Cell Migration in Three Dimensions

Preface
Contents
Contributors
Chapter 1: Cell Migration in Three Dimensions
	1 Cell Migration in Health and Disease
	2 Regulation of Cell Migration
	3 Model Systems to Investigate Cell Migration
	4 Developments and Advances in the Cell Migration Field
	References
Part I: The Cell Biology of Cell Migration
	Chapter 2: Use of Ecto-Tagged Integrins to Monitor Integrin Exocytosis and Endocytosis
		1 Introduction
		2 Materials
			2.1 Cells
			2.2 Plasmids
			2.3 Culture Media
			2.4 Plasticware
			2.5 Chloroalkane Halo Ligands
			2.6 Buffers and Reagents
			2.7 Microscopy Equipment
		3 Methods
			3.1 Design and Generation of Ecto-Tagged Integrins
			3.2 Generation of Stable Cell Lines Expressing Ecto-Tagged β1 Integrins
				3.2.1 Production of Lentiviral Particles Driving Expression of Ecto-Tagged β1 Integrin
				3.2.2 Replacing Endogenous β1 Integrins with Ecto-Tagged β1 Integrins in Murine Fibroblasts
				3.2.3 Replacing Endogenous β1 Integrins with Ecto-Tagged β1 Integrins in Human Cell Lines
			3.3 Validation of Ecto-Tagged Integrin Function
			3.4 Imaging Exocytosis of Ecto-Tagged Integrins
				3.4.1 Imaging Exocytosis of pHluorin-β1 Integrins by Live TIRFM
				3.4.2 Imaging Newly Exocytosed Ecto-Halo-β1 Integrins
			3.5 Using Ecto-Halo-β1 Integrins to Investigate Integrin Endocytosis and Recycling
				3.5.1 Labeling Surface and Intracellular Pools of Ecto-Halo-β1 Integrins with Distinct Halo Ligands and Following Exchange
				3.5.2 Imaging Endocytosis of Ecto-Halo-β1 Integrins Using HILO-TIRF
				3.5.3 Imaging the Recycling of Ecto-Halo-β1 Integrins Using Reducible Halo Ligands
		4 Notes
		References
	Chapter 3: Probing the ER-Focal Adhesion Link During Cell Migration
		1 Introduction
		2 Materials
			2.1 Cell Culture and Transfection
			2.2 Microscopy and Image Analysis
		3 Methods
			3.1 Migration Assay
			3.2 Live-Cell Imaging
			3.3 Image Analysis
		4 Notes
		References
	Chapter 4: Mapping the Localization of Proteins Within Filopodia Using FiloMap
		1 Introduction
		2 Materials
		3 Methods
			3.1 Measuring Line Intensity Profiles in Fiji
				3.1.1 Installing FiloMap in Fiji
				3.1.2 Analyzing Images in Fiji
				3.1.3 Your Result Folder
			3.2 Combining Filopodium Intensity Profiles in  R
				3.2.1 Preparing RStudio Desktop to Use FiloMap
				3.2.2 Combining Filopodia in  R
				3.2.3 Your Result Folder
			3.3 Compiling Multiple Filopodia Maps to Create a Heatmap
				3.3.1 Combining Multiple Filopodia  Maps
				3.3.2 Creating a Heatmap in  R
		4 Notes
		References
	Chapter 5: Extended Methods for 2D Confinement
		1 Introduction
		2 Materials
			2.1 Nanogrooved Surfaces
				2.1.1 Nanogroove Fabrication
				2.1.2 Surface Treatment
				2.1.3 Cell Confinement
			2.2 Collagen Inclusion
				2.2.1 Surface Treatment
				2.2.2 Preparation of Collagen Gels
				2.2.3 Gel Reticulation and Confinement
			2.3 Fixation under Confinement
				2.3.1 Preparation of Confinement Rings
				2.3.2 Surface Treatment
				2.3.3 Preparation of Agarose Gels
				2.3.4 Agarose Cell Confinement
				2.3.5 Cell Fixation and Removal of Gel
		3 Methods
			3.1 Cell Confinement Combined with Nanogrooved Surfaces
				3.1.1 Fabrication of the Nanogrooved Coverslips (Fig. 2E)
				3.1.2 Surface Treatment
				3.1.3 Cell Confinement
			3.2 Dynamic Cell Confinement Combined with Collagen Inclusion
				3.2.1 Surface Treatment
				3.2.2 Preparation of the Collagen Gel Solution
				3.2.3 Gel Reticulation and Confinement (Fig. 3a)
			3.3 Cell Fixation Under Agarose Confinement
				3.3.1 Preparation of 3D Printed Confinement Rings
				3.3.2 Surface Treatment
				3.3.3 Preparation of Agarose Gels (Fig. 4b-c)
				3.3.4 Agarose Cell Confinement (Fig. 4b-c)
				3.3.5 Cell Fixation and Removal of Agarose Gel
		4 Notes
		References
	Chapter 6: Visualization of Exosome Release and Uptake During Cell Migration Using the Live Imaging Reporter pHluorin_M153R-CD...
		1 Introduction
		2 Materials
			2.1 Generating Stable Cell Lines
			2.2 Live Cell Imaging
			2.3 Image Analyses
		3 Methods
			3.1 Viral Particle Production
			3.2 Target Cell Infection
			3.3 Observation of Exosome Secretion/Deposition During Cell Migration
			3.4 Quantitation of Exosome Secretion/Deposition
			3.5 Observation of MVB Trafficking and Fusion During Cell Migration
			3.6 Image Analysis of Exosome Secretion Movies
			3.7 Observation of Exosome Uptake During Cell Migration
			3.8 Quantitation of Exosome Uptake During Cell Migration from Images
		4 Notes
		References
	Chapter 7: Approaches to Determine Nuclear Shape in Cells During Migration Through Collagen Matrices
		1 Introduction
		2 Materials
			2.1 Embedding of Single Cells
			2.2 Fixation and Labeling
			2.3 Imaging and Analysis
		3 Methods
			3.1 Embedding of Cells in Rat Tail Collagen
			3.2 Fixation and Labeling (Optional)
			3.3 Imaging
			3.4 Fiji-Based Image Segmentation and Quantification of Single Images
			3.5 Analysis of Nuclear Shapes
			3.6 Fiji-Based Image Segmentation and Quantification of Image Sequences
			3.7 Analysis of Nuclear Shape Changes over Time
		4 Notes
		References
Part II: Developmental Model Systems to Assess Cell Migration During Morphogenesis
	Chapter 8: Dissecting Collective Cell Behavior in Migrating Testis Myotubes in Drosophila
		1 Introduction
		2 Materials
			2.1 Culture Media
				2.1.1 Preparing Agarose Dishes
			2.2 Collecting and Timing Prepupae
			2.3 Dissecting Testes
				2.3.1 Mounting for Long-Term Imaging
				2.3.2 Mounting for Short-Term Imaging
			2.4 Live-Cell Imaging and Laser Ablation
			2.5 Preparation and Staining of Testes
				2.5.1 Preparation of Pupal Testes
				2.5.2 Preparation of Adult Testes
				2.5.3 Fixation and Staining
		3 Methods
			3.1 Preparing Agarose Dishes
			3.2 Collecting and Timing Prepupae
			3.3 Dissecting Testes from 33-h APF Drosophila Pupae
				3.3.1 Mounting for Long-Term Imaging (>2 h, 25x Magnification)
				3.3.2 Mounting for Short-Term Imaging and Laser Cell Ablation (<2 h, 63x Magnification)
			3.4 Live-Cell Imaging and Laser-Induced Cell Ablation
				3.4.1 Long-Term Imaging (>2 h, 25x Magnification)
				3.4.2 Short-Term Imaging (<2 h, 63x Magnification)
				3.4.3 Laser-Induced Cell Ablation
			3.5 Preparation and Staining of Pupal and Adult Testes
				3.5.1 Preparation of Pupal Testes
				3.5.2 Preparation of Adult Testes
				3.5.3 Fixation
				3.5.4 Phalloidin and DAPI Staining
				3.5.5 Antibody Staining
		4 Notes
		References
	Chapter 9: Whole-Mount In Situ Hybridization for Detection of Migrating Zebrafish Endodermal Cells
		1 Introduction
		2 Materials
			2.1 Fixation of Zebrafish Embryos
				2.1.1 Collection and Fixation
				2.1.2 Removal of Chorion, Storage, and Permeabilization
			2.2 Endoderm-Specific Probe Synthesis
			2.3 Whole-Mount In Situ Hybridization (WISH)
				2.3.1 Hybridization (Day 1)
				2.3.2 High-Stringency Washes (Day 2)
				2.3.3 Detection (Day 3)
			2.4 Clearing and Mounting
			2.5 Flat Mounting
		3 Methods
			3.1 Fixation of Zebrafish Embryos
				3.1.1 Collection and Fixation of Staged Zebrafish Embryos
				3.1.2 Manual Removal of the Chorion, Storage, and Permeabilization of Embryos
			3.2 Endoderm-Specific Digoxigenin-11-UTP (DIG)-Labeled Probe Synthesis
			3.3 Whole-Mount In Situ Hybridization
				3.3.1 Hybridization with Endoderm-Specific Probe (Day 1)
				3.3.2 Stringent Washing (Day 2)
				3.3.3 Detection (Day 3)
			3.4 Imaging Embryos as Whole Mounts in Glycerol
			3.5 Flat Mounting sox17 or foxa2-Stained Embryos for Quantification
		4 Notes
		References
	Chapter 10: Live-Imaging Analysis of Epithelial Zippering During Mouse Neural Tube Closure
		1 Introduction
		2 Materials
			2.1 Mouse Lines
			2.2 Embryo Dissection
			2.3 Embryo Pre-Culture
			2.4 Embryo Mounting
			2.5 Live Imaging and Analysis
			2.6 Whole-Mount Immunofluorescence
		3 Methods
			3.1 Mouse Lines and Timed Matings
			3.2 Embryo Dissection
			3.3 Pre-Culture of Embryos in Rolling Conditions
			3.4 Preparation for Live Imaging
			3.5 Embryo Mounting for Static Culture
			3.6 Live Imaging of the Zippering Region
			3.7 Post-acquisition Processing
			3.8 Segmentation and Morphometric Analysis of Zippering
		4 Notes
		References
	Chapter 11: Time-Lapse Imaging and Morphometric Analysis of Tracheal Development in Drosophila
		1 Introduction
		2 Materials
			2.1 Embryo Labeling
				2.1.1 Fixation of Embryos
				2.1.2 Immunostaining and Embryo Mounting
			2.2 Tracheal Cell Live Imaging
			2.3 Tracheal Terminal Cell Larval Analysis
		3 Methods
			3.1 Embryo Labeling for Immunofluorescence Microscopy
				3.1.1 Embryo Fixation
				3.1.2 Immunostaining and Embryo Mounting
			3.2 Tracheal Cell Live Imaging
			3.3 Tracheal Terminal Cell Larval Analysis
			3.4 Morphometric Analysis
				3.4.1 Tube Quantification
				3.4.2 Quantification of Cellular Parameters
				3.4.3 Analysis and Quantification of Terminal Cell Morphology and Branching
		4 Notes
		References
	Chapter 12: A Guide Toward Multi-scale and Quantitative Branching Analysis in the Mammary Gland
		1 Introduction
		2 Materials
			2.1 Genetic Lineage Tracing
			2.2 Mammary Gland Dissection
			2.3 Whole-Mount Preparation
			2.4 Image Visualization and Analysis
		3 Methods
			3.1 Genetic Lineage Tracing to Mark Contribution of Cell Population(s) of Interest
			3.2 Mammary Gland Dissection
			3.3 Whole-Mount Preparation
			3.4 Whole-Mount Confocal Imaging
			3.5 Image Processing
			3.6 Branch Analysis and Clone Quantification
			3.7 Modeling of Branching and Cellular Dynamics Based on Whole-Gland Image Analysis
		4 Notes
		References
	Chapter 13: Analysis of Integrin-Dependent Melanoblast Migration During Development
		1 Introduction
		2 Materials
			2.1 Melanocyte Culture and Immunofluorescence
				2.1.1 Setting up Primary Culture
				2.1.2 Labeling and Imaging Melanocyte Focal Adhesions
			2.2 Whole-Mount Imaging of Melanoblasts
				2.2.1 Embryo Dissection
				2.2.2 Embryo Staining and Imaging
			2.3 Ex Vivo Migration of Melanoblasts
				2.3.1 Embryo Dissection
				2.3.2 Skin Mounting and Imaging
		3 Methods
			3.1 Melanocyte Primary Culture and Immunofluorescence
				3.1.1 Setting up Primary Culture
				3.1.2 Labeling and Imaging Melanocyte Focal Adhesions
			3.2 Fixed Whole-Mount Imaging of Melanoblasts in Mouse Embryos
				3.2.1 Embryo Dissection
				3.2.2 Embryo Staining and Imaging
			3.3 Ex Vivo Migration of Melanoblasts
				3.3.1 Embryo Dissection
				3.3.2 Skin Mounting and Imaging
		4 Notes
		References
Part III: Cell Migration in Cancers and the Tumor Microenvironment
	Chapter 14: Invadopodia Methods: Detection of Invadopodia Formation and Activity in Cancer Cells Using Reconstituted 2D and 3D...
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Gelatin Degradation Assay
				2.2.1 Resuspension of Fluorescent Gelatin
				2.2.2 Fluorescence Labeling of Gelatin
				2.2.3 Coverslip Preparation for Immunostaining
				2.2.4 MatTek Preparation for Live-Cell Imaging
			2.3 Collagen Degradation Assay
				2.3.1 Coverslip Preparation and Coating
				2.3.2 Labeling of Collagen
			2.4 Fixation and Immunolabeling
		3 Methods
			3.1 Cell Culture and Cell Plating
			3.2 Invadopodia Knockdown as Negative Control of Invadopodia Formation and Matrix Degradation
			3.3 Transient Transfection of Plasmids
			3.4 Gelatin Degradation Assay for Immunofluorescence Microscopy
				3.4.1 Dissolution of Commercial Fluorescent-Conjugated Gelatin
				3.4.2 Gelatin Coupling to Fluorophore
				3.4.3 Coverslip Coating with Gelatin
				3.4.4 Gelatin Coating of Glass-Bottom MatTek Dish for Live-Cell Imaging
			3.5 Collagen-Based Matrix for Immunofluorescence Microscopy
				3.5.1 Collagen Labeling
				3.5.2 Preparation of Fluorescently Labeled Collagen Solution
				3.5.3 Polymerization of a Type-I Collagen Layer for 2D Assays
				3.5.4 MatTek Coating with Collagen for 2D Live-Cell Imaging
				3.5.5 Coverslip Coating with a 3D Collagen Drop
				3.5.6 MatTek Coating with Bilayered Collagen Sandwich for 3D Assay
			3.6 Immunolabeling of Invadopodia Markers and Collagen Cleavage
				3.6.1 Immunolabeling of Invadopodia Markers in Fixed Cells
				3.6.2 Immunolabeling of MMP-Cleaved Collagen
			3.7 Image Acquisition and Analysis
				3.7.1 Image Acquisition and Analysis of Gelatin Degradation and Invadopodia Parameters
				3.7.2 Live-Cell Imaging of Invadopodia Dynamics in Cells Plated on Gelatin
				3.7.3 Image Acquisition and Analysis of Invadopodia Formation and Collagen Cleavage on a 2D Collagen Layer
				3.7.4 Image Acquisition and Analysis of Cell Invasion and Collagen Cleavage into a 3D Collagen  Gel
				3.7.5 Live-Cell Imaging of Cells Plated into 3D Collagen Gel and Bilayered Type-I Collagen Sandwich (3D Assays)
		4 Notes
		References
	Chapter 15: Analysis of Energy-Driven Leader-Follower Hierarchy During Collective Cancer Cell Invasion
		1 Introduction
		2 Materials
			2.1 Generation of Tumor Spheroids
			2.2 3D Culture for Tumor Spheroid Invasion
			2.3 Imaging and Analysis of Leader-Follower Dynamics
			2.4 Imaging and Analysis of Glucose Uptake
			2.5 Imaging and Analysis of Intracellular ATP:ADP Ratio
		3 Methods
			3.1 Generation of Tumor Spheroids
			3.2 3D Culture for Tumor Spheroid Invasion
			3.3 Imaging and Analysis of Leader-Follower Turnover
				3.3.1 Imaging Leader-Follower Turnover
				3.3.2 Analysis of Leader-Follower Turnover
			3.4 Imaging and Analysis of Glucose Uptake
				3.4.1 Imaging Glucose Uptake Along Strands
				3.4.2 Analysis of Glucose Uptake Along Strands
			3.5 Imaging and Analysis of ATP:ADP Ratio
				3.5.1 Imaging ATP:ADP Ratio Dynamics
				3.5.2 Calibration of ATP:ADP Ratio with Intracellular pH (See Note 26)
				3.5.3 Calculation of ATP:ADP Ratio
				3.5.4 Analysis of ATP:ADP Ratio During Collective Invasion
		4 Notes
		References
	Chapter 16: Visualizing and Quantifying mRNA Localization at the Invasive Front of 3D Cancer Spheroids
		1 Introduction
		2 Materials
			2.1 Formation of Invasive 3D Spheroids
			2.2 Fixation and RNA FISH
			2.3 Imaging and Image Analysis
		3 Methods
			3.1 Formation of Invasive 3D Spheroids
				3.1.1 Formation of Hanging Droplets
				3.1.2 Embedding Spheroids into Matrigel
			3.2 Fixation and RNA FISH
			3.3 Imaging and Image Analysis
		4 Notes
		References
	Chapter 17: Multimodal Techniques to Study Tumor Growth, Basement Membrane Breaching, and Invasion in 3D Matrices
		1 Introduction
		2 Materials
			2.1 Propagation of Organoids
				2.1.1 Thawing and Passaging
				2.1.2 Freezing
			2.2 Transfer of Established Organoids
				2.2.1 Isolating Organoids from BME Gels with Dispase
				2.2.2 Isolating Organoids from BME Gels Without Dispase
				2.2.3 Embedding Organoids (from BME to Collagen I)
				2.2.4 Embedding of Organoids in BME (BME to BME)
			2.3 Fixation
				2.3.1 Fixation with PFA and Glutaraldehyde
				2.3.2 Fixation with  PFA
			2.4 Immunostaining for Confocal Microscopy
			2.5 Protein and RNA Extraction
				2.5.1 Protein Extraction
				2.5.2 Western  Blot
				2.5.3 RNA Extraction
				2.5.4 cDNA Synthesis
				2.5.5 RT-qPCR
		3 Methods
			3.1 Propagation of Cancer-Derived Organoid Culture
				3.1.1 Passaging (Fig. 1a)
				3.1.2 Freezing
			3.2 Isolation and Embedding of Organoids in Collagen I or  BME
				3.2.1 Isolating Organoids from Cultures Using Cold Medium (Fig. 2a, b)
				3.2.2 Isolating Organoids from Cultures Using Dispase II (See Note 5) (Fig. 2a, c)
				3.2.3 Preparation of 3D Collagen I  Gels
				3.2.4 Embedding Organoids in 3D Collagen I (Fig. 2a)
				3.2.5 Embedding of Organoids Back to BME (See Note 10)
			3.3 Fixation and Immunostaining for Confocal Microscopy
				3.3.1 Fixation with PFA and Glutaraldehyde (Fig. 3a)
				3.3.2 Fixation with PFA (See Note 12) (Fig. 3b)
			3.4 (Immuno)staining for Confocal Microscopy (See Notes 13 and 14)
			3.5 Extraction of Organoids for Protein and RNA Isolation
				3.5.1 Using Dispase II and Collagenase  I
				3.5.2 Without Enzymes (See Note 18) (Fig. 4b)
				3.5.3 Western  Blot
				3.5.4 RNA Isolation
				3.5.5 cDNA Synthesis
				3.5.6 RT-qPCR
		4 Notes
		References
	Chapter 18: Analysis of Collective Migration Patterns Within Tumors
		1 Introduction
		2 Materials
			2.1 Intestinal Tumor Extraction and Imaging
				2.1.1 Tumor Dissection
				2.1.2 Tumor Embedding
				2.1.3 Tumor Slicing
				2.1.4 Tumor Slice Mounting and Imaging
			2.2 Quantitative Analysis of Tumor Cell Migration
		3 Methods
			3.1 Extraction and Live Imaging of Intestinal Tumors
				3.1.1 Tumor Dissection
				3.1.2 Tumor Embedding
				3.1.3 Tumor Slicing
				3.1.4 Tumor Slice Mounting and Imaging
			3.2 Quantitative Analysis of Tumor Cell Migration
				3.2.1 Pre-processing of Live Imaging  Data
				3.2.2 Segmentation of Nuclei Using StarDist
				3.2.3 Tracking Individual Nuclei Using TrackMate
				3.2.4 Extraction of Migration Dynamics
				3.2.5 Particle Image Velocimetry
				3.2.6 Extraction of Additional Cell and Tissue Parameters
		4 Notes
		References
	Chapter 19: An In Vivo Model to Study Cell Migration in XYZ-T Dimension Followed by Whole-Mount Re-evaluation
		1 Introduction
		2 Materials
			2.1 Experimental Animals (See Note 1)
			2.2 Dorsal Skinfold Transplantation
				2.2.1 Dorsal Skinfold Chamber (Fig. 3a)
				2.2.2 Surgical Procedure (Fig. 3b)
			2.3 Intravital 4D Evaluation
			2.4 Whole-Mount Staining
		3 Methods
			3.1 Dorsal Skinfold Transplantation
				3.1.1 Dissecting Tumor from Donor Mouse
				3.1.2 Surgical Procedure
				3.1.3 Housing and  Care
			3.2 Intravital 4D Evaluation
			3.3 Whole-Mount Staining Procedure
		4 Notes
		References
Part IV: Blood Vessel Formation and Interactions
	Chapter 20: Angiogenesis Invasion Assay to Study Endothelial Cell Invasion and Sprouting Behavior
		1 Introduction
		2 Materials
			2.1 Matrigel Plug Preparation and Cell Seeding
				2.1.1 Preparation of Matrigel Plugs
				2.1.2 Cell Preparation
			2.2 Image Acquisition of Unstained Samples
				2.2.1 Phase Contrast and Time-Lapse Video Microscopy
			2.3 Whole-Mount Immunofluorescent Staining
				2.3.1 Sample Fixation
				2.3.2 Immunofluorescent Staining
				2.3.3 Image Acquisition of Stained Samples
			2.4 Data Quantification
		3 Methods
			3.1 Matrigel Plug Preparation and Cell Seeding
				3.1.1 Preparation of Matrigel Plugs
				3.1.2 Cell Preparation
				3.1.3 Acquire Large Images of the Samples (See Notes 12 and 13)
				3.1.4 Perform Time-Lapse Video Microscopy of the Samples
			3.2 Labeling of Matrigel Plugs for Whole-Mount Immunofluorescence Microscopy
				3.2.1 Sample Fixation
				3.2.2 Sample Staining
				3.2.3 Image Acquisition
			3.3 Data Quantification
				3.3.1 Sprout Numbers
				3.3.2 Total Sprout Length
				3.3.3 Average Sprout Length
				3.3.4 Real-Time Analysis of Tip Cell Protrusion Dynamics
				3.3.5 Real-Time Analysis of Cell Motility
				3.3.6 Observation of Stained Samples in 3D
				3.3.7 Analysis of Filopodia and Podosomes in Angiogenic Sprouts
		4 Notes
		References
	Chapter 21: Live-Cell Labeling and Artificial Intelligence Approaches for High-Resolution XYZT Imaging of Cytoskeletal Dynamic...
		1 Introduction
		2 Materials
			2.1 HUVEC Culture
			2.2 Live-Cell Labeling
			2.3 Scratch Assay
			2.4 Sprouting Assay
			2.5 Live-Cell Imaging
			2.6 Staining of Fixed Cells and Sprouts
			2.7 Imaging of Fixed Cells
			2.8 Image Enhancement by AI and Analysis
			2.9 Image Segmentation and Cell Tracking
		3 Methods
			3.1 HUVEC Culture
			3.2 Live-Cell Labeling with Fluorogenic Probes
			3.3 Scratch Assay
			3.4 Sprouting Assay
			3.5 Live-Cell Imaging
				3.5.1 Live Imaging of Cell Migration in Scratch Assays
				3.5.2 Live Imaging of Sprouting Angiogenesis in 3D
			3.6 Staining of Live-Labeled Cells After Fixation
				3.6.1 Staining of Monolayers and Scratch Assays
				3.6.2 Staining of Sprouted Cells in Fibrin Gels
			3.7 Imaging of Fixed Cells
				3.7.1 Imaging of Coverslips and Plates for Scratch Assays
				3.7.2 Imaging of Sprouting in Fibrin Gels
			3.8 Image Processing Using Enhance.Ai
			3.9 Image Segmentation and Cell Tracking
				3.9.1 Automated Tracking
				3.9.2 Manual Tracking
		4 Notes
		References
	Chapter 22: Analysis of mRNA Subcellular Distribution in Collective Cell Migration
		1 Introduction
		2 Materials
			2.1 Preparation and Trypsinization of Cells
			2.2 Fibrin Bead Assay
			2.3 Scratch Wound Assay
			2.4 Fixation and Permeabilization
			2.5 smFISH Probe Hybridization
			2.6 Immunofluorescence
			2.7 Mounting
		3 Methods
			3.1 Stellaris smFISH Probe Design
			3.2 Preparation of Cells
				3.2.1 HPF Cell Culture
				3.2.2 HUVEC Culture
			3.3 Coating Cytodex 3 Microcarrier Beads with HUVECs for Fibrin Gel Assay (Day -10)
				3.3.1 Trypsinization and Counting of HUVECs
				3.3.2 Preparation of Microcarrier Beads
				3.3.3 Coating Beads with HUVECs
			3.4 Embedding Beads in Fibrin Gel and Seeding HPFs (Day-9)
				3.4.1 Prepare Fibrinogen Solution
				3.4.2 Mix Coated Beads with Fibrinogen Solution
				3.4.3 Making Fibrin  Gels
				3.4.4 Seed HPFs on Top of  Gels
			3.5 Scratch Wound Assay
				3.5.1 Seeding HUVECs (Day -2)
				3.5.2 Creating the Scratch Wound (Day -1)
			3.6 Fixation and Permeabilization of Samples (Day -1)
				3.6.1 Trypsinization of HPFs from Gibrin  Gels
				3.6.2 Fixation and Permeabilization of Scratch Wounds and Fibrin  Gels
			3.7 smFISH Probe Hybridization (Day 0)
				3.7.1 Probe Hybridization in Fibrin  Gels
				3.7.2 Probe Hybridization in Scratch Wounds
			3.8 smFISH Washes (Day 1)
				3.8.1 Washing Scratch Wounds
				3.8.2 Washing Fibrin  Gels
			3.9 Immunofluorescence
				3.9.1 Immunofluorescence of Sprouts in Fibrin Gels
				3.9.2 Immunofluorescence in Scratch Wounds
			3.10 Final Washes and Mounting
				3.10.1 Scratch Wound Final Washes and Mounting
				3.10.2 Fibrin Gel Final Washes and Mounting
		4 Notes
		References
	Chapter 23: A Bioengineered Model for Studying Vascular-Pericyte Interactions of the Placenta
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Device Fabrication
			2.3 Device Seeding and Culture of Microvessels
			2.4 Imaging and Morphology Quantification
		3 Methods
			3.1 Cell Culture
				3.1.1 Thawing of the Cells
				3.1.2 Splitting of the Cells
			3.2 Device Fabrication
				3.2.1 Fabrication of the Negative Mold
				3.2.2 Fabrication of the PDMS Devices
				3.2.3 Device Bonding and Coating
			3.3 Device Seeding and Culture of Microvessels
				3.3.1 Preparation of Fibrin Gel Components
				3.3.2 Seeding and Culture
			3.4 Imaging and Morphology Quantification
				3.4.1 Image Acquisition
				3.4.2 Image Analysis
				3.4.3 Morphology Calculation and Quantification
		4 Notes
		References
	Chapter 24: Analysis of Vascular Morphogenesis in Zebrafish
		1 Introduction
		2 Materials
			2.1 General Vascular Assessment
				2.1.1 Preparing Embryos
				2.1.2 Mounting and Imaging
			2.2 Manipulations to Study Cell Migration
				2.2.1 Microinjections
				2.2.2 Drug Treatments
			2.3 Analysis
		3 Methods
			3.1 General Vascular Assessment During Embryonal Development
				3.1.1 Preparing Embryos for Live Imaging
				3.1.2 Mounting and Imaging
				3.1.3 ISV Imaging
				3.1.4 CtA Imaging
				3.1.5 CCV Imaging
			3.2 Manipulation Techniques to Study Endothelial Cell Migration
				3.2.1 Microinjections
				3.2.2 Gain of Function/Vascular Specific Overexpression
				3.2.3 Loss of Function/Gene Knockdown or Genetic Knockout
				3.2.4 Drug Treatments
			3.3 Analysis
				3.3.1 Analysis of Tip and Stalk Cell Competition During ISV Formation
				3.3.2 Analysis of Sprouting Angiogenesis During CtA Formation
				3.3.3 Analysis of Collective Endothelial Cell Migration Behavior During CCV Formation
		4 Notes
		References
	Chapter 25: Analysis of Monocyte Recruitment During Inflammation by Intravital Imaging
		1 Introduction
		2 Materials
			2.1 Mouse Surgery Materials for Intravital Microscopy
			2.2 Intravital Fluorescence Microscopy
			2.3 Analysis of Videos and Images
		3 Methods
			3.1 Surgical Procedure for Intravital Microscopy Studies in Cremaster Muscle
			3.2 Set Up The Intravital Microscope for Microcirculation Visualization
			3.3 Analysis of Monocyte Recruitment During Inflammation
			3.4 Adhesion: Quantification of Adherent Monocytes
			3.5 Rolling
				3.5.1 Quantification of Monocyte Rolling  Flux
				3.5.2 Quantification of Monocyte Rolling Velocity
			3.6 Crawling
				3.6.1 Quantification of Crawling Monocytes
				3.6.2 Analysis of Crawling Monocyte Velocity
				3.6.3 Analysis of Crawling Monocyte Trajectories: Crawling Pathways and Confinement Ratio
			3.7 Migration
				3.7.1 Quantification of Migrated Monocytes
				3.7.2 Analysis of Migrated Monocyte Velocity
		4 Notes
		References
Index