High Content Screening

1 - HIGH CONTENT SCREENING [Seite 3]
1.1 - CONTENTS [Seite 9]
1.2 - Preface [Seite 21]
1.3 - Contributors [Seite 23]
1.4 - SECTION I ESSENTIALS OF HIGH CONTENT SCREENING [Seite 29]
1.4.1 - 1. Approaching High Content Screening and Analysis: Practical Advice for Users [Seite 31]
1.4.1.1 - 1.1 Introduction [Seite 31]
1.4.1.2 - 1.2 What is HCS and Why Should I Care? [Seite 32]
1.4.1.3 - 1.3 How does HCS Compare with Current Assay Methods? [Seite 33]
1.4.1.4 - 1.4 The Basic Requirements to Implement HCS [Seite 36]
1.4.1.4.1 - 1.4.1 Cell Banking [Seite 37]
1.4.1.4.2 - 1.4.2 Plating, Cell Density, and the Assay Environment [Seite 38]
1.4.1.4.3 - 1.4.3 Compound Addition and Incubation [Seite 39]
1.4.1.4.4 - 1.4.4 Post-Assay Processing [Seite 39]
1.4.1.4.5 - 1.4.5 HCS Imaging Hardware [Seite 40]
1.4.1.4.6 - 1.4.6 HCS Analysis Software [Seite 41]
1.4.1.4.7 - 1.4.7 Informatics [Seite 41]
1.4.1.5 - 1.5 The Process [Seite 43]
1.4.1.6 - 1.6 An Example Approach [Seite 44]
1.4.1.7 - 1.7 Six Considerations for HCS Assays [Seite 46]
1.4.1.7.1 - 1.7.1 Garbage In, Garbage Out (GIGO) [Seite 46]
1.4.1.7.2 - 1.7.2 This Is Not a Plate Reader [Seite 47]
1.4.1.7.3 - 1.7.3 Understand Your Biology [Seite 48]
1.4.1.7.4 - 1.7.4 Subtle Changes Can Be Measured and Are Significant [Seite 48]
1.4.1.7.5 - 1.7.5 HCS Workflow - Flexibility is the Key [Seite 49]
1.4.1.7.6 - 1.7.6 HCS is Hard - How Do I Learn It and Become Proficient at It? [Seite 49]
1.4.1.8 - References [Seite 50]
1.4.2 - 2. Automated High Content Screening Microscopy [Seite 53]
1.4.2.1 - 2.1 Introduction [Seite 53]
1.4.2.2 - 2.2 Automated HCS Imaging Requirements [Seite 54]
1.4.2.3 - 2.3 Components of Automated Imaging Platforms [Seite 54]
1.4.2.3.1 - 2.3.1 Fluorescence Imaging and Multiplexing [Seite 54]
1.4.2.3.2 - 2.3.2 Light Sources [Seite 56]
1.4.2.3.3 - 2.3.3 Optical Designs: Confocal Versus Wide-Field [Seite 56]
1.4.2.3.4 - 2.3.4 Objectives [Seite 57]
1.4.2.3.5 - 2.3.5 Detectors [Seite 57]
1.4.2.3.6 - 2.3.6 Autofocus [Seite 57]
1.4.2.3.7 - 2.3.7 Environmental Controls and On-Board Liquid Handling Capabilities [Seite 58]
1.4.2.4 - 2.4 Imaging Platform Software [Seite 59]
1.4.2.5 - 2.5 Data Storage and Management [Seite 60]
1.4.2.6 - 2.6 Selecting an HCS Platform [Seite 60]
1.4.2.7 - 2.7 Comparison of a SAPK Activation HCS Assay Read on an ArrayScan(®) 3.1, an ArrayScan(®) V(Ti), and an IN Cell 3000 Automated Imaging Platform [Seite 61]
1.4.2.8 - References [Seite 68]
1.4.3 - 3. A Primer on Image Informatics of High Content Screening [Seite 71]
1.4.3.1 - 3.1 Background [Seite 71]
1.4.3.2 - 3.2 HCS Image Processing [Seite 74]
1.4.3.2.1 - 3.2.1 Image Pre-Processing [Seite 74]
1.4.3.2.2 - 3.2.2 Cell Detection, Segmentation, and Centerline Extraction [Seite 76]
1.4.3.2.2.1 - 3.2.2.1 Cell Detection [Seite 76]
1.4.3.2.2.2 - 3.2.2.2 Particle Detection [Seite 78]
1.4.3.2.2.3 - 3.2.2.3 Cell Segmentation [Seite 80]
1.4.3.2.2.4 - 3.2.2.4 Centerline/Neurite Extraction [Seite 85]
1.4.3.2.3 - 3.2.3 Cell Tracking and Registration [Seite 88]
1.4.3.2.3.1 - 3.2.3.1 Simple Matching Algorithm [Seite 88]
1.4.3.2.3.2 - 3.2.3.2 Mean Shift [Seite 90]
1.4.3.2.3.3 - 3.2.3.3 Kalman Filter [Seite 90]
1.4.3.2.3.4 - 3.2.3.4 Mutual Information [Seite 91]
1.4.3.2.3.5 - 3.2.3.5 Fuzzy-System-Based Tracking [Seite 92]
1.4.3.2.3.6 - 3.2.3.6 Parallel Tracking [Seite 94]
1.4.3.2.4 - 3.2.4 Feature Extraction [Seite 94]
1.4.3.2.4.1 - 3.2.4.1 Features Extracted from Markov Chain Modeling of Time-Lapse Images [Seite 95]
1.4.3.3 - 3.3 Validation [Seite 95]
1.4.3.4 - 3.4 Information System Management [Seite 97]
1.4.3.5 - 3.5 Data Modeling [Seite 98]
1.4.3.5.1 - 3.5.1 Novel Phenotype Discovery Using Clustering [Seite 98]
1.4.3.5.2 - 3.5.2 Gene Function Study Using Clustering [Seite 100]
1.4.3.5.3 - 3.5.3 Screening Hits Selection and Gene Scoring for Effectors Discovery [Seite 102]
1.4.3.5.3.1 - 3.5.3.1 Fuzzy Gene Score Regression Model [Seite 103]
1.4.3.5.3.2 - 3.5.3.2 Experimental Results [Seite 104]
1.4.3.5.4 - 3.5.4 Metabolic Networks Validated by Using Genomics, Proteomics, and HCS [Seite 104]
1.4.3.5.5 - 3.5.5 Connecting HCS Analysis and Systems Biology [Seite 105]
1.4.3.5.6 - 3.5.6 Metabolic Networks [Seite 106]
1.4.3.6 - 3.6 Conclusions [Seite 107]
1.4.3.7 - 3.7 Acknowledgments [Seite 107]
1.4.3.8 - References [Seite 108]
1.4.4 - 4. Developing Robust High Content Assays [Seite 113]
1.4.4.1 - 4.1 Introduction [Seite 113]
1.4.4.2 - 4.2 Overview of a Typical Immunofluorescence-Based High Content Assay [Seite 114]
1.4.4.2.1 - 4.2.1 Staining Protocol [Seite 115]
1.4.4.2.2 - 4.2.2 Sources of Variability [Seite 115]
1.4.4.3 - 4.3 Identifying Sources of Variability in a High Content Assay [Seite 116]
1.4.4.3.1 - 4.3.1 Verifying the Accuracy and Precision of Liquid Handling Procedures [Seite 117]
1.4.4.3.2 - 4.3.2 Deconstruction of Immunofluorescence and Cell Culture Protocols [Seite 118]
1.4.4.3.3 - 4.3.3 Control Experiments [Seite 118]
1.4.4.3.4 - 4.3.4 Protocol Optimization [Seite 120]
1.4.4.3.5 - 4.3.5 Antibody Optimization Using a Design of Experiments Framework [Seite 122]
1.4.4.3.6 - 4.3.6 Addressing Sources of Variability in Microscopy [Seite 124]
1.4.4.3.7 - 4.3.7 Optimization of Image Processing Parameters in a High Content Assay [Seite 127]
1.4.4.4 - 4.4 From Immunofluorescence to High Content: Selecting the Right Metric [Seite 129]
1.4.4.5 - 4.5 Validation of High Content Assays [Seite 130]
1.4.4.5.1 - 4.5.1 Establishing SOPs and Reagent Stocks for Cell Culture and Immunofluorescence Staining [Seite 131]
1.4.4.5.2 - 4.5.2 Linking Assay Variability to Assay Performance [Seite 132]
1.4.4.5.3 - 4.5.3 Design of Assay Quality Control Measures [Seite 133]
1.4.4.6 - 4.6 Conclusion [Seite 135]
1.4.4.7 - 4.7 Acknowledgments [Seite 136]
1.4.4.8 - References [Seite 136]
1.5 - SECTION II APPLICATIONS OF HCS IN BASIC SCIENCE AND EARLY DRUG DISCOVERY [Seite 139]
1.5.1 - 5. HCS in Cellular Oncology and Tumor Biology [Seite 141]
1.5.1.1 - 5.1 Cancer Cell Biology and HCS [Seite 141]
1.5.1.1.1 - 5.1.1 Oncology Research and the Search for Effective Anticancer Therapeutics [Seite 141]
1.5.1.1.2 - 5.1.2 A General Protocol for Establishing HCS Assays Within Oncology Research [Seite 143]
1.5.1.1.2.1 - 5.1.2.1 What is the Underlying Biology to be Evaluated in an HCS Assay? [Seite 143]
1.5.1.1.2.2 - 5.1.2.2 What Resources Are Immediately Available for Characterizing the Target or its Activity? [Seite 144]
1.5.1.1.2.3 - 5.1.2.3 How Do the Available Reagents Perform Quantitatively? [Seite 146]
1.5.1.1.2.4 - 5.1.2.4 What Multiplexing is Required for the Assay? [Seite 147]
1.5.1.2 - 5.2 The Cell Biology of Cell Death [Seite 148]
1.5.1.2.1 - 5.2.1 Cell Death Stimuli and Response Pathways [Seite 148]
1.5.1.2.2 - 5.2.2 Induction of Cell Death Signals [Seite 149]
1.5.1.2.2.1 - 5.2.2.1 Activation of Cell Death Receptors [Seite 149]
1.5.1.2.2.2 - 5.2.2.2 Mitochondrial Damage [Seite 150]
1.5.1.2.2.3 - 5.2.2.3 Mitotic Arrest, Replication Stress, and DNA Damage [Seite 150]
1.5.1.2.2.4 - 5.2.2.4 ER Stress [Seite 150]
1.5.1.2.3 - 5.2.3 Propagation of Cell Death Signals into Specific Cell Death Responses [Seite 151]
1.5.1.2.3.1 - 5.2.3.1 Apoptosis [Seite 154]
1.5.1.2.3.2 - 5.2.3.2 Mitotic Catastrophe [Seite 155]
1.5.1.2.3.3 - 5.2.3.3 Autophagy [Seite 155]
1.5.1.2.3.4 - 5.2.3.4 Necrosis [Seite 156]
1.5.1.2.3.5 - 5.2.3.5 Senescence [Seite 157]
1.5.1.2.4 - 5.2.4 Cytological and High Content Assays for Cancer Cell Death [Seite 157]
1.5.1.2.4.1 - 5.2.4.1 Detection of Moderate and Severe ER Stress in Cancer Cells [Seite 158]
1.5.1.2.4.2 - 5.2.4.2 Effects of Cytotoxic Therapeutics on Apoptosis and Necrosis of Cancer Cells [Seite 158]
1.5.1.3 - 5.3 Cell Signaling Pathways in Cancer [Seite 161]
1.5.1.3.1 - 5.3.1 Signal Transduction in Cancer [Seite 161]
1.5.1.3.2 - 5.3.2 A Multiparametric Assay for the PI3K/AKT Pathway as Representative of Quantitative Measures of Signal Transduction in Cancer Cells [Seite 163]
1.5.1.4 - 5.4 HCS in Tumor Biology [Seite 165]
1.5.1.4.1 - 5.4.1 The Biology of Tumor Growth [Seite 165]
1.5.1.4.2 - 5.4.2 An HCS Assay to Study Tumor Biology in vitro [Seite 165]
1.5.1.5 - 5.5 Conclusions [Seite 167]
1.5.1.6 - References [Seite 167]
1.5.2 - 6. Exploring the Full Power of Combining High Throughput RNAi with High Content Readouts: From Target Discovery Screens to Drug Modifier Studies [Seite 173]
1.5.2.1 - 6.1 Background: The Convergence of High Content Analysis and RNAi [Seite 173]
1.5.2.2 - 6.2 Integrating HT-RNAi and HCA in Drug Discovery: The Potential [Seite 174]
1.5.2.2.1 - 6.2.1 Technology Platform, HCA, and HT-RNAi Methodologies [Seite 174]
1.5.2.2.2 - 6.2.2 Key Applications of HT-RNAi Combined with HCA in Drug Discovery [Seite 176]
1.5.2.2.2.1 - 6.2.2.1 Target Discovery Screens [Seite 176]
1.5.2.2.2.2 - 6.2.2.2 Target Validation Studies [Seite 177]
1.5.2.2.2.3 - 6.2.2.3 Drug Mechanism of Action Screens [Seite 177]
1.5.2.3 - 6.3 Combining RNAi and HCA in One Assay - The Reality [Seite 178]
1.5.2.3.1 - 6.3.1 General Considerations [Seite 178]
1.5.2.3.1.1 - 6.3.1.1 Choice of the Right Cell Model [Seite 178]
1.5.2.3.1.2 - 6.3.1.2 Establishment of an RNAi Delivery Protocol [Seite 178]
1.5.2.3.1.3 - 6.3.1.3 Assay Optimization [Seite 179]
1.5.2.3.2 - 6.3.2 Applications: Combining HCA with HT-RNAi to Integrate Functional Validation Directly Within Target Discovery Studies [Seite 179]
1.5.2.3.2.1 - 6.3.2.1 Multipass Strategies for Systematic Screens [Seite 179]
1.5.2.3.2.2 - 6.3.2.2 Hurdles and Caveats [Seite 180]
1.5.2.3.2.3 - 6.3.2.3 Example: A Multiparametric Oncology Assay Platform [Seite 183]
1.5.2.3.3 - 6.3.3 RNAi Target Validation Studies [Seite 187]
1.5.2.3.3.1 - 6.3.3.1 Functional Profiling [Seite 188]
1.5.2.3.3.2 - 6.3.3.2 Transcriptional Profiling [Seite 188]
1.5.2.3.3.3 - 6.3.3.3 Cytological Profiling [Seite 188]
1.5.2.3.3.4 - 6.3.3.4 Pathway Profiling [Seite 188]
1.5.2.3.3.5 - 6.3.3.5 Target Titration [Seite 188]
1.5.2.3.4 - 6.3.4 RNAi Drug Modifier Screens [Seite 189]
1.5.2.4 - 6.4 HCA-Based RNAi Studies - The Future [Seite 192]
1.5.2.5 - 6.5 Acknowledgments [Seite 194]
1.5.2.6 - References [Seite 194]
1.5.3 - 7. Leveraging HCS in Neuroscience Drug Discovery [Seite 197]
1.5.3.1 - 7.1 High Content Screening and Drug Discovery [Seite 197]
1.5.3.2 - 7.2 The Neuron and Neuronal Morphology [Seite 197]
1.5.3.2.1 - 7.2.1 What Does Morphology Tell Us About Neuronal Function? [Seite 198]
1.5.3.3 - 7.3 Methods for Measuring Neuronal Morphology [Seite 200]
1.5.3.3.1 - 7.3.1 Traditional Methods [Seite 200]
1.5.3.3.2 - 7.3.2 Available HCS Systems for Neuronal Morphology Measurements and Evolution of Technology [Seite 201]
1.5.3.3.3 - 7.3.3 Methods for Imaging Neurons and Types of Morphologic Measurement [Seite 204]
1.5.3.4 - 7.4 Small Molecule Screening for Neurite Outgrowth [Seite 206]
1.5.3.5 - 7.5 RNAi in Neuroscience and HCA [Seite 207]
1.5.3.6 - 7.6 Measurement of Signal Transduction in Neurons [Seite 208]
1.5.3.7 - 7.7 High Content Screening in Complex CNS Models [Seite 209]
1.5.3.8 - 7.8 Methods Used in Neuronal HCS [Seite 210]
1.5.3.8.1 - 7.8.1 Preparation of Neuronal Culture Samples for HCS Morphology Analysis [Seite 210]
1.5.3.8.2 - 7.8.2 Culture Fixing [Seite 211]
1.5.3.8.3 - 7.8.3 Immunocytochemistry [Seite 211]
1.5.3.8.4 - 7.8.4 Neurite Morphology Measurement and Analysis [Seite 212]
1.5.3.9 - References [Seite 213]
1.5.4 - 8. Live Brain Slice Imaging for Ultra High Content Screening: Automated Fluorescent Microscopy to Study Neurodegenerative Diseases [Seite 217]
1.5.4.1 - 8.1 Introduction and Background [Seite 217]
1.5.4.2 - 8.2 Live Brain Slice Model to Study Huntington's Disease [Seite 219]
1.5.4.3 - 8.3 Imaging Platforms [Seite 219]
1.5.4.4 - 8.4 Center of Well (COW) for Image Processing [Seite 222]
1.5.4.5 - 8.5 Generic Protocol for the Cellomics ArrayScan VTI [Seite 225]
1.5.4.6 - 8.6 Data and Results [Seite 225]
1.5.4.7 - 8.7 Discussion [Seite 229]
1.5.4.8 - References [Seite 231]
1.5.5 - 9. High Content Analysis of Human Embryonic Stem Cell Growth and Differentiation [Seite 233]
1.5.5.1 - 9.1 Introduction [Seite 233]
1.5.5.2 - 9.2 Cell Culture Methods [Seite 234]
1.5.5.2.1 - 9.2.1 Maintaining Pluripotency [Seite 234]
1.5.5.2.2 - 9.2.2 Cardiomyocyte Differentiation [Seite 235]
1.5.5.2.3 - 9.2.3 Neuronal Differentiation [Seite 235]
1.5.5.3 - 9.3 Statistical Wavelet-Based Analysis of Images for Stem Cell Classification [Seite 235]
1.5.5.3.1 - 9.3.1 Motivation for Algorithm Development [Seite 235]
1.5.5.3.2 - 9.3.2 Measuring Amorphous Biological Shapes [Seite 237]
1.5.5.3.3 - 9.3.3 Texture and Borders as Biological Features [Seite 238]
1.5.5.3.4 - 9.3.4 Texture Analysis [Seite 238]
1.5.5.4 - 9.4 Molecular Analysis of Pluripotency and Cell Proliferation in Undifferentiated Stem Cells [Seite 242]
1.5.5.4.1 - 9.4.1 Methods [Seite 243]
1.5.5.4.2 - 9.4.2 Analysis of Pluripotency and Cell Proliferation in Undifferentiated Stem Cells [Seite 243]
1.5.5.5 - 9.5 Analysis of Cardiomyocyte Differentiation [Seite 246]
1.5.5.6 - 9.6 Analysis of Neuronal Differentiation [Seite 247]
1.5.5.6.1 - 9.6.1 Methods [Seite 247]
1.5.5.6.2 - 9.6.2 Analysis of Neurectodermal Intermediates in Early Differentiated hESC [Seite 248]
1.5.5.6.3 - 9.6.3 Analysis of Neuronal Processes [Seite 249]
1.5.5.7 - References [Seite 249]
1.6 - SECTION III HCS IN DRUG DEVELOPMENT [Seite 253]
1.6.1 - 10. HCS for HTS [Seite 255]
1.6.1.1 - 10.1 Introduction [Seite 255]
1.6.1.2 - 10.2 HCS for Orphan GPCRs and Transfluor [Seite 256]
1.6.1.3 - 10.3 HCS for Multiparameter Cytotoxicity Screening [Seite 264]
1.6.1.4 - 10.4 Discussion [Seite 271]
1.6.1.5 - 10.5 Summary [Seite 274]
1.6.1.6 - References [Seite 274]
1.6.2 - 11. The Roles of High Content Cellular Imaging in Lead Optimization [Seite 277]
1.6.2.1 - 11.1 Introduction [Seite 277]
1.6.2.2 - 11.2 Statistical Validation of Assays [Seite 278]
1.6.2.3 - 11.3 High Content Cellular Imaging is a Diverse Assay Platform [Seite 279]
1.6.2.4 - 11.4 Use of High Content Cellular Imaging for Oncology Research at Eli Lilly [Seite 283]
1.6.2.4.1 - 11.4.1 Cell Cycle and High Content Cellular Imaging [Seite 283]
1.6.2.4.2 - 11.4.2 Advantages of High Content Cellular Imaging [Seite 284]
1.6.2.4.2.1 - 11.4.2.1 Rare Cell Populations [Seite 284]
1.6.2.4.2.2 - 11.4.2.2 End Point Multiplexing [Seite 285]
1.6.2.4.2.3 - 11.4.2.3 Advantages of Multiplexing [Seite 287]
1.6.2.5 - 11.5 The Future of High Content Cellular Imaging in Lead Optimization [Seite 289]
1.6.2.6 - 11.6 Acknowledgments [Seite 292]
1.6.2.7 - References [Seite 292]
1.6.3 - 12. Using High Content Analysis for Pharmacodynamic Assays in Tissue [Seite 297]
1.6.3.1 - 12.1 Introduction [Seite 297]
1.6.3.1.1 - 12.1.1 Preclinical Models [Seite 297]
1.6.3.1.2 - 12.1.2 Pharmacokinetics/Pharmacodynamics (PK/PD) [Seite 298]
1.6.3.1.3 - 12.1.3 PK/PD Approaches in Practice [Seite 299]
1.6.3.2 - 12.2 Designing a High Content Assay for Use in Tissues [Seite 300]
1.6.3.2.1 - 12.2.1 Preliminary Biomarker Characterization [Seite 300]
1.6.3.2.2 - 12.2.2 Development and Validation of HC Assays in Tissue [Seite 301]
1.6.3.3 - 12.3 Technical Challenges in Establishing High Content Assays for Tissue [Seite 302]
1.6.3.3.1 - 12.3.1 Logistical Challenges in Tissue Staining and Acquisition [Seite 302]
1.6.3.3.2 - 12.3.2 Plane-of-Focus and Plane-of-Section Issues [Seite 303]
1.6.3.3.3 - 12.3.3 Heterogeneity in Tissue Samples [Seite 305]
1.6.3.3.4 - 12.3.4 Automated Detection of Areas of Interest [Seite 307]
1.6.3.3.5 - 12.3.5 Segmentation and Background Issues in High Content Assays [Seite 310]
1.6.3.3.6 - 12.3.6 Variability in Staining [Seite 312]
1.6.3.4 - 12.4 Case Study: Design and Validation of a High Content Assay for Biomarker X [Seite 314]
1.6.3.5 - 12.5 Conclusions [Seite 317]
1.6.3.6 - 12.6 Acknowledgments [Seite 318]
1.6.3.7 - References [Seite 318]
1.6.4 - 13. High Content Analysis of Sublethal Cytotoxicity in Human HepG2 Hepatocytes for Assessing Potential and Mechanism for Chemical- and Drug-Induced Human Toxicity [Seite 321]
1.6.4.1 - 13.1 Introduction [Seite 321]
1.6.4.1.1 - 13.1.1 Past Failure of Cytotoxicity Assessments [Seite 321]
1.6.4.1.2 - 13.1.2 Development of a Novel Cellomic Cytotoxicity Model [Seite 323]
1.6.4.1.3 - 13.1.3 Parameters Monitored in the Cellomic Cytotoxicity Model [Seite 324]
1.6.4.1.4 - 13.1.4 Materials and Methods [Seite 328]
1.6.4.2 - 13.2 Results from High Content Analysis of Human Toxicity Potential [Seite 329]
1.6.4.3 - 13.3 Discussion [Seite 335]
1.6.4.3.1 - 13.3.1 Applications of the Cellomic Cytotoxicity Model [Seite 335]
1.6.4.3.2 - 13.3.2 Limitations of the Cellomic Cytotoxicity Model [Seite 336]
1.6.4.3.3 - 13.3.3 Future Studies [Seite 337]
1.6.4.4 - 13.4 Acknowledgments [Seite 337]
1.6.4.5 - 13.5 Appendix: Detailed Methods [Seite 337]
1.6.4.5.1 - 13.5.1 Materials [Seite 337]
1.6.4.5.2 - 13.5.2 Methods: Cell Culture [Seite 338]
1.6.4.5.3 - 13.5.3 Subculture of HepG2 Cells [Seite 338]
1.6.4.5.4 - 13.5.4 Poly-D-Lysine Coating [Seite 339]
1.6.4.5.5 - 13.5.5 Drug Treatment Protocol for Three-Day Plates [Seite 339]
1.6.4.5.6 - 13.5.6 Drug Solubility [Seite 340]
1.6.4.5.7 - 13.5.7 Preparing the Drug Plate [Seite 340]
1.6.4.5.8 - 13.5.8 Indicator Dye Loading Procedure [Seite 340]
1.6.4.5.9 - 13.5.9 KSR Protocol: Fluorescence Settings [Seite 341]
1.6.4.5.9.1 - 13.5.9.1 Data Capture [Seite 342]
1.6.4.5.9.2 - 13.5.9.2 Assay Protocol Settings [Seite 342]
1.6.4.5.9.3 - 13.5.9.3 Plate Protocol Settings [Seite 343]
1.6.4.5.9.4 - 13.5.9.4 Quality Control [Seite 343]
1.6.4.6 - References [Seite 343]
1.7 - SECTION IV DATA MANAGEMENT, DATA ANALYSIS, AND SYSTEMS BIOLOGY [Seite 345]
1.7.1 - 14. Open File Formats for High Content Analysis [Seite 347]
1.7.1.1 - 14.1 Introduction [Seite 347]
1.7.1.2 - 14.2 The Data Problem in Biology: Why is it so Hard? [Seite 347]
1.7.1.3 - 14.3 High Content Data in Biology: A Definition [Seite 348]
1.7.1.4 - 14.4 The Difference Between a File Format and a Minimum Specification [Seite 349]
1.7.1.5 - 14.5 File Formats: Open vs Closed [Seite 349]
1.7.1.6 - 14.6 File Formats: Balancing Flexibility with Standards [Seite 351]
1.7.1.7 - 14.7 Supporting a Successful File Format [Seite 351]
1.7.1.8 - 14.8 Commercial Realities: How Users and Developers Can Define File Formats [Seite 352]
1.7.1.9 - 14.9 OME-XML and OME-TIFF: Moving Towards a Standard Format For High Content Biological Data [Seite 352]
1.7.1.9.1 - 14.9.1 Metadata Support for High Throughput Assays [Seite 354]
1.7.1.10 - 14.10 Data Model and File Format Integration: Towards Usable Tools [Seite 355]
1.7.1.11 - 14.11 Conclusions [Seite 355]
1.7.1.12 - 14.12 Acknowledgments [Seite 356]
1.7.1.13 - References [Seite 356]
1.7.2 - 15. Analysis of Multiparametric HCS Data [Seite 357]
1.7.2.1 - 15.1 Cytological Classification and Profiling [Seite 357]
1.7.2.1.1 - 15.1.1 Multiparametric HCS Data and Cytological Profiling [Seite 357]
1.7.2.1.2 - 15.1.2 Cytological Features [Seite 358]
1.7.2.1.3 - 15.1.3 Using Cytological Features in Assays [Seite 359]
1.7.2.2 - 15.2 Setting Up Cytological Profiling Studies [Seite 361]
1.7.2.2.1 - 15.2.1 Planning for a Cytological Classification Experiment [Seite 361]
1.7.2.2.2 - 15.2.2 Feature Extraction by Image Analysis and Export of Data for Analysis [Seite 363]
1.7.2.2.3 - 15.2.3 Example Studies that Use Cytological Profiling to Study Small Molecule Inhibitors and siRNAs [Seite 364]
1.7.2.3 - 15.3 Sources of Variability and Corrections [Seite 364]
1.7.2.3.1 - 15.3.1 Detection and Elimination of Plate and Sample Outliers from a Data Set [Seite 364]
1.7.2.3.2 - 15.3.2 Visualization of Plate-Level Features to Assess Data Quality [Seite 365]
1.7.2.3.3 - 15.3.3 Normalization and Scaling of Data [Seite 368]
1.7.2.3.4 - 15.3.4 Post-Normalization Analysis of Data Quality [Seite 369]
1.7.2.4 - 15.4 General Analysis Considerations [Seite 369]
1.7.2.4.1 - 15.4.1 Choosing the Appropriate Analysis Level: Well or Cell [Seite 370]
1.7.2.4.1.1 - 15.4.1.1 Cell Cycle Analysis [Seite 370]
1.7.2.4.1.2 - 15.4.1.2 Perturbations Where the Cell Is an Effective Experimental Block [Seite 370]
1.7.2.4.2 - 15.4.2 Statistical Summaries for Cell-Level Features [Seite 371]
1.7.2.4.3 - 15.4.3 Feature Relationships, Redundancy, and Selection [Seite 371]
1.7.2.5 - 15.5 Data Analysis Methods [Seite 374]
1.7.2.5.1 - 15.5.1 Feature Transformation [Seite 374]
1.7.2.5.2 - 15.5.2 Linear Modeling of Feature Responses [Seite 375]
1.7.2.5.3 - 15.5.3 Unsupervised Clustering Methods [Seite 376]
1.7.2.5.4 - 15.5.4 Supervised Classification Methods [Seite 379]
1.7.2.6 - 15.6 Software for HCS Data Analysis [Seite 380]
1.7.2.7 - 15.7 Conclusions [Seite 380]
1.7.2.8 - References [Seite 381]
1.7.3 - 16. Quantitative and Qualitative Cellular Genomics: High Content Analysis as an End Point for HT-RNAi Phenotype Profiling Using GE's IN Cell Platform [Seite 383]
1.7.3.1 - 16.1 Cellular Genomics [Seite 383]
1.7.3.2 - 16.2 Enabling Technologies to Facilitate Cellular Genomics: RNA Interference [Seite 385]
1.7.3.3 - 16.3 High Throughput RNAi (HT-RNAi) [Seite 386]
1.7.3.3.1 - 16.3.1 Platforms and Screening Infrastructure [Seite 386]
1.7.3.3.2 - 16.3.2 Establishing Methods for Successful HT-RNAi [Seite 386]
1.7.3.4 - 16.4 High Content Analysis (HCA) for High Throughput Phenotype Profiling [Seite 390]
1.7.3.4.1 - 16.4.1 IN Cell Analyzer 1000 [Seite 391]
1.7.3.4.2 - 16.4.2 IN Cell Analyzer 3000 [Seite 391]
1.7.3.4.3 - 16.4.3 HCA Assay Suites [Seite 391]
1.7.3.4.4 - 16.4.4 Fixed-Cell Assays [Seite 394]
1.7.3.4.5 - 16.4.5 Live-Cell Assays [Seite 394]
1.7.3.5 - 16.5 Future Directions [Seite 396]
1.7.3.6 - References [Seite 396]
1.7.4 - 17. Optimal Characteristics of Protein-Protein Interaction Biosensors for Cellular Systems Biology Profiling [Seite 399]
1.7.4.1 - 17.1 Introduction [Seite 399]
1.7.4.2 - 17.2 Challenge of Cellular Systems Biology (CSB) [Seite 400]
1.7.4.3 - 17.3 Optimal Characteristics of Protein-Protein Interaction Biosensors (PPIBs) [Seite 401]
1.7.4.4 - 17.4 Example of a PPIB and Cellular Systems Biology Profiling [Seite 403]
1.7.4.4.1 - 17.4.1 Testing a First-Generation p53-HDM2 PPIB Based on Full Length and Protein Fragments [Seite 403]
1.7.4.4.2 - 17.4.2 Overexpression of a Labeled p53 Fusion Protein Modulates Multiple Cellular Systems: Testing a Critical Potential Problem [Seite 407]
1.7.4.4.3 - 17.4.3 An Optimized p53-HDM2 PPIB [Seite 408]
1.7.4.5 - 17.5 Summary and Prospects [Seite 412]
1.7.4.6 - 17.6 Acknowledgments [Seite 413]
1.7.4.7 - References [Seite 413]
1.8 - Index [Seite 417]