Measuring biological responses with automated microscopy

Preface......Page 8
Introduction......Page 40
Design and Construction of Dynamic Cell Cycle Sensors......Page 42
Validation of Sensors......Page 44
Cell Cycle Analysis by Automated High-Throughput Imaging for Drug Profiling and Target Validation......Page 48
Acknowledgments......Page 56
References......Page 57
High-Content Fluorescence-Based Screening for Epigenetic Modulators......Page 60
Introduction......Page 61
Epigenetic Regulators of Gene Expression as Drug Targets......Page 62
Rationale for the Development of Cell-Based Assays to Screen for Epigenetic Modulators......Page 63
Choice of Cell Line, Reporter System, and Selection Marker for High Content Transcriptional Assays......Page 64
Development and Basic Characterization of Cell-Based Fluorescent Assays to Screen for Epigenetic Modulators......Page 65
Setup and Optimization of Primary Screens......Page 67
Evaluation of Hit Compounds......Page 71
Available Secondary Screens for the Evaluation of Candidate Epigenetic Modulators......Page 72
General Conclusions and Perspectives......Page 73
References......Page 74
Nuclear Receptor Biology......Page 76
Rationale for the Development of Translocation Assays for Nuclear Receptor Ligand Discovery......Page 77
Design of Translocating Nuclear Receptor Chimeras......Page 78
Protocol for the Design of Translocating Chimeras......Page 80
Construction of Mammalian Cell Lines Stably Expressing GFP-Tagged Chimeric Receptors......Page 82
Simple Protocol for Evaluation of Known Ligands......Page 83
Protocol for Manual Evaluation of Dose Responsiveness and EC50 Calculations......Page 84
Automated Measurements of Nuclear Translocation......Page 86
Perspectives and Future Applications......Page 87
References......Page 88
The Ligand-Independent Translocation Assay: An Enabling Technology for Screening Orphan G Protein-Coupled Receptors by Arrestin Recruitment......Page 89
Introduction......Page 90
Overview of the LITe Assay......Page 92
Early Validation of oGPCRs in Transfluor......Page 94
Validation of oGPCR Stable Cell Lines for Transfluor Screening Campaigns......Page 96
Quality Control for Transfluor Screening Campaigns......Page 98
References......Page 101
High-Content Screening of Known G Protein-Coupled Receptors by Arrestin Translocation......Page 102
G Protein-Coupled Receptors (GPCRs), G Protein-Coupled Receptor Kinases (GRKs), and Arrestins......Page 103
ArrestinGFP Translocation Assay......Page 105
Evaluation of Response......Page 106
Clone Selection and Expansion......Page 108
Identification of Positive Clones......Page 109
Choosing the Best Clone for the Screen......Page 110
Scale-up of Cells......Page 112
Liquid-Handling Assay Protocol......Page 113
Harvesting High Content Data......Page 114
Acknowledgments......Page 116
References......Page 117
Introduction......Page 118
Overview......Page 120
Kinetics of Receptor Internalization......Page 121
Overview......Page 124
Data Analysis......Page 125
Assay Characterization......Page 126
Assay Application......Page 128
Assay Protocol......Page 130
Assay Characterization......Page 131
Screening......Page 132
Acknowledgments......Page 136
References......Page 137
Introduction......Page 138
Technology......Page 141
Image Analysis Algorithm......Page 144
EC50 Fitting and Scoring......Page 147
Tissue Preparation......Page 148
Single Target Expression Profiling: Real Time Quantitative PCR......Page 149
Development of Stable Cell Line......Page 150
Materials......Page 152
Image Analysis Algorithm......Page 154
Example of Experimental Findings......Page 155
Concluding Remarks......Page 157
References......Page 158
Introduction......Page 160
High-Throughput Confocal Cellular Imaging Systems......Page 161
GPCR Internalization Assays......Page 162
Fluorophore-Labeled Ligand Internalization......Page 165
Ligand Cointernalization Protocol......Page 166
Image Analysis and Quantification......Page 168
Materials......Page 169
Imaging......Page 170
Conclusions and Outlook......Page 172
References......Page 176
Introduction......Page 179
Reagents and Materials......Page 181
Primary Preosteoblast Cell Culture and Compound Screening......Page 182
Immunofluorescent Staining......Page 183
Imaging and Quantitation of Nuclear Translocation......Page 184
References......Page 187
A Live Cell, Image-Based Approach to Understanding the Enzymology and Pharmacology of 2-Bromopalmitate and Palmitoylation......Page 189
Introduction......Page 190
Protein Prenylation......Page 191
Protein Acylation......Page 192
Palmitate Turnover......Page 193
Enzymes for Depalmitoylation......Page 194
Role of Palmitoylation in Development......Page 195
Cysteines: Primary Sites of Palmitoylation......Page 197
Palmitoyl Acyl Transferases (PATs)......Page 198
Links between Palmitoylation and Disease......Page 199
Monomeric Fluorescent Proteins: A Critical Feature for Studying Palmitoylation......Page 200
High-Throughput Microscopy (HTM)/High-Content Screening (HCS) to Study Lipid-Modified Proteins......Page 202
Morphometric Analysis of Palmitoylation with HCS......Page 203
GAP43:YFP is Palmitoylated, Localized to the Plasma Membrane, and Is a Stereotypical Reporter of the Cellular Capacity for Palmitoylation......Page 204
Determination of the Residence Half-Life of Palmitate on GAP43:YFP Using HCS......Page 206
Thora Can Measure Precisely the Subcellular Distribution of GAP43:YFP: IC50 of 2BP......Page 207
Cytotoxic Effects of Antagonists of Palmitoylation......Page 210
Creation of Stable Reporter Cell Lines......Page 213
Choice of Cell Type......Page 214
Troubleshooting......Page 215
Image Organization and Analysis......Page 217
References......Page 218
High-Throughput Microscopy (HTM)......Page 227
Steroid Nuclear Receptors and Coregulators......Page 228
Coating with Poly-D-Lysine......Page 230
Formaldehyde Fixation and Immunolabeling of Cells......Page 231
Immunolabeling......Page 232
Reagents and Supplies......Page 233
Nuclear Receptor Coregulator SRC-3......Page 234
Imaging, Data Extraction and Filtering, and Data Analysis......Page 235
Androgen Receptor......Page 237
Cell Culture, Treatment, and Labeling......Page 239
Imaging, Data Extraction, and Data Analysis......Page 240
Estrogen Receptor......Page 242
Cell Culture, Treatment, and Labeling......Page 245
Imaging, Data Extraction and Filtering, and Data Analysis......Page 246
References......Page 248
Introduction......Page 250
Semiconductor Quantum Dots as Biological Probes......Page 251
Single Molecule Imaging in Living Cells......Page 252
Single Quantum Dot Tracking of Membrane Proteins......Page 254
Antibodies and Quantum Dots......Page 255
Single Quantum Dot Tracking of Intracellular Proteins......Page 256
Culture Reagents and Buffers......Page 257
Purification of Quantum Dots Streptavidin Conjugates (QD-SAVs)......Page 258
Optical Microscope......Page 259
Single Molecule Imaging in Live Cells......Page 260
Measurement of the Point Spread Function......Page 262
Blinking......Page 263
Mean Square Displacement Function......Page 264
References......Page 265
Introduction......Page 0
Introduction......Page 268
Microscope......Page 269
Image Database Module......Page 272
Image Segmentation and Connected Component Analysis Module......Page 273
Multiparametric Scoring of Changes in Treated versus Control Cells......Page 274
Chemical Compounds Screens......Page 275
Protocol for Cell-Based FA Perturbation Screening......Page 276
siRNA Screen......Page 277
A cDNA screen for Identifying Novel Structural Cellular Proteins Based on Localization of YFP-Fusion Proteins......Page 279
Protocol for cDNA Screening......Page 281
Screening for Genes Affecting Cell Migration......Page 282
Protocol for Motility Assay......Page 283
Acknowledgments......Page 284
References......Page 285
Introduction......Page 287
Design and Construction of Adenoviral Sensors for Cellular Assays......Page 288
Application of Adenoviral Sensors to High-Content Analysis......Page 293
EGFP-Glucocorticoid Receptor (GR) Translocation Sensor......Page 294
Assay Procedure......Page 296
Assay Analysis......Page 297
Nuclear Factor of Activated T Cells (NFAT) Nitroreductase Reporter Gene Sensor......Page 298
Assay Procedure......Page 299
Assay Analysis......Page 300
Conclusions and Future Perspectives......Page 301
References......Page 302
Cell-Based Assays Using Primary Endothelial Cells to Study Multiple Steps in Inflammation......Page 305
Selection of a Suitable Cell System......Page 306
Selection of the Optimal Probe......Page 307
Cell Seeding and Stimulation......Page 309
Fixing and Staining Solutions......Page 310
Image Acquisition......Page 311
E-Selectin and VCAM-1 Expression Assays......Page 312
The NF-kappaB Translocation Assay Produced Data with High Confidence......Page 313
NF-kappaB Translocation Depends on IkappaB Kinase (IKK) and Proteasome Activity......Page 314
Assay Principle......Page 315
E-Selectin Expression Has Multiple Activators......Page 317
Assay Principle......Page 318
Concluding Remarks and Future Perspectives......Page 320
References......Page 321
Introduction......Page 323
General Considerations......Page 324
Monitoring Cell Cycle Progression......Page 326
Cell Culture......Page 328
Cyclin-B1-GFP Live Cell Assay......Page 329
Phosphohistone H3 Immunfluorescence Assay for Detecting Late G2 and Mitotic Populations......Page 330
Visualizing Apoptosis by Fluorescence Microscopy......Page 331
Apoptosis Detection Using Antibodies to Cleaved PARP......Page 332
Protocol......Page 333
Detecting Morphological Changes Associated with Apoptosis......Page 334
Multiplexing Cell Cycle and Apoptosis Assays......Page 335
Protocol......Page 337
Acknowledgments......Page 338
References......Page 339
General Considerations of Cell-Based Assay Technology Applied to Stem and Progenitor Cell Biology......Page 340
Promoter-Reporter Constructs......Page 341
Lentiviral Technology to Create Stable Reporter Lines......Page 342
Validation of Cell Lines......Page 343
Assay Development......Page 344
Imaging and Processing a Stem Cell Differentiation Assay......Page 346
Notes on Data Handling......Page 349
Cell Plating......Page 352
Data Handling......Page 353
References......Page 354
Introduction......Page 356
Media and Reagents......Page 358
Fluorescence Microscopy......Page 359
Measurement of Resting Ca2+ State and Stimulated [Ca2+]i Response......Page 360
Decay Kinetics of Agonist-Stimulated [Ca2+]i Response......Page 363
Conclusions from HCA and Rationale for High-Content Screening......Page 364
High-Content Screening......Page 365
Cell Plating, Labeling, and Imaging Setup......Page 366
Data Display, Analysis, and Report......Page 368
Acknowledgments......Page 371
References......Page 372
Introduction......Page 374
Overview......Page 375
Assay Protocol......Page 381
Overview......Page 382
Assay Protocol......Page 384
References......Page 386
Introduction......Page 387
Assay Procedures......Page 388
Immunofluorescence Labeling......Page 389
High-Content Analysis......Page 390
Image Acquisition and Object Identification......Page 391
Optimization of Fluorophore Labels to Minimize Channel Bleed Through......Page 392
Evaluation of Assay Reliability......Page 397
Output Parameters Related to Cell Health......Page 398
Subpopulation Analysis......Page 399
References......Page 401
Introduction......Page 403
Definition of the Cell Model......Page 405
Generation and Characterization of MK2-EGFP HeLa Cell Line......Page 406
Cellomics ArrayScan Automated Imaging Platform......Page 408
Characterization of MK2-EGFP Clones via Imaging......Page 411
Characterization of the p38 MAPK Signaling Pathway in HeLa-MK2-EGFP Cells......Page 414
Assay Development on the ArrayScan 3.1 Imaging Platform......Page 415
MK2-EGFP Translocation Assay Reproducibility and Signal Widow Evaluation......Page 417
Secondary Analysis Parameters......Page 419
Discussion......Page 423
References......Page 426
Introduction......Page 429
Definition of the Cell Model......Page 430
Cellomics ArrayScan Automated Imaging Platform......Page 433
Development of the JNK MAPK Signaling Pathway Assay......Page 434
Buffers and Working Solutions......Page 438
c-Jun Activation Signal Window and Reproducibility......Page 439
Development of the ERK MAPK Signaling Pathway Assay......Page 440
Buffers and Working Solutions......Page 445
ERK1/2 Activation Signal Window and Reproducibility......Page 446
MAPK Pathway Inhibitor Test Cassette......Page 447
p38a Inhibitor Profiling......Page 452
JNK Inhibitor Profiling......Page 453
References......Page 456
Introduction......Page 458
Cellomics ArrayScan Automated Imaging Platform......Page 459
Conversion of the 96-Well MK2-EGFP Translocation Assay to a 384-Well Format Assay on the Arrayscanreg Imager......Page 460
MK2-EGFP Translocation Assay Reproducibility and Signal Widow Evaluation......Page 464
MK2-EGFP Translocation HTS Assay for p38 Inhibitors......Page 468
Discussion......Page 474
References......Page 477
Introduction......Page 479
Quantifying Cellular Morphology Changes......Page 480
Preparation of Compounds Plates......Page 481
Microscopy......Page 482
Image Analysis......Page 483
Analysis of Quantitative Cellular Phenotypes across Cell Lines......Page 486
Comparing Attribute Changes Across Cell Types......Page 490
Signature Construction and Visualization......Page 492
Clustering and Classification of Compounds......Page 497
Classification......Page 498
Conclusions......Page 500
References......Page 505
Cellular Assay and Imaging Preparation......Page 508
Image Acquisition......Page 509
Wide-Field Versus Confocal Systems......Page 510
Objectives......Page 513
Autofocus Mechanisms......Page 514
Image Analysis......Page 515
Image Database and Data Visualization Tools......Page 517
Protocol for the Transfluor Assay to Screen for Small Molecule Inhibitors......Page 520
Protocol for Image Analysis Routine (Granularity Analysis Algorithm from IN Cell Analyzer 3000) on the Transfluor Assay (Fig. 5)......Page 521
References......Page 522
Introduction......Page 523
Assay Processing......Page 526
Cell and Compound Plate Preparation......Page 527
Automated Assay Processing......Page 529
Process Control Software......Page 532
Imaging System Instrumentation......Page 534
Imaging Parameter Optimization......Page 535
Imaging Speed Optimization......Page 537
Image and Data Analysis......Page 538
Basic Feature Extraction/Cell-Level Analysis......Page 539
Cell Classification/Well-Level Population Analysis......Page 540
Multiwell and Plate-Level Analysis......Page 541
Analysis Program Example......Page 543
Analysis Software Components......Page 544
Data Review and Quality Control......Page 546
Identifying Potential Problems That May Affect Data Integrity......Page 547
Software Tools......Page 548
Summary......Page 550
References......Page 551
Pathway Screening Using BioImage Redistribution Technology......Page 552
p53-Hdm2 Protein-Protein Interaction Assay......Page 555
Assay Protocol......Page 557
Use of High-Content Assays in RNAi Studies......Page 559
Use of siRNA-Mediated Knockdown to Validate Akt Isoform Dependency of a FKHR Redistribution Assay......Page 561
General Protocol for siRNA Transfection in 96-Well Plates......Page 564
Assay-Specific Cell-to-Cell Heterogeneity Plays a Role in Assay Quality......Page 565
Future Developments......Page 567
References......Page 568
Introduction......Page 570
cDNA Libraries......Page 571
Chemically Synthesized siRNAs......Page 573
Plasmid Short-Hairpin siRNAs......Page 574
Maintaining Large Arrayed-Well Plasmid cDNA or shRNA Clone Libraries......Page 575
Collection Replication......Page 577
Growth of Bacterial Cultures in High-Throughput Format for DNA Preparation......Page 578
Preparing DNA from 96-Well Deep-Well Block Cultures......Page 579
Normalization of Plasmid DNA......Page 581
Arraying Collections into High-Throughput Assay Plates......Page 582
To Determine the DNA Concentration......Page 583
Reagents......Page 584
Transfection Protocol (Lentiviral Packaging)......Page 586
Automated Microscopy......Page 588
High-Throughput HCS Equipment......Page 590
Automated Microtiter Plate Washers......Page 592
Fluorescent Biomarkers for HCS Applications......Page 593
Selecting Appropriate Cell Lines for HCS Assays......Page 595
Protocols for Applying Cell Fixatives......Page 596
Determining Transfection or Transduction Efficiency......Page 597
Quantitative Image Analysis......Page 598
Image Segmentation......Page 599
Summary......Page 601
References......Page 603
Introduction......Page 605
The Principle of Laser-Scanning Microplate Cytometers......Page 607
Preparation of the Library of Pharmacologically Active Compounds (LOPAC) Titration Series......Page 608
1536-Well Plate Liquid Handling......Page 609
Solutions and Materials......Page 610
High-Throughput Screening Protocol......Page 612
Validation of the 1536-Well GR Nuclear Translocation Assay......Page 614
Locus Derepression Assay......Page 615
Assay Optimization for 1536-Well Plate Format......Page 616
Solutions and Materials......Page 617
High-Throughput Screening Protocol......Page 618
beta-Arrestin:beta2-Adrenergic Receptor (betaARR:beta2AR) Protein Fragment Complementation Assay......Page 619
High-Throughput Screening Protocol......Page 621
Assay Validation......Page 624
Summary......Page 625
References......Page 626
Plate::Vision......Page 629
Optical Setup......Page 630
Readout Methods......Page 632
Absorbance Assays......Page 633
TRF Assays......Page 637
Fluorescence Intensity (FI) and Fluorescence Polarization (FP) Assays......Page 638
References......Page 639
Background......Page 640
HCS Instrumentation and Assay Design......Page 643
Advanced Reagents to Measure and Manipulate Cellular Constituents......Page 645
Informatics for Systems Cell Biology......Page 647
Cancer Model Using the Human Lung Carcinoma Cell Line (A549) Expressing Wild-Type p53......Page 649
Summary and Conclusions......Page 652
Prospectus......Page 653
References......Page 655
Introduction......Page 659
Hardware......Page 660
Software......Page 663
Viscoelasticity......Page 666
Conclusion......Page 668
Appendix......Page 669
References......Page 671
Introduction......Page 672
Operating Principle of a Streak Camera......Page 673
Measurement Principle of FLIM System......Page 675
System Calibration......Page 677
Lifetime Imaging in Cells......Page 678
FRET Imaging in Cells......Page 679
References......Page 681