Stem Cell Renewal and Cell-Cell Communication: Methods and Protocols (Methods in Molecular Biology, 2346)

✍ Scribed by Kursad Turksen (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 253
• Edition: 2nd ed. 2021
• Category: Library

✦ Synopsis

This detailed book brings together a new set of protocols to arm cell biologists with techniques that are currently being used in a number of well-established laboratories around the world. The contents represent the great strides made in the field of cell-cell communications with respect to the identification and characterization of key components of the communication apparatus, assembly and maintenance of the communications structures, and concomitantly their roles in not only tissue formation and maintenance but also regeneration and repair. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective chapters, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. 

Authoritative and practical, Stem Cell Renewal and Cell-Cell Communication: Methods and Protocols, Second Edition serves as an ideal guide for experts and newcomers alike seeking to increase our understanding of the crucial biological and physiological roles of cell-cell communications in tissue function and organismal integrity.

✦ Table of Contents

Preface
Contents
Contributors
Inference of Ligand-Receptor Pairs from Single-Cell Transcriptomics Data
1 Introduction
2 Materials
2.1 Input Data Files
2.2 Software
2.3 Hardware
3 Methods
3.1 Installation
3.2 Running in the Statistical Analysis Mode
3.3 Subsampling and Statistical Analysis
3.4 Normal Mode, Without Statistical Analysis
3.5 Visualization
3.6 Different Versions of the CellPhoneDB Database
3.7 Generating a User-Specific Database
3.8 Help Option
3.9 Interactive Web Portal
4 Notes
References
Multiple Imaging Modalities for Cell-Cell Communication via Calcium Mobilizations in Corneal Epithelial Cells
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Cells for Ca2+ Mobilization Studies
3.2 Ex Vivo In Situ Preparation of Tissue for the Study of Calcium Mobilizations
3.3 Imaging
3.4 Analysis
3.4.1 Grid-Based Modeling of Ca2+ Mobilizations
3.4.2 Cell-Based Modeling of Ca2+ Mobilizations
3.4.3 Event Probability Calculation for Ca2+ Mobilizations
3.4.4 Analysis of Cytoskeletal Changes with Calcium Mobilizations
4 Notes
References
Interactions of Hematopoietic Stem Cells with Bone Marrow Niche
1 Introduction
2 Materials
2.1 Tissues
2.2 Reagents and Supplies
2.3 Fluorescence-Conjugated Antibodies
2.4 Equipment
3 Methods
3.1 Preparing Single-Cell Suspension from Long Bones (Femur and Tibia)
3.2 Staining BM Cells and Endosteal Stromal Cells for Flow Cytometry Analysis
3.3 Staining the Endosteal Stromal Cells for Flow Cytometry Sorting
3.4 HSCs and Stromal Cells Co-culture
4 Notes
References
Ex Vivo Modeling of Hematopoietic Stem Cell Homing to the Fetal Liver
1 Introduction
2 Materials
2.1 Microfluidic Channel
2.2 Polyacrylamide Hydrogels
2.3 Collagen Gel Coating
2.4 Fetal Liver Tissue
2.5 AGM Dissociation for Isolation of Hematopoietic Stem and Progenitor Cells
2.6 Fluidics Assembly and Imaging
3 Methods
3.1 Microfluidic Device Assembly
3.1.1 Microfluidic Channel Base Slide
3.1.2 Polyacrylamide Hydrogels
3.1.3 Collagen Coating
3.2 Fetal Liver-on-a-Chip Assembly
3.3 HSC Isolation
3.4 Fluidics Assembly and Imaging
4 Notes
References
Analysis of Epithelial Architecture and Planar Spindle Orientation in the Drosophila Wing Disc
1 Introduction
2 Materials
2.1 Dissection of Wing Discs
2.2 Immunostaining of Wing Discs
2.3 Mounting Wing Discs on Slides
2.4 Imaging Wing Discs
2.5 Angle Analysis of Mitotic Spindle Orientation
3 Methods
3.1 Dissection of Larvae
3.2 Immunostaining
3.3 Mounting of Wing Imaginal Discs
3.4 Image Acquisition
3.5 Visualization of Epithelial Architecture
3.6 Measurements of Planar Spindle Orientation
4 Notes
References
A Co-culture Model to Study the Effect of Kidney Fibroblast-p90RSK on Epithelial Cell Survival
1 Introduction
2 Materials
2.1 Mice
2.2 Cell and Cell Culture Reagents
2.3 Other Instruments and Materials
3 Methods
3.1 Kidney Fibroblast Isolation
3.2 Culture of Fibroblasts
3.3 Culture of Epithelial Cells
3.4 Co-culture of Epithelial Cells and Fibroblasts
4 Notes
References
Calcium Fluorescence Recordings from Neuroepithelial Stem Cells
1 Introduction
2 Materials
2.1 Recording Apparatus
2.2 Fluorescent Ca2+ Indicators
2.3 Solutions
3 Methods
3.1 Preparation of Retinal Neuroepithelium
3.2 Loading of Ca2+ Indicator
3.3 Ca2+ Fluorescence Measurement
4 Notes
References
Ultrastructural Analysis of Cell-Cell Interactions in Drosophila Ovary
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 ROTO Processing
2.3 Resin Preparations and Embedding
2.4 Sectioning
3 Methods
3.1 Fly Culture
3.2 Sample Preparation
3.3 Processing
3.3.1 First Osmium Penetration
3.3.2 TCH Incubation
3.3.3 Second Osmium Infiltration
3.3.4 Lead Aspartate Staining
3.3.5 Dehydration
3.3.6 Resin Infiltration
3.4 Embedding
3.5 Sectioning
3.5.1 Trimming the Block
3.5.2 Ultramicrotome
3.5.3 Section Preparation and Collection
3.5.4 Imaging
4 Notes
References
TIRF Microscopy as a Tool to Determine Exosome Composition
1 Introduction
2 Materials
2.1 Cells
2.2 Reagents
2.3 Media and Solutions/Buffers
2.4 Equipment
2.5 Software
3 Methods
3.1 CD4+ T-Cell Purification from Human Peripheral Blood Lymphocytes (huPBLs)
3.2 Generation of Lymphoblasts from Human PBLs: PHA or SEE-Specific
3.3 Isolation of EVs from Cell Culture
3.3.1 Production and Isolation of EVs (Fig. 1a)
3.3.2 Quantification and Characterization of EVs
3.4 Coating of Coverslips and Coverslip-Bottom Dishes
3.4.1 Preparation of Poly-L-Lysine-Coated Surfaces
3.4.2 Preparation of Antibody-Coated Surfaces
3.5 Sample Preparation for Imaging
3.5.1 Preparation of Samples on Poly-L-Lysine-Coated Surfaces (Fig. 1b)
3.5.2 Preparation of Samples on Capture Antibodies-Coated Surfaces(Fig. 1c)
3.6 Imaging and Analysis Under TIRF Microscope
4 Notes
References
Rapid Visualization of Intracellular Vesicle Events During Synaptic Stimulation
1 Introduction
2 Materials
2.1 Primary Cells and Cell Lines
2.2 Reagents
2.3 Media
2.4 Equipment
2.5 Software
3 Methods
3.1 Isolation of T Cells
3.1.1 Isolation of Human PBLs
3.1.2 Isolation of Mouse CD4+ T Cells
3.2 Generation of SEE-Specific or PHA Lymphoblasts from Human PBLs
3.3 Transfection of T Cells
3.3.1 Electroporation of Lymphoblastoid Cell Lines and Human SEE T Lymphoblasts
3.3.2 Nucleofection of Human T Lymphoblasts
3.3.3 Nucleofection of Mouse CD4+ T Cells
3.4 Coating of Coverslip-Bottom Chambers
3.4.1 Preparation of poly-L-Lys-Coated Surfaces
3.4.2 Preparation of Stimulating Surfaces
3.5 Live Imaging Vesicular Traffic Studies
3.5.1 Preparation of T Cells
3.5.2 Image Acquisition
3.5.3 Image Analysis LAS X Software (See Fig. 1)
3.5.4 Image Analysis Imaris Bitplane Software (See Fig. 2)
4 Notes
References
Monitoring of Active Notch Signaling in Mouse Bladder Urothelium
1 Introduction
2 Materials
2.1 Mice
2.2 Tissue Fixation Reagents and Components
2.3 Paraffin Embedding Reagents and Equipment
2.4 Immunostaining Reagents
2.5 Other Equipment for Immunostaining
2.6 Microscopy and Imaging Set Up
2.7 Image Processing and Analysis
3 Methods
3.1 Tissue fixation
3.2 Paraffin Embedding and Sectioning Onto Slides
3.3 Immunostaining on Paraffin Sections for Detection of Nuclear Notch ICD Levels
3.3.1 Deparaffinization and Rehydration of Tissue
3.3.2 Antigen Retrieval
3.3.3 Blocking
3.3.4 Primary Antibody Binding
3.3.5 Secondary Antibody Binding
3.3.6 Tyramide Signal Amplification
Cover-Slipping
Imaging
4 Notes
References
Examining Local Cell-to-Cell Signalling in the Kidney Using ATP Biosensing
1 Introduction
2 Materials
2.1 Tissue Culture
2.2 Instrumentation
2.3 Balanced Salt Solution (BSS)
2.4 Biosensors
3 Methods
3.1 Tissue Culture
3.2 Initial Rig Preparation
3.3 Experiment Preparation
3.4 Biosensor Polarization
3.5 Experiment
3.6 Analysis
4 Notes
References
Isolation and Assessment of Pancreatic Islets Versus Dispersed Beta Cells: A Straightforward Approach to Examine Cell-Ce
1 Introduction
2 Materials
2.1 Islet Isolation
2.2 Islet Dispersion
2.3 Calcium Imaging
2.4 Islet Cell Staining
3 Methods
3.1 Islet Isolation
3.2 Islet Dispersion
3.3 Calcium Imaging
3.4 Islet Cell Staining
4 Notes
References
Promoter Pull-Down Assay: A Biochemical Screen for DNA-Binding Proteins
1 Introduction
2 Materials
2.1 Probe Preparation
2.2 Buffers and Reagents
2.3 Cell Lysis
2.4 Pull-Down
2.5 SDS-PAGE Gel
3 Methods
3.1 Cell Culture
3.2 Probe Preparation
3.3 Dynabead Preparation
3.4 Probe Immobilization
3.5 Bead Equilibration
3.6 Cell Lysis
3.7 Preclearing the Lysate
3.8 Dynabead Incubation with Lysate
3.9 Elution
3.10 Analysis of Eluted Proteins
4 Notes
References
Purification of the Vibrio Quorum-Sensing Transcription Factors LuxR, HapR, and SmcR
1 Introduction
2 Materials
2.1 Cell Culture Reagents
2.2 Buffers and Reagents
2.3 Cell Lysis
2.4 Protein Purification
2.5 SDS-PAGE Gel
2.6 Autoinduction Media (Optional)
3 Methods
3.1 Protein Overexpression (See Note 2)
3.2 Cell Lysis
3.3 Protein Purification: Native Purification (See Note 7)
3.4 Protein Purification: Affinity Tag Option 1 - His-Tag (See Note 10)
3.5 Protein Purification: Affinity Tag Option 2 - IMPACT (See Note 11)
3.6 Protein Purification: Size Exclusion Chromatography (SEC) (See Note 13)
4 Notes
References
Preserving Cytonemes for Immunocytochemistry of Cultured Adherent Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Fixation, Permeabilization and Block
2.3 Immunocytochemistry
2.4 Mounting Coverslips
3 Methods
3.1 Cell Culture
3.2 Fixation, Permeabilization and Blocking
3.3 Antibody Binding
3.4 Mounting
4 Notes
References
Fluorescent Labeling of Connexin with As Complex and X-Y Coordinate Registration of Target Single Cells Based on a Triangle St
1 Introduction
2 Materials
2.1 HeLa Cell Culture
2.2 Transfection
2.3 Fluorescent Dyes for Diffusion Marker
2.4 Chelating Reagent for a TC Tag and Washing Buffer
2.5 Polystyrene Plate
2.6 Cell Fixation Reagents
3 Methods
3.1 Preparation of an Insert DNA: A Cx43-TC Chain
3.2 Isolation of a Plasmid Vector pDsRed Monomer C1
3.3 Linearization of the Plasmid Vector
3.4 Ligation of Cx43-TC Chain and the Vector to Generate pCMV-Cx43-TC
3.5 Transfection of HeLa Cells and FlAsH Staining
3.6 Analysis of GJ Plaque Localization and Gap Junctional Dye Diffusion
3.7 Preparation of a Culture Dish with a Coordinate Chip
3.8 Correlation of X-Y Coordinates Determined by Light and Electron Microscopy
4 Notes
References
Chemical and Voltage Gating of Gap Junction Channels Expressed in Xenopus Oocytes
1 Introduction
2 Materials
2.1 Solutions Used During Oocyte Preparation and in Electrophysiological Experiments
3 Methods
3.1 Oocyte Preparation
3.2 Inhibition of Native-Connexin Expression
3.3 Inhibition of Calmodulin (CaM) Expression
3.4 Expression of CaM Mutant
3.5 Oocyte Injection of Connexin cRNAs and Oocyte Pairing
3.6 Measurement of Gap Junctional Conductance by Double Voltage Clamp
3.7 Measurement of Trans Junctional Voltage Gating
3.8 Oocyte Chamber Perfusion and Uncoupling Protocol
3.9 Bubble-Trap Device
4 Notes
References
The Analysis of Gap Junctional Intercellular Communication Among Osteocytes in Chick Calvariae by Fluorescence Recovery After
1 Introduction
1.1 Osteocyte Network
1.2 Fluorescence Recovery After Photobleaching
2 Materials
2.1 Animals
2.2 General Reagents
2.3 Equipment
3 Methods
3.1 Tissue Collection and Calcein AM Loading
3.2 FRAP Experiment on an Osteocyte
3.3 Data Analyses
4 Notes
References
Flow Cytometry Evaluation of Gap Junction-Mediated Intercellular Communication Between Cytotoxic T Cells and Target Tumor Cell
1 Introduction
2 Materials
2.1 Common Reactive and Equipment
2.2 Melanoma-Associated Antigen (gp10025-33)-Specific CTL Generation
2.3 Wild-Type Naive CD8+ T Cell Generation
2.4 Calcein-Acceptor Tumor Cells
2.5 Calcein-Donor Cell Labeling
2.6 Calcein-Acceptor Cell Labeling
2.7 Co-incubation of Calcein-Donor and Calcein-Acceptor Cells
2.8 Flow Cytometry
3 Methods
3.1 Melanoma Antigen gp100-Specific CTL Generation
3.2 Wild-Type Naive CD8+ T Cell Obtaining
3.3 Calcein-Acceptor Tumor Cells Obtaining
3.4 Calcein-Donor Cell Labeling
3.5 Calcein-Acceptor Cell Labeling
3.6 Co-incubation of Calcein-Donor and Calcein-Acceptor Cells
3.7 Flow Cytometry Analysis
4 Notes
References
Index