Reactive Oxygen Species: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Introduction to In Silico Modeling to Study ROS Dynamics
1 Introduction
1.1 Computational Modeling
1.2 Modeling Purposes
1.3 The Scale of a Model
1.4 Overview of Modeling Approaches
2 Materials
2.1 Software Tools for Biologists
3 Methods
3.1 Review of Computational Models in ROS Biology
3.2 Protocol of Model Development Processes
3.2.1 Defining the Scope of a Model
3.2.2 Specifying Input, Elements, and Output
3.2.3 Equation Rate Settings
3.2.4 Parameter Calibration and Identification
3.2.5 Validation of Model Structure and Output
3.2.6 Verification of Model-Generated Hypotheses or Predictions
4 Notes
References
Chapter 2: Assessment of ROS Production in the Mitochondria of Live Cells
1 Introduction
2 Materials
3 Methods
3.1 Preparation of the Imaging Material
3.2 Confocal Imaging of Mitochondrial ROS Production and Lipid Peroxidation
3.2.1 Measurements of Mitochondrial ROS Production with MitoSOX Red
3.2.2 Measurements of Mitochondrial ROS Production with MitoTracker Red CM-H2XRos
3.2.3 Measurement of Mitochondrial Lipid Peroxidation with BODIPY™ 581/591 C11
References
Chapter 3: Measurement of Superoxide Production in Acute Hypoxia by Fixed-Cell Microscopy
1 Introduction
2 Materials
3 Methods
3.1 Superoxide Detection in Hypoxia
3.2 Superoxide Detection in Normoxia
3.3 Microscopy Imaging and Quantification
4 Notes
References
Chapter 4: A Photodynamic Tool to Promote a Sustained, ROS-Dependent Growth of Human Hair Follicles in Ex Vivo Culture
1 Introduction
2 Materials
2.1 Hair Follicle Maintenance
2.2 ROS Production and Inhibition
2.3 ROS Detection
3 Methods
3.1 Hair Extraction and Culturing
3.2 ROS Production and Monitoring
3.3 ROS Quantification
3.4 Hair Growth Monitoring and Quantification
4 Notes
References
Chapter 5: Singlet Oxygen and Protochlorophyllide Detection in Arabidopsis thaliana
1 Introduction
2 Materials
3 Methods
3.1 Detection of 1O2 Using SOSG
3.2 Direct Visualization of Pchlide Accumulation
3.3 Extraction and Quantification of Pchlide
4 Notes
References
Chapter 6: Characterization of Oxidative Lipidomics and Autophagy Induction in Chlamydomonas reinhardtii Under Abiotic Stress
1 Introduction
2 Materials
2.1 Chlamy- domonas Culture
2.2 Protein Extraction and Immunodetection of Autophagy Marker ATG8
2.3 Lipid Extraction, Chromatography, and Visualization of Lipid Bodies
3 Methods
3.1 Protein Extraction, Electrophoresis, and ATG8 Immunoblotting
3.2 Extraction of Lipids from Chlamydomonas
3.3 Chromato­graphic Separation of Lipids by TLC
3.4 Visualization of Lipid Droplets in Chlamydomonas
4 Notes
References
Chapter 7: Fluorescence Detection of Increased Reactive Oxygen Species Levels in Saccharomyces cerevisiae at the Diauxic Shift
1 Introduction
2 Materials
2.1 Yeast Cell Culture
2.2 Yeast Growth Measurements and Assessment of Cell Numbers
2.3 H2-DCFDA Treatment and Fluorescence Measurements
3 Methods
3.1 Growth of S. cerevisiae Cells in YPAD Medium
3.2 Monitoring Growth and Assessment of Yeast Cell Number
3.3 H2-DCFDA Treatment and Measurement of Fluorescence
4 Notes
References
Chapter 8: Flow Cytometry and Confocal Microscopy for ROS Evaluation in Fish and Human Spermatozoa
1 Introduction
1.1 Why Is It Important to Analyze ROS in Spermatozoa?
1.2 When Is It Important to Analyze ROS in Spermatozoa?
1.3 How Could ROS Be Evaluated in the Spermatozoa?
2 Materials
2.1 Preparatory Work (General)
2.2 Preparatory Work (Flow Cytometry)
2.3 Preparatory Work (Confocal Microscopy)
2.4 Sampling (Human Samples)
2.5 Sampling (Teleost Samples)
2.6 Sperm Sample Evaluation and Preparation
2.7 DCF Incubation
2.8 ROS Evaluation (Flow Cytometry)
2.9 ROS Evaluation (Confocal Microscopy)
3 Methods
3.1 Preparatory Work Before the Experiment (General)
3.2 Preparatory Work Before the Experiment (Flow Cytometry Approach)
3.3 Preparatory Work Before the Experiment (Confocal Microscopy Approach)
3.4 Sampling (Human Samples)
3.5 Sampling (Teleost Samples)
3.6 Sperm Sample Evaluation and Preparation
3.7 DCF Incubation
3.8 ROS Evaluation (Flow Cytometry Approach)
3.9 ROS Evaluation (Confocal Microscopy Approach)
4 Notes
References
Chapter 9: Measurement of Reactive Oxygen Species in Semen Samples Using Chemiluminescence
1 Introduction
2 Materials
2.1 Sample Collection
2.2 Equipment
2.3 Reagents
3 Methods
3.1 Sample Preparation
3.2 Luminometer Setup
3.3 Analysis of Results
4 Notes
References
Chapter 10: Methods to Unravel Pathways of Reactive Oxygen Species in the Photodynamic Inactivation of Bacteria
1 Introduction
2 Materials
2.1 Stock Solution of PS
2.2 Stock Solutions of Scavengers
2.3 Preparation of Culture Media
3 Methods
3.1 Preparation of S. aureus Cultures
3.2 Photosensitized Inactivation of S. aureus
3.3 In Vitro Photodynamic Mechanism of Action
3.3.1 Effect of Media on the Photoinactivation of S. aureus
Anoxic Conditions
Cell Suspensions in D2O
3.3.2 Effect of ROS Scavengers on the Photoinactivation of S. aureus
Cell Suspension in the Presence of NaN3
Cell Suspension in the Presence of DABCO
Cell Suspension in the Presence of d-Mannitol
3.3.3 O2(1Δg) Detection in Microbial Cells
Photooxidation of DMA in S. aureus Cells
Photooxidation of DMA in S. aureus Cells After a Washing Step
Photooxidation of DMA in S. aureus Cells in the Presence of NaN3
4 Notes
References
Chapter 11: Revealing ROS Production by Antibiotics and Photosensitizers in Biofilms: A Fluorescence Microscopy Approach
1 Introduction
2 Materials
2.1 Buffer and Growth Media
2.2 Imaging Chambers
3 Method
3.1 Culture Preservation
3.2 Screening of the Minimum Inhibitory Concentration of the Antibiotic/Photosensitizers in Planktonic Cells
3.3 Biofilm Formation
3.4 Detection of ROS
3.5 Antibiotic Treatment
3.6 PS Treatment
3.7 Real-Time Imaging of Fluorescence
3.8 Imaging Processing
4 Notes
References
Chapter 12: EPR Spectroscopy as a Method for ROS Quantification in the Skin
1 Introduction
2 Materials
2.1 Excised Porcine Skin
2.2 Chemicals
2.3 Irradiation Unit
2.4 EPR Spectrometer
2.5 Software
3 Methods
3.1 Skin Preparation for EPR Measurements
3.2 Measurements of Radicals Induced by UVA Irradiation by the X-Band EPR Spectrometer MS5000
3.3 Calculation of Radicals Induced by UVA Irradiation
3.4 Step-by-Step Guide for Calculating the Cumulative Radical Production
4 Notes
References
Chapter 13: Detection of Superoxide Radical in Adherent Living Cells by Electron Paramagnetic Resonance (EPR) Spectroscopy Using Cyclic Nitrones
1 Introduction
2 Materials
2.1 Cell Culture
2.2 EPR Spectroscopy
3 Methods
3.1 Cell Culture
3.2 EPR Analysis
4 Notes
References
Chapter 14: Singlet Oxygen Quantum Yield Determination Using Chemical Acceptors
1 Introduction
2 Materials
3 Methods
3.1 Mathematical Analysis Approach
3.2 1O2Quantum Yield Determination
4 Notes
References
Chapter 15: A General Method to Establish the Relative Efficiency of Different Sonosensitizers to Generate ROS for SDT
1 Introduction
1.1 Sonosensiti
1.2 Reactive Pathways (Inhibitors or Target Molecules)
1.2.1 Equations from the Kinetic Scheme
2 Materials
3 Methods
3.1 Photosynthesis of DPAO2 (Sref, Sonosensitizer of Reference)
3.2 Sonolysis of DPAO2 or Other Sonosensitizers
3.3 The Sonosensitizer Efficiency
3.3.1 Measurement of the Absolute Sonosensitizer Efficiency (φ1O2)
3.3.2 Measurement of the Relative Stationary Singlet Oxygen Concentration Generated by Different Sonosensitizer (Relative Efficiency)
4 Notes
References
Chapter 16: Lipid Peroxidation Assay Using BODIPY-Phenylbutadiene Probes: A Methodological Overview
1 Introduction
2 Lipid Peroxidation
3 BODIPY-Based Methods
4 Accumulation, Oxidation, and Localization Sites
5 Methodological Design
6 Delayed Detection of Peroxidative Damage
7 Pitfalls from Probe Properties
7.1 Color Changes
7.2 Fluorescence Fading
8 Conclusion
References
Chapter 17: The Use of Fluorescent Probes to Detect ROS in Photodynamic Therapy
1 Introduction
1.1 Fluorescence Detection Methods for Different ROS
1.2 Singlet Oxygen
1.3 Hydroxyl Radicals
1.4 Hydrogen Peroxide
1.5 Superoxide
1.6 Scavengers and Quenchers
2 Materials
3 Methods
3.1 Singlet Oxygen Detection Using SOSG
3.2 Hydroxyl Radical Detection Using 4-Hydroxyphenyl-Fluorescein (HPF)
3.3 Hydrogen Peroxide Detection Using Amplex Red
3.4 Superoxide Detection Using XTT Reduction Assay
4 Notes
References
Index