دانلود کتاب Mitochondrial Bioenergetics: Methods and Protocols (Methods in Molecular Biology, 2878)

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Preface
Contents
Contributors
Chapter 1: Evaluation of Respiration with a Clark-Type Electrode in Isolated Mitochondria, Intact and Permeabilized Cells, and...
1 Introduction
2 Materials
2.1 Cell Culture/Preparation and Permeabilization
2.1.1 Electropermeabilized Cells-Neutrophils (Adapted from)
2.1.2 Saponin-Permeabilization-Muscle Fibers (Adapted from)
2.1.3 Digitonin-Permeabilization-Lymphocytes (Adapted from)
2.2 Isolation of Mitochondrial Fraction (from Three Different Organs)
2.3 Intact Cells (Adapted from)
2.4 Embryonic Lungs from Chicken (Adapted from)
2.5 Oxygen Consumption Assays Targeted to Mitochondria-Solutions
2.5.1 Permeabilized Animal Cells
2.5.2 Isolated Mitochondria
2.6 Oxygen Consumption Assays in Intact Cells and Tissues-Solutions
2.7 Oxygen Electrode
2.7.1 Oxygen Electrodes and Polarographic Systems
2.7.2 Oxygen Permeable Membranes
3 Methods
3.1 Cell Culture and Permeabilization
3.1.1 Electropermeabilized Cells-Neutrophils (Adapted from)
3.1.2 Saponin-Permeabilization-Muscle Fibers (Adapted from)
3.1.3 Digitonin-Permeabilization-Lymphocytes (Adapted from)
3.2 Isolation of Mitochondrial Fractions
3.2.1 Mitochondrial Isolation from the Brain (Adapted from)
3.2.2 Mitochondrial Isolation from the Heart (Adapted from)
3.2.3 Mitochondrial Isolation from the Liver (Adapted from)
3.3 Intact Mice Sertoli Cells Culture and Preparation (Adapted from)
3.4 Embryonic Chicken Lung Dissection and Explantation (Adapted from)
3.5 Oxygen Electrodes Preparation and Maintenance
3.6 Modulators of Mitochondrial Complexes
3.7 Modulators of Energy Metabolism
3.8 Oxygen Consumption Assays
3.8.1 Permeabilized Animal Cells (Adapted from)
3.8.2 Isolated Mitochondria (Adapted from)
3.8.3 Intact Cells (Adapted from)
3.8.4 Embryonic Chick Lung Explants (Adapted from)
3.9 Respiratory Parameters and Calculations of Respiratory Rates
3.9.1 Exogenously Fed Mitochondria
3.9.2 Intact Cell Respiratory Parameters
3.9.3 Tissue/Explants Respiratory Parameters
4 Notes
5 Conclusion
References
Chapter 2: High-Resolution Respirometry Methodology for Bioenergetic and Metabolic Studies in Intact Brain Slices
1 Introduction
2 Materials
2.1 Reagents and Solutions
2.2 Media
2.2.1 Substrate, Inhibitor, and Uncoupler Stock Solutions
2.2.2 Sample Holder
2.2.3 Preparation of Hippocampal Slices (Rat and Mouse)
3 Methods
3.1 Preparation of Hippocampal Slices
3.2 Preparation of O2k for Experiments
3.3 High-Resolution Respirometry
3.3.1 Substrate-Uncoupler-Inhibitor Titration (SUIT) Protocol for Intact Brain Slices
3.3.2 Evaluation of Tissue Viability
3.3.3 Propidium Iodide Staining
3.4 Concluding Remarks
4 Notes
References
Chapter 3: Plate-Based Measurement of Respiration by Isolated Mitochondria and by Intact Cells
1 Introduction
2 Materials
2.1 Mitochondrial Isolation from Rat Skeletal Muscle
2.2 Mitochondrial Oxygen Consumption and Acidification Measurement
2.3 Mitochondrial Compound Preparation
2.4 Cell Culturing
2.5 Intact Cells Oxygen Consumption and Extracellular Acidification Measurements
2.6 Intact Cells Compound Preparation
3 Methods
3.1 Isolating Mitochondria from Rat Skeletal Muscle
3.2 Preparing the XF Assay Cartridge for Isolated Mitochondrial Measurement
3.3 Setting Up an Isolated Mitochondrial Sample Plate for the XF Assay
3.4 Respiratory Control Assay of Isolated Mitochondria
3.5 Electron Flow Assay in Isolated Mitochondria
3.6 Intact Cell Respiratory Control Assay
3.7 Intact Cell Respiratory Control Assay
4 Notes
References
Chapter 4: Metabolic Phenotyping of Synaptic Mitochondria Using MitoPlates and Synaptoneurosomes
1 Introduction
2 Materials
2.1 Preparation of Synaptoneurosomes
2.2 Measurement of Absorbance with MitoPlates
2.3 Determination of Protein Concentration
3 Methods
3.1 Isolation of Synaptoneurosomes
3.2 Mitochondrial Function Assay with MitoPlates
3.3 Protein Concentration Assessment
4 Notes
References
Chapter 5: Characterization of the Mitochondria Function and Metabolism in Skin Fibroblasts Using the Biolog MitoPlate S-1
1 Introduction
2 Materials
2.1 Equipment and Consumables
2.2 Cell Culture Medium Preparation for NHDF Cells
2.3 Preparation of Saponin (24x) Stock Solution
2.4 Preparation of 1x Biolog MAS for Cell (Re)suspension
3 Methods
3.1 Dissolving the Substrates in the MitoPlate S-1
3.2 Preparation of the Cell Suspension
3.3 MitoPlate S-1 Assay Using the Biolog OmniLog
3.3.1 Planning a Batch (See Note 7)
3.3.2 Loading a Batch
3.3.3 Unloading Mito Plates
3.4 Step by Step Analysis of OmniLog-Generated Data
3.4.1 File List
3.4.2 Loading the Files
3.4.3 Calculating Kinetic Parameters
3.5 Dataframe Assembly
3.6 Data Analysis with Google Sheets
3.6.1 Organizing Data with Pivot Tables
3.6.2 Creating Heatmaps with Conditional Formatting
3.6.3 Identifying and Excluding Outliers
4 Notes
References
Chapter 6: Measurement of Mitochondrial ROS Formation
1 Introduction
2 Materials
2.1 Measurement of ROS in Isolated Mitochondria
2.2 Measurement of ROS in Intact Cells
3 Methods
3.1 Measurement of ROS in Isolated Mitochondria
3.1.1 Isolation of Mouse Heart Mitochondria
3.1.2 Generation of H2O2 Calibration Curve
3.1.3 Measurement of H2O2 Formation in Isolated Mitochondria with Amplex Red
3.2 Measurement of ROS in Intact Cells
3.2.1 Measurement of ROS Formation with Small Molecule Fluorescent Probes
3.2.2 Measurement of ROS Production with Genetically Encoded Fluorescent Probe HyPer7
4 Notes
References
Chapter 7: Mitochondrial Membrane Potential (ΔΨ) Fluctuations Associated with the Metabolic States of Mitochondria
1 Introduction
1.1 Mitochondrial Membrane Potential (Δψ)
1.2 Estimation of Δψ
1.3 Estimation of Δψ Using Cationic Fluorescent Dyes
2 Materials
2.1 Reagents and Buffers
2.2 Isolation of Mitochondrial Fraction Solutions
2.3 Preparation of the TPP+ Electrodes
2.4 Measurement of ΔΨ Using TMRM
3 Methods
3.1 Isolation of Rat Liver Mitochondria
3.2 ΔΨ Fluctuations Associated to the Phosphorylation-Dephosphorylation Cycle
3.2.1 Effect of FCCP or KCN
3.2.2 Effect of Phosphate
3.2.3 Effect of Nigericin
3.2.4 Energization of Mitochondrial Membrane by ATP
3.3 Effects of Inhibitors of the TCA Cycle on Membrane Potential
3.3.1 Effect of Salicylate or Br-Succinimide
3.3.2 Effect of Malonyl-CoA
3.3.3 Effect of CoASH
3.4 Effects of Transportable Metabolites on ΔΨ
3.4.1 Effect of Carboxyactractyloside
3.4.2 Effect of Ruthenium Red
3.4.3 Effect of Oligomycin A
3.4.4 Effect of Co-transport
3.5 Effect of Oxidants on the Induction of the Mitochondrial Permeability Transition
3.5.1 Effect of NEM, DTT, or CyA
3.6 Measurement of ΔΨ Using TMRM in Isolated Rat Liver Mitochondria Using a Microplate Reader
3.7 Measurement of ΔΨ Using TMRM in Mammalian Cells Using Flow Cytometry
3.7.1 Cell Staining with TMRM
3.7.2 Flow Cytometry Analysis of Δψ
4 Notes
References
Chapter 8: The Relation Between Mitochondrial Membrane Potential and Reactive Oxygen Species Formation
1 Introduction
2 Materials
2.1 Isolation of Crude Mitochondria from Mouse Brain
2.2 Cell Cultures
2.3 Measurement of Reactive Oxygen Species Production in Isolated Mitochondria
2.4 Measurement of Hydrogen Peroxide Production in Isolated Mitochondria by Peroxidase/Amplex Red Assay
2.5 Measurement of the Mitochondrial Transmembrane Potential in Isolated Mitochondria
2.6 Determination of Protein Concentration in Isolated Mitochondria
2.7 Measurement of the Oxygen Consumption in Ehrlich Ascites Tumor Cells
2.8 Fluorometric Measurement of the Mitochondrial Membrane Potential in Ehrlich Ascites Tumor Cells
2.9 Fluorometric Measurement of Reactive Oxygen Species Production in Ehrlich Ascites Tumor Cells
2.10 Measurement of Mitochondrial Membrane Potential in HeLa Cells Using a Confocal Microscope
2.11 Measurement of Reactive Oxygen Species Production in HeLa Cells Using a Confocal Microscope
2.12 Measurement of Mitochondrial Superoxide Production in HeLa Cells Using a Confocal Microscope
2.13 Measurement of Cytosolic Calcium in HeLa Cells Using a Confocal Microscope
2.14 Measurement of the NAD(P)H-Dependent Dehydrogenases Activity in Human Fibroblasts by Resazurin Assay
2.15 Measurement of the Mitochondrial Transmembrane Potential in Human Fibroblasts
2.16 Measurement of Mitochondrial Superoxide Level in Human Fibroblasts
2.17 Measurement of the Cytosolic Superoxide Level in Human Fibroblasts
2.18 Measurement of Reactive Oxygen Species Production in Human Fibroblasts
2.19 Sulforhodamine B (SRB) Assay for Determination of Cellular Protein Content After Bioenergetics and ROS Levels Measurements
3 Methods
3.1 Isolation of Crude Mouse Brain Mitochondria for Measurement of ΔΨ and ROS Production
3.2 Measurement of Reactive Oxygen Species Production in Isolated Brain Mitochondria Using CM-H2DCFDA
3.3 Hydrogen Peroxide Measurement in Isolated Brain Mitochondria by Peroxidase/Amplex Red Assay
3.4 Measurement of the Mitochondrial Membrane Potential in Isolated Brain Mitochondria Using Safranine O
3.5 Measurement of Protein Concentration in Isolated Brain Mitochondria
3.6 Measurement of Oxygen Consumption in Ehrlich Ascites Tumor Cells
3.7 Fluorometric Measurement of Mitochondrial Membrane Potential in Ehrlich Ascites Tumor Cells Using TMRM
3.8 Fluorometric Measurement of Reactive Oxygen Species Production in Ehrlich Ascites Tumor Cells Using CM-H2DCFDA
3.9 Measurement of Mitochondrial Membrane Potential in HeLa Cells Using a Confocal Microscope
3.10 Measurement of Reactive Oxygen Species Production in HeLa Cells Using a Confocal Microscope
3.11 Measurement of Mitochondrial Superoxide Level in HeLa Cells Using a Confocal Microscope
3.12 Simultaneous Measurement of the Mitochondrial Membrane Potential and Cytosolic Calcium in HeLa Cells Using a Confocal Mic...
3.13 Simultaneous Measurement of the Mitochondrial Membrane Potential and Reactive Oxygen Species Production in HeLa Cells Usi...
3.14 Measurement of the Mitochondrial Membrane Potential in Human Fibroblasts Using JC-1
3.15 Measurement of the NAD(P)H-Dependent Dehydrogenases Activity in Human Fibroblasts by Resazurin Assay
3.16 Measurement of Mitochondrial Superoxide Level in Human Fibroblasts Using MitoSox Red
3.17 Measurement of Cytosolic Superoxide Level in Human Fibroblasts Using DHE
3.18 Measurement of Reactive Oxygen Species Production in Human Fibroblasts Using CM-H2DCFDA
3.19 Sulforhodamine B Assay (SRB) for Determination of Cellular Protein Content After Bioenergetics and ROS Levels Measurements
4 Notes
References
Chapter 9: Creating Physiological Cell Environments In Vitro: Adjusting Cell Culture Media Composition and Oxygen Levels to In...
1 Introduction
1.1 Importance of In Vitro Models
1.2 In Vitro and Ex Vivo Models in Biomedicine
1.3 Human Dermal Fibroblasts as a Valuable Cell Model for Pre-clinical Research
1.4 Importance and Impact of Cell Culture Medium in Mitochondrial Research
1.5 Inducing Metabolic Remodeling by Adjusting Extracellular Glucose Levels
1.6 Mimicking the Tumor Microenvironment In Vitro
2 Materials
2.1 Inducing NHDF Metabolic Remodeling by Adjusting Extracellular Glucose Levels
2.1.1 NHDF Culture and Assays
2.2 Mimicking the Tumor Microenvironment In Vitro
2.2.1 Measuring Pericellular O2 Levels During Hypoxia Experiments
2.2.2 Tumor Microenvironment Medium (TMEM)
2.2.3 Routine Cell Culture of Mouse Pancreatic Ductal Carcinoma Cells (KCPY) in TMEM
2.2.4 Rapid Quantification of Glucose Using the GlucCell Glucose Monitor During an Experiment
2.2.5 Monitoring Glucose, L-Lactate, L-Glutamine and L-Glutamate Concentrations During an Experiment
2.2.6 Continuous pH Monitoring During an Experiment
3 Methods
3.1 Inducing NHDF Metabolic Remodeling by Adjusting Extracellular Glucose Levels
3.1.1 NHDF Thawing Procedure
3.1.2 Maintenance of NHDF Cultures
3.1.3 Cell Freezing Procedure
3.1.4 Adaptation of NHDF Cells to Low Glucose and OXPHOS-Promoting Media
3.1.5 Procedures to Confirm NHDF Metabolic Reconfiguration
Real-Time Oxygen Consumption Rate (OCR) Monitoring Using Resipher (Lucid Scientific)
Normalization-Nuclei Count
3.2 Mimicking the Tumor Microenvironment In Vitro
3.2.1 Preparation of TMEM Stock Solutions and Fresh TMEM
Stock Solution 1: 100x Amino Acid Stock, 200 mL
Stock Solution 2: (1000x), 100 mL
Stock Solution 3: (1000x), 20 mL
Stock Solution 4: (500x), 40 mL
Stock Solution 5: (100x), 100 mL
Stock Solution 6: (50x), 200 mL
Stock Solution 7: (500x), 200 mL
Fresh TMEM (Complete), 500 mL
3.2.2 The Effect of Cell Seeding Density on Pericellular Oxygen Levels During Hypoxia
3.2.3 Thawing KPCY Cells into TMEM
3.2.4 Monitoring Media Metabolite Levels Using GlucCellTM and YSI Bioanalyzer
Monitoring Glucose Concentrations Using GlucCell
Monitoring Glucose, Lactate, Glutamine, and Glutamate Concentrations Using a YSI 2900 Biochemistry Analyzer
3.2.5 Real-Time pH Monitoring in Cultured Cells
4 Notes
4.1 Glucose
References
Chapter 10: Imaging Mitochondrial Axonal Transport in Human Induced Pluripotent Stem Cell-Derived Neurons
1 Introduction
2 Materials
2.1 Maintenance of Human Induced Pluripotent Stem Cells (iPSCs)
2.2 Medium Spiny Neurons (MSNs) Differentiation and Maintenance
2.3 Mitochondrial Labeling with Mito-DsRed2
3 Methods
3.1 Medium Spiny Neurons Induction and Patterning
3.2 Labelling of Mitochondria
3.3 Imaging Transport of Mitochondria
3.4 Analysis of Mitochondria Transport
4 Note
References
Chapter 11: Understanding Mitochondrial and Lysosomal Dynamics by Fluorescent Microscopy
1 Introduction
2 Materials
2.1 Cell Culture, Mitochondrial and Lysosomal Staining, and Image Acquisition
2.2 Software for Image Analyses
3 Methods
3.1 Cell Culture, Mitochondrial, and Lysosomal Staining
3.2 Image Acquisition by Spinning Disk Confocal Microscope
3.3 Images Processing and Analysis
3.3.1 2D Analysis of Mitochondrial Morphology and Network Connectivity
3.3.2 3D Analysis of Mitochondrial Morphology and Network Connectivity
3.3.3 2D and 3D Analyses of Lysosomal Morphology
4 Notes
References
Chapter 12: Mitochondrial Morphofunctional Profiling in Primary Human Skin Fibroblasts Using TMRM and Mitotracker Green Co-sta...
1 Introduction
2 Materials
2.1 Cells
2.2 Medium
2.3 Solutions
2.4 Reagents
2.5 Instruments and Equipment
2.6 Software
3 Methods
3.1 Cell Staining and Epifluorescence Microscopy Imaging
3.2 Image Processing and Quantification of Mitochondrial Morphology
4 Notes
References
Chapter 13: Absolute Quantification of Cellular and Cell-Free Mitochondrial DNA Copy Number from Human Blood and Urinary Sampl...
1 Introduction
2 Materials
2.1 Blood Collection and Storage
2.1.1 Collection of Whole Blood
2.1.2 Separation of PBMCs and Plasma from Whole Blood
2.1.3 Separation of Serum from Whole Blood
2.2 Urine Collection and Storage
2.2.1 Separation of Urinary Pellet and Supernatant from Whole Urine
2.3 DNA Extraction
2.3.1 DNA Extraction from Whole Blood and PBMCs
2.3.2 DNA Extraction from Plasma and Serum
2.3.3 DNA Extraction from Whole Urine
2.3.4 DNA Extraction from Urinary Supernatant
2.4 Reference Standards Preparation
2.5 qPCR
2.6 Equipment Used
3 Methods
3.1 Collection and Storage of Samples
3.1.1 Collection of Whole Blood
3.1.2 Collection of Urine
3.2 Extraction of DNA from Blood Samples
3.2.1 DNA Isolation from Whole Blood and PBMCs
3.2.2 DNA Isolation from Plasma and Serum
3.3 DNA Extraction from Urine Samples
3.3.1 DNA Extraction from Whole Urine
3.3.2 DNA Extraction from Urinary Supernatant
3.4 Pre-treatment/Fragmentation of DNA (See Note 5)
3.5 Preparation of Reference Standards for Absolute Quantification
3.6 Real-Time qPCR
3.7 How to Analyze Your Results and Check Specificity/Accuracy of the Experiment
3.8 Calculation of the Sample Size to Adequately Power the Study
3.9 Conclusion
4 Notes
References
Chapter 14: Isolation and Analysis of Mitochondrial Small RNAs from Rat Liver Tissue and HepG2 Cells
1 Introduction
2 Materials
2.1 Isolation of Mitochondrial Fraction
2.1.1 Mitochondrial Isolation from Liver Tissue
2.2 Mitochondrial Isolation from HepG2 Cells (Based on Ref.)
2.3 Immunoblotting
2.4 RNA Isolation
2.5 RNA Quality Control
2.6 miRNA Expression Profiles
2.7 Identification of Stable Reference MicroRNAs
3 Methods
3.1 Isolation of Mitochondrial Fraction
3.1.1 Mitochondrial Isolation from Liver Tissue (Adapted from Ref.)
3.1.2 Mitochondrial Isolation from HepG2 Cells (Adapted from Ref.)
3.2 Immunoblotting
3.3 RNA Isolation
3.4 RNA Quality Control
3.5 miRNA Expression Analysis
3.6 Identification of Potential Reference Genes
4 Notes
References
Chapter 15: Methodologies for Mitochondrial Omic Profiling During Spaceflight
1 Introduction
2 Materials
2.1 Plasma RNA Purification and Quality Check
2.2 Single-Cell Multi-omic Analysis of Astronaut Peripheral Blood Mononuclear Cells (PBMCs)
2.2.1 Blood Collection and PBMCs Isolation
2.2.2 Nuclei Isolation
2.2.3 Single-Cell Multi-Ome Library Preparation
2.2.4 Sequencing
2.3 293 T, A549, and THP-1 Cell Culturing
2.4 Inflammasome Reconstitution System in A549- or 293 T-Cells
2.5 Detection of Cell-Free/Cytosolic mtDNA and NLRP3-I-Activated IL-1β Reagents
2.5.1 Immunoblotting Reagents / Antibodies/ELISAs
2.5.2 Cell-Free/Cytosolic mtDNA Detection Reagents
2.5.3 Reagents in Common
2.5.4 Drugs
3 Methods
3.1 Recovery and Aliquoting Plasma Sample for Human Studies
3.2 Dissolving Plasma into TRIzol-LS for Human Studies
3.3 Recovery and Aliquoting of Plasma for Mouse Studies
3.4 Dissolving Plasma into TRIzol-LS for Mouse Studies
3.5 RNA Purification from Plasma Dissolved in TRIzol
3.6 RNA Sequencing Library Preparation
3.7 Small RNA Sequencing Library Preparation
3.8 Specific Mitochondrial Analysis on Bulk RNA-Seq Data
3.9 Isolating PBMCs from Astronauts
3.10 Single-Cell Multi-ome (GEX + ATAC) Library Preparation
3.11 Sequencing Single-Cell Multi-Ome Libraries
3.12 Specific Mitochondrial Analysis on Single-Cell Multi-Ome Data
3.12.1 Alignment
3.12.2 Processing Single-Cell Multi-ome Data
3.12.3 Gene Set Enrichment Analysis (GSEA) of the Mitochondrial Pathways
3.13 Detection of Mitochondrial-Released mtDNA and the NLRP3-I-Activated IL-1β Using In Vitro and In Vivo Models
3.13.1 Cell Culture of 293 T, A549 and THP-1 Cells
3.13.2 Detection of mtDNA in Cytoplasmic-Enriched Cell Fractions
3.13.3 Detection of Cell-Free-mtDNA in Bronchoalveolar Lavage Fluid (BALF) and Serum
3.14 Detection of NLRP3-I-Activated IL-1β in A549- or 293 T-Cells Expressing an Inflammasome Reconstitution System
3.14.1 Detection of Cleaved Caspase-1 Via Immunoblotting
3.14.2 Detection of Secreted IL-1B in A549- or 293 T-Cells Expressing an Inflammasome Reconstitution System Via ELISA
3.14.3 Detection of Secreted IL-1β from Mouse or Human BALF/Serum Via ELISA
3.14.4 Detection of Secreted IL-1B from THP1-Monocyte-Derived Macrophages
4 Notes
References
Index