Monitoring Vesicular Trafficking in Cellular Responses to Stress

Name: Monitoring Vesicular Trafficking in Cellular Responses to Stress
Brand: Academic Press
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Academic Press

Published on 30. June 2021

222 pages

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978-0-12-823545-4 (ISBN)

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Content

Intro
Monitoring Vesicular Trafficking in Cellular Responses to Stress - Part A
Copyright
Contents
Contributors
Vesicular trafficking in cellular responses to stress
Acknowledgments
Conflict of interest statement
References
Chapter 1: Monitoring TFEB translocation
1. Introduction
2. Disposable material
3. Equipment
4. Reagents
5. Cell culture and passage
6. Immunofluorescence
7. Concluding remarks
8. Notes
Acknowledgment
References
Chapter 2: Monitoring lipophagy in kidney epithelial cells in response to shear stress
1. Introduction
2. Materials
2.1. Cell culture
2.2. Shear stress devices
2.3. Oil red O staining
2.4. Immunofluorescence
2.5. Colorimetric/fluorometric assay to quantify the amount of triglycerides
2.6. Drugs
3. Methods
3.1. Number and lipid droplets size measurement
3.1.1. Lipid droplets quantification using ICY software
3.1.2. How to measure lipid droplets size using ICY software
3.2. Colocalization between LDs and autophagic markers
3.3. Triglycerides measurement assay
4. Conclusion
5. Notes
Acknowledgments
References
Chapter 3: Live cell imaging of LC3 dynamics
1. Introduction
2. Material
2.1. Disposables
2.2. Equipment
2.3. Reagents
3. Methods
3.1. Cell culture routine
3.2. Cell seeding
3.3. Cell treatment
3.4. Live cell imaging
3.5. Image analysis and features extraction
3.6. Data interpretation
4. Concluding remarks
5. Notes
Acknowledgments
Conflict of interest
References
Chapter 4: Autophagy assessment in circulating leukocytes
1. Introduction
2. Material
2.1. Disposables
2.2. Equipment
2.3. Reagents
3. Induction of autophagy by starvation in mice and blood collection
4. Leukocytes isolation, fixation and permeabilization
5. Sample preparation and immunostaining
6. Image flow cytometric acquisition and data analysis
7. Notes
8. Concluding remarks
Acknowledgments
References
Chapter 5: Measuring lysosomal size and frequency by electron microscopy
1. Introduction
1.1. Rationale
1.2. Terminology and organelle classification-Or: Make yourself familiar with your object of desire through organelle mapping
1.3. Preview on the described sample preparation methods-Pros and cons
1.4. Geometrical considerations
2. Material
2.1. Disposables and reagents
2.1.1. Essentials
2.1.2. Options
2.2. Equipment
2.2.1. Essentials
2.2.2. Options
3. Workflow
3.1. Cell culture
3.2. Sample preparation
3.3. EM-imaging, data acquisition and analysis
4. Concluding remarks
Notes
Acknowledgment
References
Chapter 6: Autophagic flux assessment by immunoblot
1. Introduction
2. Material
2.1. Disposables
2.2. Equipment
2.3. Reagents
2.4. Osteosarcoma cell culture
2.5. Autophagy activation or inhibition of osteosarcoma
2.6. Sample preparation
2.7. Immunoblotting
2.8. Data analysis
3. Concluding remarks
4. Notes
Acknowledgments
Declaration of interests
References
Chapter 7: Assessing autophagic flux in yeast
1. Introduction
2. GFP liberation assay
2.1. Principle
2.2. Equipment, disposables and materials
2.3. Reagents
2.4. Experimental procedure
2.4.1. Strains and culture conditions
2.4.2. Cell extract and sample preparation for immunoblotting
2.4.3. Immunoblotting
2.4.4. Fluorescence microscopy
3. ALP activity assay
3.1. Principle
3.2. Equipment, disposables and materials
3.3. Reagents
3.4. Experimental procedure
3.4.1. Strains and culture conditions
3.4.2. Cell extract and sample preparation
3.4.3. Enzymatic reaction
4. Concluding remarks
5. Notes
Acknowledgments
Conflict of interest
References
Chapter 8: Multiple analysis of mitochondrial metabolism, autophagy and cell death
1. Introduction
2. Cell death assays
2.1. Lactate dehydrogenase (LDH) release assay
2.1.1. Materials, reagents and equipment
2.1.2. Protocol
2.1.2.1. Reagent preparation for CytoTox 96 assay kit
2.1.2.2. Assay
2.2. Propidium iodide staining assay
2.2.1. Materials, reagents and equipment
2.2.2. Protocol
3. Autophagy monitoring
3.1. Autophagic biomarker evaluation by western-blot
3.1.1. Materials, reagents and equipment
3.1.1.1. Materials and reagents
3.1.1.2. Equipment
3.1.2. Protocol
3.2. Autophagosome formation using GFP-LC3 plasmid transfection
3.2.1. Equipment, materials and reagents
3.2.1.1. Materials and reagents
3.2.1.2. Equipment
3.2.2. Protocol
4. Mitochondrial network analysis
4.1. Equipment, materials and reagents
4.1.1. Materials and reagents
4.1.2. Equipment
4.2. Experimental setup
5. Real time bioenergetic profile analysis
5.1. Equipment, materials and reagents
5.1.1. Materials and reagents
5.1.2. Equipment
5.2. Experimental setup
5.2.1. XF Cell Mito Stress Test
5.2.2. XF Glycolysis Stress Test
Notes
Acknowledgments
References
Chapter 9: Quantitative determination of mitophagy by fluorescence microscope
1. Introduction
2. Material
2.1. Disposables
2.2. Equipment
2.3. Reagents
3. GFP-LC3 and MitoTracker red approach
3.1. Preparation
3.2. Cell culture and treatment
3.3. MitoTracker red CMXRos
3.4. Fixation
3.5. Observation
3.6. Quantification of mitophagy
4. Concluding remarks
Acknowledgments
References
Chapter 10: Quantifying phagosomal HOCl at single immune-cell resolution
1. Introduction
2. Protocol
2.1. Assembly and characterization of cHOClate
2.1.1. Materials/equipment
2.1.2. Sample preparation
2.2. Surface labeling of zymosan to z-cHOClate
2.2.1. Additional materials and equipment
2.2.2. Sample preparation
2.3. In vitro pH and HOCl measurements
2.3.1. Additional materials
2.3.2. Image acquisition
2.3.3. pH measurements
2.3.4. HOCl measurements
2.3.5. Image analysis
2.4. In cellulo pH and HOCl clamping
2.4.1. Additional materials
2.4.2. Cell culture
2.4.3. Imaging experiments
2.4.4. pH clamping
2.4.5. HOCl clamping
2.5. In cellulo kinetics
2.5.1. Additional materials
2.5.2. In cellulo HOCl dynamics
2.6. Detection of differential MPO levels in innate immune cells
2.6.1. Additional materials
2.6.2. Isolation of human monocytes and neutrophils
2.6.3. Isolation of human peripheral blood monocytes
2.6.4. Isolation of human peripheral blood neutrophils
2.6.5. Differential of human monocytes to macrophages (HMDMs)
2.6.6. Isolation and differentiation of mouse monocytes (BMDMs)
2.6.7. Isolation of mouse adipose tissue macrophages
2.6.8. Isolation of peritoneal macrophages
2.6.9. Opsonization of z-cHOClate
2.6.10. pH and HOCl measurements
2.6.11. Fluorescence microscopy
2.6.12. Mapping HOCl levels in innate immune cells
3. Summary
Acknowledgments
References
Chapter 11: The intracellular metabolome of starving cells
1. Introduction
2. Materials
2.1. Disposables
2.2. Equipment
2.3. Software
2.4. Consumables
2.5. Reagents
3. Methods
3.1. Sample preparation
3.2. Targeted and untargeted analysis
3.3. Widely-targeted analysis of intracellular metabolites with gas chromatography (GC) coupled to triple quadrupole (QQQ ...
3.4. Targeted analysis of bile acids by high performance liquid chromatography (HPLC) coupled to a triple quadrupole (QQQ ...
3.5. Targeted analysis of polyamines by ion pairing high performance liquid chromatography (HPLC) coupled to a triple qua ...
3.6. Targeted analysis of short chain fatty acids by high performance liquid chromatography (HPLC) coupled to a triple qu ...
3.7. Targeted analysis of oxylipins by ion pairing high performance liquid chromatography (HPLC) coupled to a 6500+ QTRAP ...
3.8. Targeted analysis of lipids by high performance liquid chromatography (HPLC) coupled to a 6500+ QTRAP Mass spectrometer
3.9. Targeted analysis of sugar phosphates by ion pairing high-performance liquid chromatography (HPLC) coupled to a trip ...
3.10. Untargeted analysis of intracellular metabolites by ultra-high performance liquid chromatography (UHPLC) coupled to ...
3.11. Untargeted analysis of intracellular metabolites by ultra-high performance liquid chromatography (UHPLC) coupled to ...
3.12. Post-acquisition processing of targeted acquisition
3.13. Post-acquisition processing of the untargeted acquisition
3.14. Stable isotope labeling
3.15. Targeted analysis of isotope labeled metabolites
3.16. Labeling experiment by targeted analysis of by gas chromatography (GC) coupled to a triple quadrupole (QQQ) mass sp ...
4. Quality control
4.1. Signal to noise monitoring
4.2. Quality control pool
4.3. Chromatographic system rinsing and storage
5. Concluding remarks
6. Notes
References
Chapter 12: Quantitative determination of autophagy flux by probes
1. Introduction
2. Material
2.1. Disposables
2.2. Equipment
2.3. Reagents
2.4. Breast cancer cell culture
2.5. Sample preparation and immunostaining
2.6. Image acquisition and data analysis
3. Concluding remarks
4. Notes
References
Chapter 13: Assessment of mammalian endosomal microautophagy
1. Introduction
2. Biochemical procedures for the assessment of eMI activity in mammals
2.1. Isolation of LE/MVB from mouse liver
2.2. Detection of endogenous eMI substrates in isolated LE/MVBs
2.3. In vitro reconstitution of eMI with isolated LE/MVBs
3. Image-based procedures for assessment of eMI activity in cultured cells
3.1. Generation of cells stably expressing eMI reporters
3.2. Assessing KFERQ-selective and in bulk eMI in reporter-expressing cells
4. Criteria to identify a protein as potential eMI substrate
5. Concluding remarks
Acknowledgments
References
Chapter 14: Measuring oxidation within LC3-associated phagosomes that optimizes MHC class II restricted antigen presentation
1. Introduction
2. Materials
2.1. Disposables
2.2. Equipment
2.3. Reagents
3. Protocol I: Measurement of the oxidation level within LAPosomes in human macrophages
3.1. Human macrophage differentiation from monocytes isolated from peripheral blood mononuclear cells (PBMCs)
3.2. Zymosan coating with OxyBURST to stimulate human macrophages
3.2.1. Zymosan coating with OxyBURST
3.2.2. Stimulation of human macrophages with Zymosan-OxyBURST
3.3. Immunostaining of human macrophages stimulated with Zymosan-OxyBURST
3.4. Confocal laser scanning microscopy and data analysis of LAPosome oxidation
3.4.1. Acquisition
3.4.2. Analysis of the oxidation level within a LAPosome
4. Protocol II: Antigen presentation assay using whole blood CD4+ T cells
4.1. Selection of human macrophages and their autologous CD4+ T cells
4.2. Antigen presentation assay using whole blood memory CD4+ T cells
5. Concluding remarks
6. Notes
Acknowledgments
References

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Monitoring Vesicular Trafficking in Cellular Responses to Stress

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