Hydrogen Peroxide and Cell Signaling, Part A

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METHODS IN ENZYMOLOGY

Contributors

Preface

Methods in Enzymology

Section 1: Hydrogen Peroxide Detection Methods

Chapter One. Photooxidation of Amplex Red to Resorufin: Implications of Exposing the Amplex Red Assay to Light

Abbreviations

1 Introduction

2 Amplex Red Assay and Its Possible Artifacts due to Light Exposure

3 Experimental Considerations

Acknowledgments

References

Chapter Two. Boronate-Based Fluorescent Probes: Imaging Hydrogen Peroxide in Living Systems

1 Introduction

2 Fluorescent Boronate-Based Hydrogen Peroxide Probes

3 Examples of Probes and Their Usage

4 Imaging Endogenous H₂O₂ Using Fluorescent Probes

5 Conclusions

Acknowledgments

References

Chapter Three. Visualization of Intracellular Hydrogen Peroxide with HyPer, a Genetically Encoded Fluorescent Probe

1 Introduction

2 Experimental Design

3 Materials

4 Procedure

Acknowledgments

References

Chapter Four. In Vivo Imaging of H₂O₂ Production in Drosophila

1 Introduction

2 Materials

3 Methods

4 Image and Data Analysis

5 Data Interpretation

Acknowledgments

References

Chapter Five. Single Fluorescent Probe Distinguishes Hydrogen Peroxide and Nitric Oxide in Cell Imaging

1 Introduction

2 A Guide to Single Fluorescent Probe Distinguishes H₂O₂ and NO

3 Fluorescence Imaging of Endogenously Produced H₂O₂ and NO in RAW 264.7 Macrophages Cells

4 Summary

Acknowledgments

References

Chapter Six. Electrochemical Biosensors for On-Chip Detection of Oxidative Stress from Cells

1 Introduction

2 Experimental Components and Procedures

3 Methods of Calibrating Enzyme Sensors

4 Monitoring ROS Production from Cells Using Electrochemistry

5 Summary

References

Chapter Seven. Electrochemical Detection of H₂O₂ Formation in Isolated Mitochondria

Abbreviations

1 Introduction

2 The Electrochemical Chamber

3 The Platinized Carbon Fiber Microelectrode

4 Data Analysis for H₂O₂ Amperometric Measurements

5 H₂O₂ and O₂ Calibration Tests

6 Preparation of Isolated Mitochondria

7 Measure of H₂O₂ from Mitochondrial Suspension

8 Conclusions

Acknowledgments

References

Chapter Eight. Detection of H₂O₂ by Fluorescence Correlation Spectroscopy

1 Introduction

2 Experimental Methods

3 Comparison of Sensitivity Between the FCS-Based Method and an Amplex Red-Based Method

4 Conclusion

Acknowledgments

References

Chapter Nine. Real-Time Monitoring of Reactive Oxygen and Nitrogen Species in a Multiwell Plate Using the Diagnostic Marker Products of Specific Probes

1 Introduction

2 Experimental Methods

3 Results and Discussion

4 Summary

Acknowledgments

References

Section 2: Temporal and Spatial H₂O₂ Distribution

Chapter Ten. H₂O₂ Delivery to Cells: Steady-State Versus Bolus Addition

1 Introduction

2 Experimental Components and Considerations

3 Pilot Experiments

4 Steady-State Experimental Implementation

5 Summary

Acknowledgments

References

Chapter Eleven. Imaging H₂O₂ Microdomains in Receptor Tyrosine Kinases Signaling

1 Introduction

2 Experimental Design

3 Materials

4 Procedure

5 Transfection

6 Anticipated Results

Acknowledgments

References

Section 3: Cellular Sources of H₂O₂

Chapter Twelve. The Determination and Analysis of Site-Specific Rates of Mitochondrial Reactive Oxygen Species Production

1 Introduction

2 H₂O₂ Production Measurements in Isolated Mitochondria

3 Maximum Production Rates from Specific Sites: The Nature and Capacity of the Machinery

4 Concluding Remarks

Acknowledgments

References

Chapter Thirteen. A Microfluidic Systems Biology Approach for Live Single-Cell Mitochondrial ROS Imaging

1 Introduction

2 Microfluidic Platform

3 Microscope System and Image Analysis

4 Imaging Mitochondrial Superoxide Production

5 Imaging Mitochondrial Hydrogen Peroxide Production

6 Conclusion

7 Potential Pitfalls

References

Chapter Fourteen. Detection of Oxidative Damage in Response to Protein Misfolding in the Endoplasmic Reticulum

1 Introduction

2 UPR-Induced Oxidative Damage

3 Summary

References

Author Index

Subject Index