Mitochondria: Methods and Protocols (Methods in Molecular Biology, 2497)

✍ Scribed by Namrata Tomar (editor)

• Publisher: Humana
• Year: 2022
• Tongue: English
• Leaves: 433
• Category: Library

✦ Synopsis

This volume details comprehensive protocols and methodologies to assess mitochondrial bioenergetics and dynamics in different tissues and cells involving health and pathological states. Chapters guide readers through methods for assessment of the energy metabolism including Oxygen Consumption Rate (OCR), mitochondrial membrane potential, and measuring mitochondrial Ca²⁺ handling, and ROS emission. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting, and systematic reproducible protocols.

Authoritative and cutting-edge, Mitochondria: Methods and Protocols aims to be a foundation for future studies and to be a source of inspiration for new investigations in the field.

✦ Table of Contents

Preface
Content and General Outline of the Book
Contents
Contributors
Chapter 1: Fast Determination of Mitochondrial Metabolism and Respiratory Complex Activity in Permeabilized and Intact Cells
1 Introduction
2 Materials
2.1 Buffers for Respirometry
2.1.1 Permeabilized Cells
2.1.2 Intactg Cells
2.2 Potentiometric Medium (PM)
3 Methods
3.1 Assessment of Respiratory Complex Activity
3.1.1 Permeabilized Cells
3.1.2 Intact Cells
3.2 Determination of Mitochondrial Membrane Potential
3.3 Determination of NADH Content
4 Notes
References
Chapter 2: Unbiased Millivolts Assay of Mitochondrial Membrane Potential in Intact Cells
1 Introduction
2 Equipment
3 Materials
3.1 Fluorescence Probes
3.2 Assay Media Components
3.3 Calibrant Components
3.4 Cell Culturing Reagents (Optional)
3.5 Consumables
4 Methods
4.1 Task #1: Pilot Experiments
4.2 Task #2: Additions Design
4.3 Task #3: Potentiometric Media and Calibrants
4.4 Task #4 Assay Plate of Cultured Adherent Cells
4.5 Task #5 Alternative: Assay Plate of Immobilized Cells
4.6 Task #6: Preparation of Treatment Additions
4.7 Task#7: Final Wash of the Assay Plate
4.8 Task #8: Potentiometric Recording
4.9 Task #9: Image Analysis
4.10 Task #10: Analysis of Fluorescence Time Courses
4.11 Task #11 Automated Analysis
4.12 Accessory Task #12: Spectral Crossbleed
4.13 Accessory Task #13: Mitochondria:Cell Volume Fraction
4.14 Accessory Task #14: Apparent Activity Coefficient Ratio
5 Notes
6 Troubleshooting
References
Chapter 3: Rotenone Decreases Ischemia-Induced Injury by Inhibiting Mitochondrial Permeability Transition: A Study in Brain
1 Introduction
2 Materials
3 Methods
3.1 Animals and Experimental Protocols for Rotenone Treatment and Brain Ischemia
3.2 Isolation of Mitochondria from Rat Cortex and Cerebellum
3.3 Measurement of Calcium Retention Capacity (CRC) in Isolated Brain Mitochondria
3.4 Assessment of Mitochondrial Respiration Rate
3.5 Evaluation of the Activity of Complex I of Mitochondrial Respiratory Chain
3.6 Evaluation of the Activity of Complex II of Mitochondrial Respiratory Chain
3.7 Assessment of H2O2 Generation in Isolated Brain Mitochondria
4 Notes
References
Chapter 4: Assessment of Mitochondrial Complex II and III Activity in Brain Sections: A Histoenzymological Technique
Abbreviations
1 Introduction
2 Materials
2.1 Histoenzymology of Mitochondrial Complex II (Succinate Dehydrogenase, SDH) Activity
2.1.1 Solutions and Reagents
2.2 Histoenzymology of Mitochondrial Complex III (CoenzymeQ-Cytochrome c Oxidoreductase) Activity
2.2.1 Solutions and Reagents
3 Method
3.1 Histoenzymology of Mitochondrial Complex II (Succinate Dehydrogenase, SDH) Activity
3.2 Histoenzymology of Mitochondrial Complex III (CoenzymeQ-Cytochrome c Oxidoreductase) Activity
4 Notes
References
Chapter 5: Measurement of Mitochondrial (Dys)Function in Cellular Systems Using Electron Paramagnetic Resonance (EPR): Oxygen ...
1 Introduction
2 Materials
2.1 EPR Spectrometer
2.2 EPR Oximetry
2.3 Superoxide Measurements
2.4 Mitochondrial Isolation
2.5 Mitotoolbox
3 Methods
3.1 EPR Oximetry on Whole Cell Model
3.2 Superoxide Measurement on Whole Cell Model
3.3 Mitochondrial Isolation
3.4 Mitotoolbox: OCR and Superoxide Simultaneous Measurement
4 Notes
References
Chapter 6: Mitochondrial Calcium Handling in Isolated Mitochondria from a Guinea Pig Heart
1 Introduction
2 Materials
3 Methods
3.1 Isolation of Mitochondria from Guinea Pig Heart Using Differential Centrifugation
3.2 Measurement of Mitochondrial Respiration To Establish the Viability of the Isolated Mitochondria
3.2.1 Measurement of Calcium Retention Capacity of Mitochondria
3.2.2 Measurement of Calcium Induced ΔΨm Loss in Isolated Mitochondria
4 Notes
References
Chapter 7: Characterizing the Electron Transport Chain: Structural Approach
1 Introduction
2 Materials
2.1 Isolation of Mitochondrial Complex (See Note 1)
2.2 Gradient Gel Electrophoresis
2.3 Immunoblotting
2.4 Gel Assay
3 Methods
3.1 Isolation of Mitochondrial from Mice Tissues (See Note 3)
3.2 Gel Electrophoresis
3.3 Immunoblotting
3.4 Gel Assay
4 Notes
References
Chapter 8: Characterizing the Electron Transport Chain: Functional Approach Using Extracellular Flux Analyzer on Mouse Tissue ...
1 Introduction
2 Materials
2.1 Surgical Instruments
2.2 Media and Reagents
2.3 Bioenergetics Measurement
3 Methods
3.1 Tissue Sample Preparation
3.2 Mitochondria Isolation
3.3 Extracellular Flux Analysis
4 Notes
5 Optimization of Assay Conditions in Mitochondria Isolated from Brain and Muscle
5.1 Optimize the Amounts of Isolated Mitochondria (Figs. 1, 2, 3, 4, and 5)
5.2 Optimize the Concentration of FCCP (Fig. 6)
References
Chapter 9: Simultaneous Acquisition of Mitochondrial Calcium Retention Capacity and Swelling to Measure Permeability Transitio...
1 Introduction
2 Materials
2.1 Mitochondrial Isolation Reagents, Buffers, and Equipment
2.2 CRC Assay Reagents and Equipment
3 Methods
3.1 Mitochondrial Isolation from Mouse Liver
3.2 CRC/Swelling Assay
3.3 Data Analysis and Quantification
4 Notes
References
Chapter 10: Measuring Mitochondrial Function: From Organelle to Organism
1 Introduction
2 Materials
2.1 Phosphorus Magnetic Resonance Spectroscopy
2.2 Isolated Mitochondria
2.3 Mitochondrial Fractional Volume
2.3.1 Cytochrome c Oxidase
2.3.2 Citrate Synthase
3 Methods
3.1 Phosphorus Magnetic Resonance Spectroscopy (31PMRS)
3.1.1 Protocols
3.1.2 Quantitative Analysis
3.2 Respirometry
3.2.1 Clark-Type Electrode Respirometers
3.2.2 High-Throughput Metabolic Flux Analyzers
3.2.3 Measurement of Oxygen Consumption Rates in Respirometry
3.2.4 Mitochondrial Respiratory States
3.3 Isolated Mitochondria
3.3.1 Mitochondrial Isolation
3.3.2 Isolated Mitochondria ADP Sensitivity by Respirometry
3.4 Whole Cell Respirometry and Permeabilized Muscle Fibers
3.4.1 Whole Cell Respirometry
3.5 Permeabilized Fibers
3.6 Mitochondrial Fractional Volume
3.6.1 Cytochrome c Oxidase
Step 1: Determine the Degree and Stability of Cytochrome c Reduction (% Reduction)
Step 2: Quantify Cytochrome c Oxidase Activity Within Samples
3.6.2 Citrate Synthase
4 Notes
References
Chapter 11: Mitochondrial Toxicity of Organic Arsenicals
1 Introduction
2 Methods
References
Chapter 12: Measurements of Mitochondrial Respiration in Intact Cells, Permeabilized Cells, and Isolated Tissue Mitochondria U...
1 Introduction
2 Materials
2.1 All Assays
2.2 Mitochondrial Stress Test
2.3 Mitochondrial Coupling Assay (Permeabilized Cells)
2.4 Mitochondrial Coupling Assay (Isolated Tissue Mitochondria)
3 Methods
3.1 All Assays
3.1.1 One or More Days Before Assay
3.1.2 One Day Before Assay
3.1.3 Day of Assay
3.2 Mitochondrial Stress Test
3.2.1 One or More Days Before Assay
3.2.2 Day of Assay
3.3 Mitochondrial Coupling Assay (Permeabilized Cells)
3.3.1 One or More Days Before Assay
3.3.2 Day of Assay
3.4 Mitochondrial Coupling Assay (Isolated Mitochondria)
3.4.1 One or More Days Before Assay
3.4.2 Day of Assay
4 Notes
References
Chapter 13: Monitoring Mitochondrial Morphology and Respiration in Doxorubicin-Induced Cardiomyopathy
1 Introduction
2 Materials
2.1 Equipment
2.2 Cell Line
2.3 Reagents and Cell Culture Supplies
3 Methods
3.1 Mitochondrial Network Morphology Visualization in Dox-Treated Cardiomyocytes Through Confocal Fluorescence Microscopy
3.2 Mitochondrial Oxygen Consumption Rates Measurement and Bioenergetic Parameters Calculation in Cardiomyocytes
3.2.1 Cardiomyocytes Seeding in XF24 Cell Culture Plates and Doxorubicin Treatment
3.2.2 Sensor Cartridge Hydration and Inhibitors Loading into the Sensor Cartridge
3.2.3 Sensor Cartridge Calibration and Running the Assay
3.2.4 Mitochondrial Respiratory Parameters Analysis
4 Notes
References
Chapter 14: Creation of Yeast Models for Evaluating the Pathogenicity of Mutations in the Human Mitochondrial Gene MT-ATP6 and...
1 Introduction
2 Materials
2.1 Yeast Strains
2.2 Media
2.2.1 For Growing Yeast Strains
2.2.2 For Growing Bacteria
2.3 Site-Directed Mutagenesis of Plasmid DNA
2.4 Biolistic Transformation of Yeast Cells
2.4.1 Equipment
2.4.2 Reagents
2.5 Screening of Chemical Libraries
3 Methods
4 Notes
References
Chapter 15: In Vivo Analysis of Mitochondrial Protein Synthesis in Saccharomyces cerevisiae Mitochondrial tRNA Mutants
1 Introduction
2 Materials
2.1 Growth Media and Buffers for Labeling Reaction
2.2 SDS-PAGE Gel
3 Methods
3.1 Cells Preparation and In Vivo Labeling of Mitochondrial Proteins
3.2 Purification of Mitochondria and Mitochondrial Proteins Extraction
3.3 SDS-Polyacrylamide Gel Electrophoresis (PAGE)
3.4 Gel Preparation for Autoradiography
4 Notes
References
Chapter 16: Visualizing Mitochondrial Importability of a Protein Using the Yeast Bi-Genomic Mitochondrial-Split-GFP Strain and...
1 Introduction
2 Materials
2.1 PCR and Isothermal Assembly
2.2 BiG Mito-Split-GFP Transformation
2.3 BiG Mito-Split-GFP Growth
2.4 Epi-Fluorescence Microscopy
2.5 SDS-PAGE Electrophoresis, Western Blotting, and Immunodetection
3 Methods
3.1 Engineering the Plasmid Expressing the GFPβ11ch-Tagged Gene of Interest Using the Gibson Assembly Procedure
3.2 Transforming the BiG Mito-Split-GFP Strain
3.3 Culture of the Transformed BiG Mito-Split-GFP Strain
3.4 Visualizing Mitochondrial Importability of the GFPβ11ch-Tagged Protein
3.5 Verifying Expression of the GFPβ11ch-Tagged Protein and/or of the Mitochondrial GFPβ1-10 Fragment
4 Notes
References
Chapter 17: Analysis of Mitochondrial Performance in Lymphocytes Using Fluorescent Lifetime Imaging Microscopy
1 Introduction
2 Materials
2.1 Isolation of Lymphocytes
2.2 Cell Staining
2.3 Slide Preparation and Mounting
2.4 Microscopy and Analysis
3 Methods
3.1 Lymphocyte Isolation
3.2 Cell Staining in Solution: LIVE/DEAD Fixable Far Red Cell Stain and TMRM
3.3 Adhesion of Cells to Slide
3.4 Microscope Setup
3.5 Image Acquisition
3.6 FLIM Analysis
4 Notes
References
Chapter 18: Monitoring Mitochondrial Perturbations During Infection
1 Introduction
2 Materials
2.1 Visualization of Mitochondrial Morphology
2.2 Examination of Mitochondrial Function
2.2.1 ROS Assay
2.2.2 ATP Assay
2.3 Visualization of Mitophagy
3 Methods
3.1 Analysis and Quantification of Mitochondrial Fission and Fusion
3.2 Assessing ROS and ATP Levels
3.2.1 Examination of ROS Levels
3.2.2 Evaluation of ATP Levels
3.3 Evaluation of Cellular Mitophagy
4 Notes
References
Chapter 19: Measuring the Mitochondrial Ubiquinone (Q) Pool Redox State in Isolated Respiring Mitochondria
1 Introduction
2 Materials
2.1 Isolation of Mouse Heart Mitochondria
2.2 Assessing Respiratory Rates and Q Redox State
3 Methods
3.1 Isolation of Mouse Heart Mitochondria
3.2 Testing the Setup
3.3 Assessing Respiratory Capacity and Q Redox State
4 Notes
References
Chapter 20: Experimental Setup for Investigation of Acute Mitochondrial Oxygen Sensing in Primary Cells
1 Introduction
2 Materials
2.1 Laboratory Equipment
2.2 Cells
2.3 Chemicals
2.4 Perfusion and Temperature Control
2.5 Consumables
3 Methods
3.1 Preparations
3.2 Experimental Procedure
4 Notes
References
Chapter 21: Assessing the Redox Status of Mitochondria Through the NADH/FAD2+ Ratio in Intact Cells
1 Introduction
2 Materials
2.1 Equipment
3 Methods
3.1 Imaging
3.2 Data Analysis
4 Notes
References
Chapter 22: Monitoring Mitochondrial Membrane Potential in Live Cells Using Time-Lapse Fluorescence Imaging
1 Introduction
2 Materials
3 Methods
3.1 Seeding and Staining Skin Fibroblasts
3.2 Data Analysis
4 Notes
References
Chapter 23: Investigating Mitochondrial Ca2+ Dynamics in Isolated Mitochondria and Intact Cells: Application of Fluorescent Dy...
1 Introduction
2 Materials
2.1 Measuring Mitochondrial Ca2+ in Isolated Mitochondria Using a Fluorescence Plate Reader
2.2 Measuring Mitochondrial Ca2+ in Intact Cells Using Rhod-2, AM, or 2mtGcaMP6 with Fluorescence Microscopy
2.3 Measuring Mitochondrial Ca2+ in Intact Cells Using Mitoaequorin Using a Luminescence Plate Reader
3 Methods
3.1 Measuring Mitochondrial Ca2+ Uptake in Isolated Mitochondria Using an Extra-Mitochondrial Fluorescence Indicator
3.2 Measuring Mitochondrial Ca2+ in Intact Cells Using Rhod-2, AM, or 2mtGcaMP6 with Fluorescence Microscopy
3.3 Measuring Mitochondrial Ca2+ in Intact Cells Using the Bioluminescent Probe, Mitoaequorin
4 Notes
References
Chapter 24: A Plate Reader-Based Measurement of the Cellular ROS Production Using Dihydroethidium and MitoSOX
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 25: Analysis of Organization and Activity of Mitochondrial Respiratory Chain Complexes in Primary Fibroblasts Using Bl...
1 Introduction
2 Materials
3 Methods
3.1 Isolation of Mitochondria from Primary Fibroblasts
3.2 Solubilization of Mitochondrial Protein and BN-PAGE
3.3 In-Gel Activity Staining for CI, CII, and CIV
3.4 Immunoblotting
4 Notes
References
Chapter 26: Multiplexing Seahorse XFe24 and ImageXpress Nano Platforms for Comprehensive Evaluation of Mitochondrial Bioenerge...
1 Introduction
1.1 Rationale
1.2 Principle and Working
2 Materials
2.1 For Primary Neuronal Culture
2.2 For Seahorse XFe24
2.3 For Immunocytochemistry and Nuclear Labeling
2.4 For Imaging
2.5 For Operation and Data Analysis
3 Methods
3.1 Primary Neuronal Culture
3.1.1 Coating Cell Culture Plates (Day 0)
3.1.2 Culturing Primary Cortical Neurons (Day 1)
3.1.3 Cell Counting and Plating
3.1.4 Media Change (Day 3)
3.2 Assessing Bioenergetics in Dissociated Primary Cortical Neurons
3.2.1 Seahorse Extracellular Flux Assay Kit preparation (Hydrate Sensor Cartridge)
3.2.2 Seahorse XFe24 Cell Culture Plate Preparation
3.2.3 Drug Preparation
3.2.4 Bioenergetic Measurement
3.3 Immunocytochemistry and Nuclear Labeling
3.4 Imaging with ImageXpress Nano
3.5 Data Analysis
3.5.1 OCR and ECAR Determination
3.5.2 Neurite Length Measurement
3.6 Conclusion
4 Notes
References
Chapter 27: Computational Modeling of Mitochondria to Understand the Dynamics of Oxidative Stress
1 Introduction
2 Materials
3 Methods
3.1 Numerical Methods
3.2 Model Description for Oxidative Phosphorylation
3.2.1 Mitochondrial Complex I
3.2.2 ROS Production Across Complex I
3.2.3 Mitochondrial Complex II
3.2.4 Mitochondrial Complex III (Cytochrome Reductase)
3.2.5 Mitochondrial Complex IV
3.2.6 Mitochondrial Respiration-Driven Proton Pumps
3.2.7 ROS Generation and Regulation by Antioxidant Enzyme System
Modeling Scavengers for Mitochondrial and Extramitochondrial Superoxide Radical: Superoxide Dismutase
Modeling Scavengers for Mitochondrial and Extramitochondrial Hydrogen Peroxide
Modeling Regeneration of Mitochondrial and Extramitochondrial Glutathione (GSH) by Glutathione Reductase (GRX)
Modeling Source of Mitochondrial and Extramitochondrial NADPH
Model for Mitochondrial NADPH
Modeling Ionic Balance
Modeling the Relationship Between SNARF Fluorescence Intensity and Mitochondrial Proton Concentration
3.3 Model Validation-Simulation Results
3.3.1 Model Validation for ROS Production at Complex I
3.3.2 Model Validation for ROS Production at Complex III
3.3.3 ROS Production Attributed to Reverse Electron Transport: Is This Valid?
3.3.4 Role of Substrates (NADH and FADH2 Linked) in ROS Production in the Presence and Absence of Rotenone
3.3.5 Effect of Malonate on ROS Production
3.3.6 Elevated [Na+]i Results in Higher H2O2 Levels
3.3.7 Regulation of ROS: GSH/GSSG Ratio
3.3.8 Role of Mitochondrial Respiration-Driven Proton Pump in Mitochondrial Acidification and Recovery
4 Notes: Discussion
References
Correction to: Mitochondria
Index