Permeability Barrier: Methods and Protocols

Preface
Contents
Contributors
Use of Ussing Chambers to Measure Paracellular Permeability to Macromolecules in Mouse Intestine
	1 Introduction
	2 Materials
	3 Methods
		3.1 Setup of Ussing Chambers
		3.2 Mouse Tissue Dissection and Mounting
		3.3 FITC-Labeled Dextran 4 kDa (FD4) Addition and Flux Measurements
		3.4 Disassembly and Cleaning of the System
		3.5 Data Analysis
	4 Notes
	References
Rapid Evaluation of Intestinal Paracellular Permeability Using the Human Enterocytic-Like Caco-2/TC7 Cell Line
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Transepithelial Electrical Resistance (TEER)
		2.3 Paracellular Permeability to Macromolecules
	3 Methods
		3.1 Cell Culture
		3.2 Transepithelial Electrical Resistance (TEER)
		3.3 Paracellular Permeability to Macromolecules
	4 Notes
	References
Evaluation of Barrier Functions in Human iPSC-Derived Intestinal Epithelium
	1 Introduction
	2 Materials
	3 Methods
		3.1 Differentiation of Human iPSCs into Intestinal Organoids
		3.2 Monolayer Culture from Intestinal Organoids
		3.3 Measurement of TEER
		3.4 Permeability Assay
		3.5 Assessment of Tight Junction Formation (ZO-1 Staining)
	4 Notes
	References
Selective Regional Isolation of Brain Microvessels
	1 Introduction
	2 Materials
		2.1 Solutions for Brain Blood Vessel Isolation
		2.2 Solutions for Western Blot
		2.3 Solutions for Immunocytofluorescence
		2.4 Solutions for Scanning Electron Microscopy
	3 Methods
		3.1 Blood Vessel Isolation from Selective Brain Regions
		3.2 Protein Expression in Isolated Blood Microvessels from Specific Brain Regions: Western Blot
		3.3 Protein Immunoreactivity in Isolated Blood Microvessels from Specific Brain Regions: Immunocytofluorescence and Confocal M...
		3.4 Morphological Analysis of Pericyte Attachment in Isolated Blood Microvessels from Specific Brain Regions: Scanning Electro...
	4 Notes
	References
Estimating Brain Permeability Using In Vitro Blood-Brain Barrier Models
	1 Introduction
	2 Materials
		2.1 Materials for Differentiation of iPSC to hBMECs
		2.2 Preparation of Aliquots and Mediums
		2.3 Materials for Differentiation of iPSC to Astrocytes
			2.3.1 Reagents
			2.3.2 Mediums
		2.4 Materials for Permeability and TEER Measurement
	3 Methods
		3.1 Differentiation of iPSCs to hBMECs
		3.2 Differentiation of iPSC into Astrocytes
		3.3 Permeability and TEER Measurement
			3.3.1 For Measuring the TEER
			3.3.2 Permeability Measurement
		3.4 Advanced Microfluidic iPSCs-Derived hBMECs on Chips
	4 Notes
	References
In Vitro Human Blood-Brain Barrier Model for Drug Permeability Testing
	1 Introduction
	2 Materials
		2.1 Triple Co-culture Growth Medium Preparation
		2.2 Cell Culture
		2.3 BBB Modeling
		2.4 Characterization of the BBB Model
		2.5 Drug Permeability Testing
	3 Methods
		3.1 Cell Culture
		3.2 BBB Modeling
		3.3 Characterization of the BBB Model
			3.3.1 TEER Measurement
			3.3.2 Lucifer Yellow (LY) Permeability
			3.3.3 Immunofluorescence (IF) Staining
		3.4 Drug Permeability Testing
	4 Notes
	References
Evaluation of Blood-Brain Barrier Integrity Using Vascular Permeability Markers: Evans Blue, Sodium Fluorescein, Albumin-Alexa
	1 Introduction
		1.1 Evans Blue Dye
		1.2 Sodium Fluorescein
		1.3 Albumin-Alexa Fluor Conjugates
		1.4 Horseradish Peroxidase
	2 Materials
		2.1 Assessment of Evans Blue Dye Extravasation
		2.2 Assessment of Sodium Fluorescein Extravasation
		2.3 Assessment of Albumin-Alexa Fluor 594 Extravasation
		2.4 Assessment of Horseradish Peroxidase Extravasation
	3 Methods
		3.1 Assessment of Evans Blue Dye Extravasation
		3.2 Assessment of Sodium Fluorescein Extravasation
		3.3 Assessment of Albumin-Alexa Fluor 594 Extravasation
		3.4 Assessment of Horseradish Peroxidase Extravasation
	4 Notes
	References
Blood-Brain Barrier (BBB) Permeability and Transport Measurement In Vitro and In Vivo
	1 Introduction
	2 Materials
		2.1 Materials for BBB Permeability Measurement In Vitro
		2.2 Materials for BBB Permeability and Transport Measurement In Vivo
	3 Methods
		3.1 Apply Experimental Treatments
		3.2 Dextran Permeability Measurement
		3.3 Calculating the Permeability of the Endothelial Barrier
		3.4 Calibration of Optical Settings of Microscope for Linear Range of Concentration vs. Intensity of Fluorescence Probe Soluti...
		3.5 Determination of the Depth of Light Collection for the Imaging System
		3.6 Animal Preparation
		3.7 Image Acquisition
			3.7.1 2-D Image Collection for the Measurement of Solute Permeability of the BBB
			3.7.2 3-D Image Collection for Determination of Solute Permeability and Effective Diffusion Coefficient in Brain Tissue
		3.8 Image Analysis
		3.9 Determination of Solute Permeability of BBB (PBBB) from Collected Images
			3.9.1 Determination of Solute Permeability of BBB (PBBB) from 2-D Images
			3.9.2 Determination of Solute Permeability of BBB (PBBB) from 3-D Images
		3.10 Determination of Effective Solute Diffusion Coefficient in Brain Tissue (Deff) from 3-D Images
		3.11 Corrections for Influence of Red Blood Cells, Free Dye, and Solvent Drag on BBB Permeability
	4 Notes
	References
Assessment of Blood-Brain Barrier Permeability Using Miniaturized Fluorescence Microscopy in Freely Moving Rats
	1 Introduction
	2 Materials
		2.1 Reagents and Supplies for Surgery and Imaging
		2.2 Equipment and Tools for Surgery and Imaging
		2.3 Animals
	3 Methods
		3.1 Surgery Preparation
		3.2 Anesthesia
		3.3 Stereotaxic Cannulation Surgery
		3.4 Post-surgery Care and Monitoring
		3.5 Habituation to the Miniscope
		3.6 Imaging Session Preparation
		3.7 Fluorescence Recording Using Miniscope
		3.8 Image Analysis of Fluorescence Recordings
	4 Notes
	References
Measurement of Lung Vessel and Epithelial Permeability In Vivo with Evans Blue
	1 Introduction
	2 Materials
		2.1 Evans Blue Injection
		2.2 Tissue Harvest
		2.3 Tissue Analysis
	3 Methods
		3.1 Tail Vein Injection of Evans Blue
		3.2 Serum Collection
		3.3 BAL Fluid Collection
		3.4 Lung Tissue Collection
		3.5 Evans Blue Measurement and Analysis
	4 Notes
	References
Measurement of Airway Epithelial Permeability: Methods and Protocols
	1 Introduction
	2 Materials
	3 Methods
		3.1 Transepithelial/Transendothelial Electrical Resistance (TEER)
		3.2 TEER Measurement Methods
			3.2.1 Ohm´s Law Method
			3.2.2 Impedance Spectroscopy
			3.2.3 Other Systems Available for TEER Measurements
				Ussing Chamber
				Transwells
				ECIS (Electric Cell-Substrate Impedance Sensing)
		3.3 Two-Path Impedance Spectroscopy
		3.4 FITC (Fluorescein Isothiocyanate)
		3.5 Mannitol
		3.6 Albumin
		3.7 Lucifer Yellow
	4 Notes
	5 Conclusion
	References
Vascular Permeability Assays In Vivo
	1 Introduction
		1.1 Mechanism of Vascular Permeability
		1.2 Applications of Vascular Permeability Assessment
		1.3 Miles Assay to Measure Vascular Permeability In Vivo
			1.3.1 Working Principle of Miles Assay
			1.3.2 Limitations of the Miles Assay
		1.4 Alternate Method: Fluorescein Isothiocyanate (FITC)-Dextran Lung Permeability Assay
	2 Materials
		2.1 Miles Assay
		2.2 Fluorescein Isothiocyanate (FITC)-Dextran Lung Permeability Assay
	3 Methods
		3.1 Miles Assay, e.g., VEGF and Ang-1-Mediated VP Assessment
		3.2 Fluorescein Isothiocyanate (FITC)-Dextran Lung Permeability Assay
	4 Notes
	References
Endothelial Permeability Assays In Vitro
	1 Introduction
		1.1 Vascular Permeability
		1.2 Physiological State of Endothelial-Barrier
		1.3 Pathophysiological State of Endothelial-Barrier
		1.4 Routes of Transportation
		1.5 Regulators of Vascular Permeability
		1.6 Significance of Vascular Permeability Studies
		1.7 In Vitro Permeability Assay in Transwell Plates
		1.8 Limitations of Transwell In Vitro Permeability Assay
		1.9 Electric Cell-Substrate Impedance Sensing (ECIS) Assay to Measure EC-Barrier Function
	2 Materials
		2.1 In Vitro Permeability Assay
			2.1.1 Cell Culture
			2.1.2 Transwell Assay
		2.2 ECIS Assay
	3 Methods
		3.1 In Vitro Permeability Assay
			3.1.1 Endothelial Cell Culture
			3.1.2 Coating the Transwell Plates
			3.1.3 Seeding ECs into Transwell Plates
			3.1.4 Transwell Assay Protocol
			3.1.5 Monolayer Staining
		3.2 ECIS Protocol for EC-Barrier Impedance Assay
	4 Notes
	References
Mapping Receptor Antibody Endocytosis and Trafficking in Brain Endothelial Cells
	1 Introduction
	2 Materials
		2.1 Antibody Endocytosis from the Luminal Plasma Membrane
		2.2 Immunofluorescence Staining of Subcellular Markers
		2.3 Imaging of Samples with Confocal Microscopy
		2.4 Analysis and Quantification of Co-localization Score
	3 Methods
		3.1 Antibody Endocytosis from the Luminal Plasma Membrane
		3.2 Immunofluorescence Staining of Subcellular Markers
		3.3 Imaging of Samples with Confocal Microscopy
		3.4 Analysis of Co-localization Using ImageJ ComDet Spot Detection Plugin
		3.5 Quantification of Co-localization Score
	4 Notes
	References
An In Vitro Assay to Monitor Sertoli Cell Blood-Testis Barrier (BTB) Integrity
	1 Introduction
	2 Materials
	3 Methods
		3.1 In Preparation
		3.2 Experiment
		3.3 Image Requisition and Data Analysis
		3.4 Conclusion
	4 Notes
	References
Ussing Chamber Methods to Study the Esophageal Epithelial Barrier
	1 Introduction
	2 Materials
		2.1 Solutions
		2.2 KCl Agar Bridges
		2.3 Ussing Chambers
		2.4 Mouse Tissue Dissection and Mounting
		2.5 Circuit Analysis
		2.6 Fluorescein Flux
		2.7 Low pH Ringer
		2.8 Microelectrode Measurements in Esophageal Tissues Mounted in a Modified Ussing Chamber
		2.9 Bicarbonate Secretion Measurement in the Ussing Chamber
	3 Methods
		3.1 Solutions
		3.2 KCl Agar Bridges
		3.3 Ussing Chambers Set-Up
		3.4 Tissue Dissection and Mounting
		3.5 Circuit Analysis
		3.6 Permeability Measurements Using Fluorescein
		3.7 Acid Treatment to Simulate Esophageal Damage from Reflux
		3.8 Modified Ussing Chambers for Intracellular Measurements in the Rabbit Esophagus
		3.9 Measurement of Bicarbonate Secretion in the Pig Esophagus Using the Ussing Chamber
	4 Notes
	References
A Simple Method to Test Mechanical Strain on Epithelial Cell Monolayers Using a 3D-Printed Stretcher
	1 Introduction
	2 Materials
		2.1 3D Printing
		2.2 Cell Culture
		2.3 Immunofluorescence
	3 Methods
		3.1 3D Design and Printing of the Stretching Apparatus
		3.2 Cell Culture
		3.3 Cell Stretching and Fixing Process
		3.4 Immunofluorescence Staining
		3.5 Imaging and Analysis
	4 Notes
	References
Differentiating Between Tight Junction-Dependent and Tight Junction-Independent Intestinal Barrier Loss In Vivo
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
	3 Methods
		3.1 Probe Preparation
		3.2 Gavage (See Notes 8 and 9)
		3.3 Blood Collection and Plasma Separation
		3.4 Measuring Plasma Fluorescein and Rhodamine B Fluorescence
		3.5 Measuring Plasma Creatinine Concentration
		3.6 Calculations
		3.7 Data Interpretation
	4 Notes
	References
Establishment of Intestinal Epithelial Cell Monolayers and Their Use in Calcium Switch Assay for Assessment of Intestinal Tigh
	1 Introduction
	2 Materials
		2.1 Reagents Needed
		2.2 Materials Needed
		2.3 Equipment
		2.4 Cell Culture
	3 Methods
		3.1 Establishment of T84 Cell Monolayers
		3.2 Ca2+-Switch Assay
	4 Notes
	References
A Method to Prepare a Bioprobe for Regulatory Science of the Drug Delivery System to the Brain: An Angulin Binder to Modulate
	1 Introduction
	2 Materials
		2.1 Bacterial Strains
		2.2 Bacteria Culture Media
		2.3 Plasmids
		2.4 Protein Purification Materials
		2.5 Cell Lines
		2.6 Cell Culture Medium
		2.7 Reagents for Flow Cytometric Analysis
		2.8 Equipment for Measuring TEER
	3 Methods
		3.1 Preparation of GST-Tagged Angulin Modulating Recombinant Proteins
		3.2 Preparation of Untagged Angulin Modulating Recombinant Proteins
		3.3 Flow Cytometric Analysis
		3.4 Assay of Cell Tight Junctions
	4 Notes
	References
dSTORM Imaging and Analysis of Desmosome Architecture
	1 Introduction
	2 Materials
	3 Methods
		3.1 Buffer Preparation
		3.2 Microscope Setup
		3.3 Data Collection
		3.4 Image Reconstruction
		3.5 Analysis
	4 Notes
	References
Index