein Detection Using LI-COR Odyssey 3 Methods 3.1 Culture of Vascular Cells 3.2 Setting Up Plates of Vascular Cells for Experiments 3.3 Membrane Preparations for Western Blotting of Phospho-­Connexins 3.4 Detection of S-Nitrosylated Connexin Proteins Using the Biotin Switch Assay 3.5 Detection of Proteins on Nitrocellulose Using LI-COR Odyssey 4 Notes References Chapter 8: Preparation of Gap Junctions in Membrane Microdomains for Immunoprecipitation and Mass Spectrometry Interactome Analysis 1 Introduction 2 Materials 2.1 Mouse Liver Tissues 2.2 Tissue Homogenization and OptiPrep Membrane Fractionation 2.3 Agarose Bead Preparation 2.4 Immuno-precipitation (IP) Complex Preparation for MS/MS 2.5 Silver Staining and Sample Preparation for MS/MS 2.6 Considerations for Reducing Contamination in Downstream MS/MS Analyses (See Note 9) 2.7 Validation and Analysis of Protein Interactions 3 Methods 3.1 Tissue Homogenization and OP Membrane Fractionation (See Notes 12 and 13) 3.2 Agarose Bead Preparation (See Note 23) 3.3 Fraction Collection and Preparation for IP 3.4 Characterizing Samples by Immunoblotting 3.5 Preparation of Samples for Immunoprecipitation 3.6 Immuno-precipitation Complex Preparation for MS/MS 3.7 Silver Staining and Sample Preparation for MS/MS (Day 4) 3.8 Validation and Analysis of Protein Interactions 4 Notes References Chapter 9: Scrape Loading/Dye Transfer Assay 1 Introduction 2 Materials 3 Methods 3.1 General Procedure 3.2 Quantification of GJIC Using Morphometric Software 4 Notes References Chapter 10: Microinjection Technique for Assessment of Gap Junction Function 1 Introduction 2 Materials 2.1 Establishment of Cell Culture 2.2 Microinjection 2.3 Microscopy 3 Methods 3.1 Establishment of Cell Culture Monolayer 3.2 Preparation of Cells for Microinjection 3.3 Preparation of the Microinjection Injection Apparatus 3.4 Microinjection of Cells 3.5 Cell Analysis 4 Notes References Chapter 11: Electroporation Loading and Dye Transfer: A Safe and Robust Method to Probe Gap Junctional Coupling 1 Introduction 2 Materials 2.1 Assembly of a Parallel 2-Wire Fork-­Shaped Pt/Ir Electrode 2.2 Electroporation Loading and Time-Lapse Imaging of Dye Transfer 2.3 Analysis of Dye Spread 3 Methods 3.1 Assembly of a Parallel 2-Wire Fork-­Shaped Electrode 3.2 Electroporation Loading and Time-Lapse Imaging of Dye Transfer 3.3 Quantification of Dye Spread 4 Notes References Chapter 12: Using Fluorescence Recovery After Photobleaching to Study Gap Junctional Communication In Vitro 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Labeling with Calcein-AM 3 Methods 3.1 Cell Culture 3.2 Labeling with Calcein-AM 3.3 Setup for Fluorescence Recovery After Photobleaching 3.4 Performing the FRAP Measurement 3.5 FRAP Analysis 4 Notes References Chapter 13: Tracking Dynamic Gap Junctional Coupling in Live Cells by Local Photoactivation and Fluorescence Imaging 1 Introduction 2 Materials 2.1 Cell Preparation 2.2 Dye Loading Solution 3 Methods 3.1 Preparation of Dye-Loading Solution 3.2 Dye Loading 3.3 Imaging Assays Using the LAMP Assay 3.4 Imaging Assays Using the Two Photon Uncaging and Imaging (Infrared-­LAMP Assay) 3.5 Data Analysis of Dye Transfer Between a Coupled Cell Pair 4 Notes References Chapter 14: A Functional Assay to Assess Connexin 43-Mediated Cell-­to-­Cell Communication of Second Messengers in Cultured Bone Cells 1 Introduction 2 Materials 2.1 Cell Culture and Transfection 2.2 Luciferase Reporter Assay 3 Methods 3.1 Cell Culture and Transfection 3.2 Luciferase Reporter Assay 4 Notes References Chapter 15: Calcium Wave Propagation Triggered by Local Mechanical Stimulation as a Method for Studying Gap Junctions and Hemichannels 1 Introduction 2 Materials 2.1 BCEC Medium Preparation 2.2 BCEC Isolation 2.3 Cell Culture 2.4 Measurement of Intercellular Calcium Waves Using Mechanical Stimulation 3 Methods 3.1 Procedure of Cell Isolation 3.2 Cell Culture 3.3 Measurement of Intercellular Calcium Waves Using Mechanical Stimulation 4 Notes References Chapter 16: Establishment of the Dual Whole Cell Recording Patch Clamp Configuration for the Measurement of Gap Junction Conductance 1 Introduction 2 Materials 2.1 Solutions 2.2 Dual Whole Cell Patch Clamp Setup 2.3 Cultured Cells 3 Methods 4 Notes References Index