Content: Dynamic GFP Sensors for High-Content Analysis of the Cell Cycle High Content Fluorescence-based Screening for Epigenetic Modulators Development of Assays for Nuclear Receptor Modulators Using Fluorescently Tagged Proteins The Ligand-Independent Translocation Assay: An Enabling Technology for Screening Orphan GPCRs by Arrestin Recruitment High Content Screening of Known G Protein-Coupled Receptors by Arrestin Translocation Cell Imaging Assays for G protein-coupled Receptor Internalization: Application to High Throughput Screening High throughput confocal microscopy for beta arrestin-GFP translocation GPCR assays using the Evotec Opera G-protein coupled receptor internalization assays in the High Content Screening Format Screening for Activators of the Wnt/Fzd Pathway by Automated Fluorescent Microscopy A live-cell, image based approach to understanding the enzymology and pharmacology of 2-bromopalmitate and palmitoylation Quantitative single cell studies of nuclear receptor function using high-resolution HTM and image analyses Tracking the motion of individual proteins with semiconductor quantum dots Development and Application of Automatic High Resolution Light Microscopy for Cell Based Screens Adenoviral Sensors for High-Content Cellular Analysis Cell based assays in primary endothelial cells to study multiple steps in inflammation Development and Implementation of Multiplexed Cell-Based Imaging Assays High throughput screening for modulators of stem cell differentiation High Content Kinetic Calcium Imaging in Drug Sensitive and Drug Resistant Human Breast Cancer Cells Measurement and Analysis of Calcium Signaling in Heterogeneous Cell Cultures Mulipliex Analysis of Inflammatory Signalling Pathways using a High Content Imaging System Generation and Characterization of a Stable MK2-EGFP Cell Line and Subswquent Development of a High Content Imaging Assay on the Cellomics ArrayScan(R) Platform to Screen for p38 MAPK Inhibitors Development and implementation of three MAPK signaling pathway imaging assays to provide MAPK module selectivity profiling for kinase inhibitors; MK2-EGFP translocation, c-Jun & ERK activation. Assay Development and Case History of a 32K Biased Library High Content MK2-EGFP Translocation Screen to Identify p38 MAPK Inhibitors on the ArrayScan(R) 3.1 Imaging Platform Development and Implementation of a dual target MAPK high content assay (MK2-EGFP translocation & cJun activation) to provide LO support for kinase inhibitors Compound classification using image-based cellular phenotypes High Content Screening: Emerging Hardware and Software Technologies An Infrastructure for High Throughput Microscopy: Instrumentation, Informatics, and Integration Protein Translocation Assays: Key Tools for Accessing New Biological Information with High Throughput Microscopy High-Content Screening of Functional Genomic Libraries Fluorescent protein-based cellular assays analyzed by laser scanning microplate cytometry in 1536-well plate format High-Throughput Measurements of Biochemical Responses Using the Plate::Vision Multimode 96 Mini-lens Array Reader Systems Cell Biology Based on High Content Screening Digital Autofocus Methods for Automated Microscopy Fluorescence Lifetime Imagine Microscopy Two-dimensional distribution measurement of fluorescence lifetime
Author(s): James Inglese
Series: Methods in Enzymology 414
Edition: 1
Publisher: Academic press
Year: 2006
Language: English
Pages: 743
000 fm.pdf......Page 2
Preface......Page 3
003 TOC.pdf......Page 6
004 contributors.pdf......Page 11
005 auther index.pdf......Page 17
Introduction......Page 52
Design and Construction of Dynamic Cell Cycle Sensors......Page 54
Validation of Sensors......Page 56
Cell Cycle Analysis by Automated High-Throughput Imaging for Drug Profiling and Target Validation......Page 60
Acknowledgments......Page 68
References......Page 69
High-Content Fluorescence-Based Screening for Epigenetic Modulators......Page 73
Introduction......Page 74
Epigenetic Regulators of Gene Expression as Drug Targets......Page 75
Rationale for the Development of Cell-Based Assays to Screen for Epigenetic Modulators......Page 76
Choice of Cell Line, Reporter System, and Selection Marker for High Content Transcriptional Assays......Page 77
Development and Basic Characterization of Cell-Based Fluorescent Assays to Screen for Epigenetic Modulators......Page 78
Setup and Optimization of Primary Screens......Page 80
Evaluation of Hit Compounds......Page 84
Available Secondary Screens for the Evaluation of Candidate Epigenetic Modulators......Page 85
General Conclusions and Perspectives......Page 86
References......Page 87
Nuclear Receptor Biology......Page 89
Rationale for the Development of Translocation Assays for Nuclear Receptor Ligand Discovery......Page 90
Design of Translocating Nuclear Receptor Chimeras......Page 91
Protocol for the Design of Translocating Chimeras......Page 93
Construction of Mammalian Cell Lines Stably Expressing GFP-Tagged Chimeric Receptors......Page 95
Simple Protocol for Evaluation of Known Ligands......Page 96
Protocol for Manual Evaluation of Dose Responsiveness and EC50 Calculations......Page 97
Automated Measurements of Nuclear Translocation......Page 99
Perspectives and Future Applications......Page 100
References......Page 101
The Ligand-Independent Translocation Assay: An Enabling Technology for Screening Orphan G Protein-Coupled Receptors by Arrestin Recruitment......Page 103
Introduction......Page 104
Overview of the LITe Assay......Page 106
Early Validation of oGPCRs in Transfluor......Page 108
Validation of oGPCR Stable Cell Lines for Transfluor Screening Campaigns......Page 110
Quality Control for Transfluor Screening Campaigns......Page 112
References......Page 115
High-Content Screening of Known G Protein-Coupled Receptors by Arrestin Translocation......Page 117
G Protein-Coupled Receptors (GPCRs), G Protein-Coupled Receptor Kinases (GRKs), and Arrestins......Page 118
ArrestinGFP Translocation Assay......Page 120
Evaluation of Response......Page 121
Clone Selection and Expansion......Page 123
Identification of Positive Clones......Page 124
Choosing the Best Clone for the Screen......Page 125
Scale-up of Cells......Page 127
Liquid-Handling Assay Protocol......Page 128
Harvesting High Content Data......Page 129
Acknowledgments......Page 131
References......Page 132
Introduction......Page 133
Overview......Page 135
Kinetics of Receptor Internalization......Page 136
Overview......Page 139
Data Analysis......Page 140
Assay Characterization......Page 141
Assay Application......Page 143
Assay Protocol......Page 145
Assay Characterization......Page 146
Screening......Page 147
Acknowledgments......Page 151
References......Page 152
Introduction......Page 153
Technology......Page 156
Image Analysis Algorithm......Page 159
EC50 Fitting and Scoring......Page 162
Tissue Preparation......Page 163
Single Target Expression Profiling: Real Time Quantitative PCR......Page 164
Development of Stable Cell Line......Page 165
Materials......Page 167
Image Analysis Algorithm......Page 169
Example of Experimental Findings......Page 170
Concluding Remarks......Page 172
References......Page 173
Introduction......Page 175
High-Throughput Confocal Cellular Imaging Systems......Page 176
GPCR Internalization Assays......Page 177
Fluorophore-Labeled Ligand Internalization......Page 180
Ligand Cointernalization Protocol......Page 181
Image Analysis and Quantification......Page 183
Materials......Page 184
Imaging......Page 185
Conclusions and Outlook......Page 187
References......Page 191
Introduction......Page 194
Reagents and Materials......Page 196
Primary Preosteoblast Cell Culture and Compound Screening......Page 197
Immunofluorescent Staining......Page 198
Imaging and Quantitation of Nuclear Translocation......Page 199
References......Page 202
A Live Cell, Image-Based Approach to Understanding the Enzymology and Pharmacology of 2-Bromopalmitate and Palmitoylation......Page 205
Introduction......Page 206
Protein Prenylation......Page 207
Protein Acylation......Page 208
Palmitate Turnover......Page 209
Enzymes for Depalmitoylation......Page 210
Role of Palmitoylation in Development......Page 211
Cysteines: Primary Sites of Palmitoylation......Page 213
Palmitoyl Acyl Transferases (PATs)......Page 214
Links between Palmitoylation and Disease......Page 215
Monomeric Fluorescent Proteins: A Critical Feature for Studying Palmitoylation......Page 216
High-Throughput Microscopy (HTM)/High-Content Screening (HCS) to Study Lipid-Modified Proteins......Page 218
Morphometric Analysis of Palmitoylation with HCS......Page 219
GAP43:YFP is Palmitoylated, Localized to the Plasma Membrane, and Is a Stereotypical Reporter of the Cellular Capacity for Palmitoylation......Page 220
Determination of the Residence Half-Life of Palmitate on GAP43:YFP Using HCS......Page 222
Thora Can Measure Precisely the Subcellular Distribution of GAP43:YFP: IC50 of 2BP......Page 223
Cytotoxic Effects of Antagonists of Palmitoylation......Page 226
Creation of Stable Reporter Cell Lines......Page 229
Choice of Cell Type......Page 230
Troubleshooting......Page 231
Image Organization and Analysis......Page 233
References......Page 234
High-Throughput Microscopy (HTM)......Page 243
Steroid Nuclear Receptors and Coregulators......Page 244
Coating with Poly-D-Lysine......Page 246
Formaldehyde Fixation and Immunolabeling of Cells......Page 247
Immunolabeling......Page 248
Reagents and Supplies......Page 249
Nuclear Receptor Coregulator SRC-3......Page 250
Imaging, Data Extraction and Filtering, and Data Analysis......Page 251
Androgen Receptor......Page 253
Cell Culture, Treatment, and Labeling......Page 255
Imaging, Data Extraction, and Data Analysis......Page 256
Estrogen Receptor......Page 258
Cell Culture, Treatment, and Labeling......Page 261
Imaging, Data Extraction and Filtering, and Data Analysis......Page 262
References......Page 264
Introduction......Page 266
Semiconductor Quantum Dots as Biological Probes......Page 267
Single Molecule Imaging in Living Cells......Page 268
Single Quantum Dot Tracking of Membrane Proteins......Page 270
Antibodies and Quantum Dots......Page 271
Single Quantum Dot Tracking of Intracellular Proteins......Page 272
Culture Reagents and Buffers......Page 273
Purification of Quantum Dots Streptavidin Conjugates (QD-SAVs)......Page 274
Optical Microscope......Page 275
Single Molecule Imaging in Live Cells......Page 276
Measurement of the Point Spread Function......Page 278
Blinking......Page 279
Mean Square Displacement Function......Page 280
References......Page 281
Development and Application of Automatic High-Resolution Light Microscopy for Cell-Based Screens......Page 284
Introduction......Page 285
Microscope......Page 286
Image Database Module......Page 289
Image Segmentation and Connected Component Analysis Module......Page 290
Multiparametric Scoring of Changes in Treated versus Control Cells......Page 291
Chemical Compounds Screens......Page 292
Protocol for Cell-Based FA Perturbation Screening......Page 293
siRNA Screen......Page 294
A cDNA screen for Identifying Novel Structural Cellular Proteins Based on Localization of YFP-Fusion Proteins......Page 296
Protocol for cDNA Screening......Page 298
Screening for Genes Affecting Cell Migration......Page 299
Protocol for Motility Assay......Page 300
Acknowledgments......Page 301
References......Page 302
Adenoviral Sensors for High-Content Cellular Analysis......Page 304
Introduction......Page 305
Design and Construction of Adenoviral Sensors for Cellular Assays......Page 306
Application of Adenoviral Sensors to High-Content Analysis......Page 311
EGFP-Glucocorticoid Receptor (GR) Translocation Sensor......Page 312
Assay Procedure......Page 314
Assay Analysis......Page 315
Nuclear Factor of Activated T Cells (NFAT) Nitroreductase Reporter Gene Sensor......Page 316
Assay Procedure......Page 317
Assay Analysis......Page 318
Conclusions and Future Perspectives......Page 319
References......Page 320
Cell-Based Assays Using Primary Endothelial Cells to Study Multiple Steps in Inflammation......Page 324
Selection of a Suitable Cell System......Page 325
Selection of the Optimal Probe......Page 326
Cell Seeding and Stimulation......Page 328
Fixing and Staining Solutions......Page 329
Image Acquisition......Page 330
E-Selectin and VCAM-1 Expression Assays......Page 331
The NF-kappaB Translocation Assay Produced Data with High Confidence......Page 332
NF-kappaB Translocation Depends on IkappaB Kinase (IKK) and Proteasome Activity......Page 333
Assay Principle......Page 334
E-Selectin Expression Has Multiple Activators......Page 336
Assay Principle......Page 337
Concluding Remarks and Future Perspectives......Page 339
References......Page 340
Introduction......Page 342
General Considerations......Page 343
Monitoring Cell Cycle Progression......Page 345
Cell Culture......Page 347
Cyclin-B1-GFP Live Cell Assay......Page 348
Phosphohistone H3 Immunfluorescence Assay for Detecting Late G2 and Mitotic Populations......Page 349
Visualizing Apoptosis by Fluorescence Microscopy......Page 350
Apoptosis Detection Using Antibodies to Cleaved PARP......Page 351
Protocol......Page 352
Detecting Morphological Changes Associated with Apoptosis......Page 353
Multiplexing Cell Cycle and Apoptosis Assays......Page 354
Protocol......Page 356
Acknowledgments......Page 357
References......Page 358
Introduction......Page 359
General Considerations of Cell-Based Assay Technology Applied to Stem and Progenitor Cell Biology......Page 360
Promoter-Reporter Constructs......Page 361
Lentiviral Technology to Create Stable Reporter Lines......Page 362
Validation of Cell Lines......Page 363
Assay Development......Page 364
Imaging and Processing a Stem Cell Differentiation Assay......Page 366
Notes on Data Handling......Page 369
Cell Plating......Page 372
Data Handling......Page 373
References......Page 374
Introduction......Page 376
Media and Reagents......Page 378
Fluorescence Microscopy......Page 379
Measurement of Resting Ca2+ State and Stimulated [Ca2+]i Response......Page 380
Decay Kinetics of Agonist-Stimulated [Ca2+]i Response......Page 383
Conclusions from HCA and Rationale for High-Content Screening......Page 384
High-Content Screening......Page 385
Cell Plating, Labeling, and Imaging Setup......Page 386
Data Display, Analysis, and Report......Page 388
Acknowledgments......Page 391
References......Page 392
Introduction......Page 395
Overview......Page 396
Assay Protocol......Page 402
Overview......Page 403
Assay Protocol......Page 405
References......Page 407
Introduction......Page 408
Assay Procedures......Page 409
Immunofluorescence Labeling......Page 410
High-Content Analysis......Page 411
Image Acquisition and Object Identification......Page 412
Optimization of Fluorophore Labels to Minimize Channel Bleed Through......Page 413
Evaluation of Assay Reliability......Page 418
Output Parameters Related to Cell Health......Page 419
Subpopulation Analysis......Page 420
References......Page 422
Introduction......Page 424
Definition of the Cell Model......Page 426
Generation and Characterization of MK2-EGFP HeLa Cell Line......Page 427
Cellomics ArrayScan Automated Imaging Platform......Page 429
Characterization of MK2-EGFP Clones via Imaging......Page 432
Characterization of the p38 MAPK Signaling Pathway in HeLa-MK2-EGFP Cells......Page 435
Assay Development on the ArrayScan 3.1 Imaging Platform......Page 436
MK2-EGFP Translocation Assay Reproducibility and Signal Widow Evaluation......Page 438
Secondary Analysis Parameters......Page 440
Discussion......Page 444
References......Page 447
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......Page 450
Introduction......Page 451
Definition of the Cell Model......Page 452
Cellomics ArrayScan Automated Imaging Platform......Page 455
Development of the JNK MAPK Signaling Pathway Assay......Page 456
Buffers and Working Solutions......Page 460
c-Jun Activation Signal Window and Reproducibility......Page 461
Development of the ERK MAPK Signaling Pathway Assay......Page 462
Buffers and Working Solutions......Page 467
ERK1/2 Activation Signal Window and Reproducibility......Page 468
MAPK Pathway Inhibitor Test Cassette......Page 469
p38a Inhibitor Profiling......Page 474
JNK Inhibitor Profiling......Page 475
References......Page 478
Introduction......Page 480
Cellomics ArrayScan Automated Imaging Platform......Page 481
Conversion of the 96-Well MK2-EGFP Translocation Assay to a 384-Well Format Assay on the Arrayscanreg Imager......Page 482
MK2-EGFP Translocation Assay Reproducibility and Signal Widow Evaluation......Page 486
MK2-EGFP Translocation HTS Assay for p38 Inhibitors......Page 490
Discussion......Page 496
References......Page 499
Introduction......Page 501
Quantifying Cellular Morphology Changes......Page 502
Preparation of Compounds Plates......Page 503
Microscopy......Page 504
Image Analysis......Page 505
Analysis of Quantitative Cellular Phenotypes across Cell Lines......Page 508
Comparing Attribute Changes Across Cell Types......Page 512
Signature Construction and Visualization......Page 514
Clustering and Classification of Compounds......Page 519
Classification......Page 520
Conclusions......Page 522
References......Page 527
Introduction......Page 530
Cellular Assay and Imaging Preparation......Page 531
Image Acquisition......Page 532
Wide-Field Versus Confocal Systems......Page 533
Objectives......Page 536
Autofocus Mechanisms......Page 537
Image Analysis......Page 538
Image Database and Data Visualization Tools......Page 540
Protocol for the Transfluor Assay to Screen for Small Molecule Inhibitors......Page 543
Protocol for Image Analysis Routine (Granularity Analysis Algorithm from IN Cell Analyzer 3000) on the Transfluor Assay (Fig. 5)......Page 544
References......Page 545
Introduction......Page 546
Assay Processing......Page 549
Cell and Compound Plate Preparation......Page 550
Automated Assay Processing......Page 552
Process Control Software......Page 555
Imaging System Instrumentation......Page 557
Imaging Parameter Optimization......Page 558
Imaging Speed Optimization......Page 560
Image and Data Analysis......Page 561
Basic Feature Extraction/Cell-Level Analysis......Page 562
Cell Classification/Well-Level Population Analysis......Page 563
Multiwell and Plate-Level Analysis......Page 564
Analysis Program Example......Page 566
Analysis Software Components......Page 567
Data Review and Quality Control......Page 569
Identifying Potential Problems That May Affect Data Integrity......Page 570
Software Tools......Page 571
Summary......Page 573
References......Page 574
Pathway Screening Using BioImage Redistribution Technology......Page 575
p53-Hdm2 Protein-Protein Interaction Assay......Page 578
Assay Protocol......Page 580
Use of High-Content Assays in RNAi Studies......Page 582
Use of siRNA-Mediated Knockdown to Validate Akt Isoform Dependency of a FKHR Redistribution Assay......Page 584
General Protocol for siRNA Transfection in 96-Well Plates......Page 587
Assay-Specific Cell-to-Cell Heterogeneity Plays a Role in Assay Quality......Page 588
Future Developments......Page 590
References......Page 591
High-Content Screening of Functional Genomic Libraries......Page 593
Introduction......Page 594
cDNA Libraries......Page 595
Chemically Synthesized siRNAs......Page 597
Plasmid Short-Hairpin siRNAs......Page 598
Maintaining Large Arrayed-Well Plasmid cDNA or shRNA Clone Libraries......Page 599
Collection Replication......Page 601
Growth of Bacterial Cultures in High-Throughput Format for DNA Preparation......Page 602
Preparing DNA from 96-Well Deep-Well Block Cultures......Page 603
Normalization of Plasmid DNA......Page 605
Arraying Collections into High-Throughput Assay Plates......Page 606
To Determine the DNA Concentration......Page 607
Reagents......Page 608
Transfection Protocol (Lentiviral Packaging)......Page 610
Automated Microscopy......Page 612
High-Throughput HCS Equipment......Page 614
Automated Microtiter Plate Washers......Page 616
Fluorescent Biomarkers for HCS Applications......Page 617
Selecting Appropriate Cell Lines for HCS Assays......Page 619
Protocols for Applying Cell Fixatives......Page 620
Determining Transfection or Transduction Efficiency......Page 621
Quantitative Image Analysis......Page 622
Image Segmentation......Page 623
Summary......Page 625
References......Page 627
Introduction......Page 629
The Principle of Laser-Scanning Microplate Cytometers......Page 631
Preparation of the Library of Pharmacologically Active Compounds (LOPAC) Titration Series......Page 632
1536-Well Plate Liquid Handling......Page 633
Solutions and Materials......Page 634
High-Throughput Screening Protocol......Page 636
Validation of the 1536-Well GR Nuclear Translocation Assay......Page 638
Locus Derepression Assay......Page 639
Assay Optimization for 1536-Well Plate Format......Page 640
Solutions and Materials......Page 641
High-Throughput Screening Protocol......Page 642
beta-Arrestin:beta2-Adrenergic Receptor (betaARR:beta2AR) Protein Fragment Complementation Assay......Page 643
High-Throughput Screening Protocol......Page 645
Assay Validation......Page 648
Summary......Page 649
References......Page 650
Introduction......Page 653
Plate::Vision......Page 654
Optical Setup......Page 655
Readout Methods......Page 657
Absorbance Assays......Page 658
TRF Assays......Page 662
Fluorescence Intensity (FI) and Fluorescence Polarization (FP) Assays......Page 663
References......Page 664
Background......Page 665
HCS Instrumentation and Assay Design......Page 668
Advanced Reagents to Measure and Manipulate Cellular Constituents......Page 670
Informatics for Systems Cell Biology......Page 672
Cancer Model Using the Human Lung Carcinoma Cell Line (A549) Expressing Wild-Type p53......Page 674
Summary and Conclusions......Page 677
Prospectus......Page 678
References......Page 680
Introduction......Page 684
Hardware......Page 685
Software......Page 688
Viscoelasticity......Page 691
Conclusion......Page 693
Appendix......Page 694
References......Page 696
Introduction......Page 697
Operating Principle of a Streak Camera......Page 698
Measurement Principle of FLIM System......Page 700
System Calibration......Page 702
Lifetime Imaging in Cells......Page 703
FRET Imaging in Cells......Page 704
References......Page 706
subject index.pdf......Page 707
x MIE index.pdf......Page 720
