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Measuring Biological Responses with Automated Microscopy

1st Edition - October 18, 2006

Editor: James Inglese

Hardback ISBN:

9 7 8 - 0 - 1 2 - 1 8 2 8 1 9 - 6

eBook ISBN:

9 7 8 - 0 - 0 8 - 0 4 6 8 9 8 - 3

The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since… Read more

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The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the series contains much material still relevant today-truly an essential publication for researchers in all fields of life sciences.

Contributors to Volume 414
- Publisher Summary
Preface
- Publisher Summary
Contents of Previous Volumes
[1]: Dynamic Green Fluorescent Protein Sensors for High-Content Analysis of the Cell Cycle
- Abstract
- Introduction
- Design and Construction of Dynamic Cell Cycle Sensors
- Validation of Sensors
- Cell Cycle Analysis by Automated High-Throughput Imaging for Drug Profiling and Target Validation
- Conclusions and Future Directions
- Acknowledgments
[2]: High-Content Fluorescence-Based Screening for Epigenetic Modulators
- Abstract
- Introduction
- Epigenetic Regulators of Gene Expression as Drug Targets
- Rationale for the Development of Cell-Based Assays to Screen for Epigenetic Modulators
- Methodological Considerations
- General Conclusions and Perspectives
[3]: Development of Assays for Nuclear Receptor Modulators Using Fluorescently Tagged Proteins
- Abstract
- Introduction
- Methods
- Perspectives and Future Applications
[4]: The Ligand-Independent Translocation Assay: An Enabling Technology for Screening Orphan G Protein-Coupled Receptors by Arrestin Recruitment
- Abstract
- Introduction
- Overview of the LITe Assay
- Utility of the LITe Assay
- Conclusion
- Acknowledgments
[5]: High-Content Screening of Known G Protein-Coupled Receptors by Arrestin Translocation
- Abstract
- Introduction
- Stable Expression of a Known GPCR for the ArrestinGFP Translocation Assay
- Screening Known Receptors with Transfluor Technology
- Conclusion
- Acknowledgments
[6]: Cell Imaging Assays for G Protein-Coupled Receptor Internalization: Application to High-Throughput Screening
- Abstract
- Introduction
- Monitoring GPCR Trafficking via Receptor–GFP
- Monitoring GPCR Trafficking via Receptor–Arrestin–GFP
- Case I: MRG-X1 Receptor
- Case Study 2: NK1 Receptor Trafficking
- Acknowledgments
[7]: High-Throughput Confocal Microscopy for β-Arrestin–Green Fluorescent Protein Translocation G Protein-Coupled Receptor Assays Using the Evotec Opera
- Abstract
- Choice of Orphans
- Transfluor
- Technology
- Image Analysis Algorithm
- EC₅₀ Fitting and Scoring
- Assay Methods
- Screen Metrics and Results
- Concluding Remarks
[8]: G Protein-Coupled Receptor Internalization Assays in the High-Content Screening Format
- Abstract
- Introduction
- High-Throughput Confocal Cellular Imaging Systems
- GPCR Internalization Assays
- Internalization Assay and Image Analysis Protocols
- Conclusions and Outlook
- Acknowledgment
[9]: Screening for Activators of the Wingless Type/Frizzled Pathway by Automated Fluorescent Microscopy
- Abstract
- Introduction
- Use of Primary Human Preosteoblasts
- Reagents and Materials
- Preparation of L Cell Control- and Wnt3A-Conditioned Medium
- Primary Preosteoblast Cell Culture and Compound Screening
- Immunofluorescent Staining
- Imaging and Quantitation of Nuclear Translocation
- Data Analysis
- Acknowledgments
[10]: A Live Cell, Image-Based Approach to Understanding the Enzymology and Pharmacology of 2-Bromopalmitate and Palmitoylation
- Abstract
- Introduction
- Types of Protein Lipid Modifications
- Palmitate Turnover
- Enzymes for Depalmitoylation
- Role of Palmitoylation in Development
- Cysteines: Primary Sites of Palmitoylation
- Palmitoyl Acyl Transferases (PATs)
- Links between Palmitoylation and Disease
- Using Fluorescent Proteins to Study Protein Lipid Modifications
- Monomeric Fluorescent Proteins: A Critical Feature for Studying Palmitoylation
- High-Throughput Microscopy (HTM)/High-Content Screening (HCS) to Study Lipid-Modified Proteins
- Morphometric Analysis of Palmitoylation with HCS
- HCS Machine Vision Algorithms to Quantify Reporter Density on the Plasma Membrane
- GAP43:YFP is Palmitoylated, Localized to the Plasma Membrane, and Is a Stereotypical Reporter of the Cellular Capacity for Palmitoylation
- Determination of the Residence Half-Life of Palmitate on GAP43:YFP Using HCS
- Thora Can Measure Precisely the Subcellular Distribution of GAP43:YFP: IC₅₀ of 2BP
- Determination of the Compatibility of the Cellular Reporter System with Dimethyl Sulfoxide (DMSO)
- Cytotoxic Effects of Antagonists of Palmitoylation
- Considerations for Designing HCS, Cell-Based Assays with an Emphasis on Palmitoylation
- Troubleshooting
- System Error Identification
- Data Tracking
- Image Organization and Analysis
- Conclusions
[11]: High-Resolution, High-Throughput Microscopy Analyses of Nuclear Receptor and Coregulator Function
- Abstract
- High-Throughput Microscopy (HTM)
- Steroid Nuclear Receptors and Coregulators
- General Methods
- Nuclear Receptor Coregulator SRC-3
- Protein Expression Level Assay
- Protein Translocation and Nuclear Variance Assays
- Foci Identification and Chromatin Remodeling Assay
- Conclusions
[12]: Tracking Individual Proteins in Living Cells Using Single Quantum Dot Imaging
- Abstract
- Introduction
- Semiconductor Quantum Dots as Biological Probes
- Single Molecule Imaging in Living Cells
- Single Quantum Dot Tracking of Membrane Proteins
- Solutions and Materials
- Methods
- Single Quantum Dot Tracking of Intracellular Proteins
- Solutions and Materials
- Methods
- Imaging of Single Conjugated Quantum Dot in Living Cells
- Analysis of Single QD trajectories
- Image Processing and Extraction of Single QD Trajectories
- Mean Square Displacement Function
- Statistical Errors
- Acknowledgments
[13]: Development and Application of Automatic High-Resolution Light Microscopy for Cell-Based Screens
- Abstract
- Introduction
- Technical Description
- Biological Screens
- Prospectives
- Acknowledgments
[14]: Adenoviral Sensors for High-Content Cellular Analysis
- Abstract
- Introduction
- Design and Construction of Adenoviral Sensors for Cellular Assays
- Validation of Adenoviral Sensors
- Application of Adenoviral Sensors to High-Content Analysis
- EGFP-Glucocorticoid Receptor (GR) Translocation Sensor
- Reagent Preparation
- Assay Procedure
- Assay Analysis
- Nuclear Factor of Activated T Cells (NFAT) Nitroreductase Reporter Gene Sensor
- Reagent Preparation
- Assay Procedure
- Assay Analysis
- Conclusions and Future Perspectives
- Acknowledgments
[15]: Cell-Based Assays Using Primary Endothelial Cells to Study Multiple Steps in Inflammation
- Abstract
- Cellular Mechanism of the Inflammatory Response
- Cell-Based Assays Used to Monitor the Effects of Proinflammatory Cytokines
- Materials and Methods Common to All Three Assays
- Image Analysis
- Statistical Analysis
- Assay Data
- Concluding Remarks and Future Perspectives
- Acknowledgments
[16]: Development and Implementation of Multiplexed Cell-Based Imaging Assays
- Abstract
- Introduction
- General Considerations
- Monitoring Cell Cycle Progression
- Protocols for Cell Cycle Analysis
- Visualizing Apoptosis by Fluorescence Microscopy
- Multiplexing Cell Cycle and Apoptosis Assays
- Conclusions
- Acknowledgments
[17]: High-Throughput Screening for Modulators of Stem Cell Differentiation
- Abstract
- Introduction
- Assay Design
- Assay Development
- Methods
- Conclusion
- Appendix
- Acknowledgments
[18]: High-Content Kinetic Calcium Imaging in Drug-Sensitive and Drug-Resistant Human Breast Cancer Cells
- Abstract
- Introduction
- High-Content Analysis of Ca²⁺ Dynamics by Fluorescence Microscopy
- Conclusions from HCA and Rationale for High-Content Screening
- High-Content Screening
- Acknowledgments
[19]: Measurement and Analysis of Calcium Signaling in Heterogeneous Cell Cultures
- Abstract
- Introduction
- Characterization of Calcium Signaling in Rat Cortical Cultures
- Subpopulation Analysis of Calcium Signaling in Cocultures
- Acknowledgments
[20]: Multiplex Analysis of Inflammatory Signaling Pathways Using a High-Content Imaging System
- Abstract
- Introduction
- Assay Procedures
- Concluding Remarks
- Acknowledgments
[21]: Generation and Characterization of a Stable MK2-EGFP Cell Line and Subsequent Development of a High-Content Imaging Assay on the Cellomics ArrayScan Platform to Screen for p38 Mitogen-Activated Protein Kinase Inhibitors
- Abstract
- Introduction
- Definition of the Cell Model
- Generation and Characterization of MK2-EGFP HeLa Cell Line
- Cellomics ArrayScan Automated Imaging Platform
- Characterization of MK2-EGFP Clones via Imaging
- Characterization of the p38 MAPK Signaling Pathway in HeLa-MK2-EGFP Cells
- Assay Development on the ArrayScan 3.1 Imaging Platform
- Standard Operating Procedure for the MK2-EGFP Translocation Assay
- MK2-EGFP Translocation Assay Reproducibility and Signal Widow Evaluation
- p38 Inhibitor Data
- Secondary Analysis Parameters
- Discussion
[22]: Development and Implementation of Three Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway Imaging Assays to Provide MAPK Module Selectivity Profiling for Kinase Inhibitors: MK2-EGFP Translocation, c-Jun, and ERK Activation
- Abstract
- Introduction
- Definition of the Cell Model
- Cellomics ArrayScan Automated Imaging Platform
- Development of the JNK MAPK Signaling Pathway Assay
- c-Jun Activation Protocol
- c-Jun Activation Signal Window and Reproducibility
- Development of the ERK MAPK Signaling Pathway Assay
- ERK1/2 Activation Protocol
- ERK1/2 Activation Signal Window and Reproducibility
- MAPK Pathway Inhibitor Test Cassette
- p38 Inhibitor Hit Assessment
- p38a Inhibitor Profiling
- JNK Inhibitor Profiling
- Discussion
[23]: Assay Development and Case History of a 32K-Biased Library High-Content MK2-EGFP Translocation Screen to Identify p38 Mitogen-Activated Protein Kinase Inhibitors on the ArrayScan 3.1 Imaging Platform
- Abstract
- Introduction
- Cellomics ArrayScan Automated Imaging Platform
- Conversion of the 96-Well MK2-EGFP Translocation Assay to a 384-Well Format Assay on the Arrayscan® Imager
- MK2-EGFP Translocation Assay Reproducibility and Signal Widow Evaluation
- Standardized Operation Procedure for the MK2-EGFP Translocation Assay
- MK2-EGFP Translocation HTS Assay for p38 Inhibitors
- Discussion
[24]: Compound Classification Using Image-Based Cellular Phenotypes
- Abstract
- Introduction
- Quantifying Cellular Morphology Changes
- Cell Culture, Compound Addition, and Image Acquisition
- Image and Data Reduction
- Analysis of Quantitative Cellular Phenotypes across Cell Lines
- Clustering and Classification of Compounds
- Conclusions
- Acknowledgments
[25]: High-Content Screening: Emerging Hardware and Software Technologies
- Abstract
- Introduction
- Cellular Assay and Imaging Preparation
- Image Acquisition
- Image Analysis
- Image Database and Data Visualization Tools
- Conclusion
- Protocols
[26]: An Infrastructure for High-Throughput Microscopy: Instrumentation, Informatics, and Integration
- Abstract
- Introduction
- Assay Processing
- Image Acquisition
- Image and Data Analysis
- Data Review and Quality Control
- Summary
- Acknowledgments
[27]: Protein Translocation Assays: Key Tools for Accessing New Biological Information with High-Throughput Microscopy
- Abstract
- Pathway Screening Using BioImage Redistribution Technology
- p53-Hdm2 Protein–Protein Interaction Assay
- Use of High-Content Assays in RNAi Studies
- Use of siRNA-Mediated Knockdown to Validate Akt Isoform Dependency of a FKHR Redistribution Assay
- Assay-Specific Cell-to-Cell Heterogeneity Plays a Role in Assay Quality
- Future Developments
[28]: High-Content Screening of Functional Genomic Libraries
- Abstract
- Introduction
- Development of Large-Scale Genomic Libraries
- Maintaining Large Arrayed-Well Plasmid cDNA or shRNA Clone Libraries
- Collection Replication
- Growth of Bacterial Cultures in High-Throughput Format for DNA Preparation
- Preparing DNA from 96-Well Deep-Well Block Cultures
- Normalization of Plasmid DNA
- Arraying Collections into High-Throughput Assay Plates
- High-Throughput Transfections
- High-Throughput Retroviral/Lentivial Packaging
- Instrumentation Required for Functional Genomics Screening
- Automated Microscopy
- High-Throughput HCS Equipment
- Fluorescent Biomarkers for HCS Applications
- Preparing Samples for Automated Microscopy
- Determining Transfection or Transduction Efficiency
- Quantitative Image Analysis
- Summary
[29]: Fluorescent Protein-Based Cellular Assays Analyzed by Laser-Scanning Microplate Cytometry in 1536-Well Plate Format
- Abstract
- Introduction
- The Principle of Laser-Scanning Microplate Cytometers
- General Methods
- GR-GFP Nuclear Translocation Assay
- Locus Derepression Assay
- β-Arrestin:β₂-Adrenergic Receptor (βARR:β2AR) Protein Fragment Complementation Assay
- Summary
- Acknowledgments
[30]: High-Throughput Measurements of Biochemical Responses Using the Plate::Vision Multimode 96 Minilens Array Reader
- Abstract
- Introduction
- Instrumentation
- Measurements of Biochemical Responses
- Acknowledgments
[31]: Systems Cell Biology Based on High-Content Screening
- Abstract
- Background
- The Systems Cell Biology Toolbox
- Example Systems Cell Biology Profile
- Summary and Conclusions
- Prospectus
- Acknowledgments
[32]: Digital Autofocus Methods for Automated Microscopy
- Abstract
- Introduction
- Hardware
- Software
- Viscoelasticity
- Multiple-Field Scans
- Conclusion
- Appendix
[33]: Fluorescence Lifetime Imaging Microscopy: Two-Dimensional Distribution Measurement of Fluorescence Lifetime
- Abstract
- Introduction
- Operating Principle of a Streak Camera
- Configuration of the FLIM System
- Streak Image
- Measurement Principle of FLIM System
- System Calibration
- Lifetime Imaging in Cells
- FRET Imaging in Cells
- Acknowledgments
Author Index
- Publisher Summary
Subject Index
- Publisher Summary