Bookedited by Sheila Spada, Lorenzo Galluzzi.
Contents:
Intro
Extracellular Vesicles
Copyright
Contents
Contributors
Preface
Extracellular vesicles: An exciting and rapidly expanding field of investigation
Acknowledgments
Disclosures
References
Chapter One: Genetic labeling of extracellular vesicles for studying biogenesis and uptake in living mammalian cells
1. Introduction
2. Equipment and materials
2.1. Equipment
2.2. Cells and culture medium
2.3. Reagents and chemicals
3. Protocol
3.1. Cell culture
3.2. Generation of transient and stable CD63-GFP/VSVG-GFP expressing cells 3.3. Exosome preparation from conditioned medium
3.4. Cell and exosome imaging
3.5. Cellular uptake of exosomes by confocal microscopy and flow cytometry
4. Concluding remarks
5. Notes
Acknowledgments
References
Chapter Two: Fluorescent labeling of extracellular vesicles
1. Introduction
1.1. Uptake studies
1.2. Biodistribution studies
1.3. Characterization studies
2. Fluorescent labeling
2.1. EV Labeling approaches
2.1.1. Surface marker labeling of EVs
2.1.2. Lipid membrane labeling of EVs
2.1.3. Luminal labeling of EVs 2.2. Post-labeling clean-up
2.2.1. Differential ultracentrifugation
2.2.2. Density gradient centrifugation
2.2.3. Size exclusion chromatography
2.2.4. Filtration
3. Materials, equipment and reagents
4. Protocols
4.1. Sample labeling
4.2. Removing excess dye
4.3. Coverslip preparation
4.4. Fluorescent imaging
4.5. Size assessment using NTA
5. Pros and cons
5.1. Surface marker labeling
5.2. Lipid membrane labeling
5.3. Luminal labeling
6. Conclusion
References Chapter Three: Use of antibody arrays to probe exosome and extracellular vesicle mediated functional changes in cells
1. Introduction
1.1. Exosomes and extracellular vesicles
1.2. Antibody arrays
2. Methods
2.1. Cell culture
2.2. Isolation of exosome fraction
2.3. Labeling of exosome fraction
2.4. Treatment of cells
2.5. Antibody arrays
3. Notes
4. Concluding remarks
References
Chapter Four: Analysis of individual extracellular vesicles by imaging flow cytometry
1. Introduction
2. Single EV analyses
2.1. Equipment
2.2. Materials 2.3. Protocol optimization
2.4. Instrument calibration
3. Protocol
3.1. Preparation of antibody solution
3.2. Sample preparation
3.3. Controls
3.4. Sample acquisition with the ISX
4. Data analysis
4.1. Defining of masks to analyze the raw image file data
4.2. Using the feature manager to determine single events
4.3. Data presentation and calculation of subset concentrations
5. Summary
Acknowledgments
References
Chapter Five: Imaging intercellular interaction and extracellular vesicle exchange in a co-culture model of chronic lymph ...
1. Introduction