Genetic Toxicology Testing

A Laboratory Manual
 
 
Academic Press
  • 1. Auflage
  • |
  • erschienen am 28. Mai 2016
  • |
  • 460 Seiten
 
E-Book | ePUB mit Adobe DRM | Systemvoraussetzungen
E-Book | PDF mit Adobe DRM | Systemvoraussetzungen
978-0-12-801006-8 (ISBN)
 

Genetic Toxicology Testing: A Laboratory Manual presents a practical guide to genetic toxicology testing of chemicals in a GLP environment. The most commonly used assays are described, from laboratory ad test design to results analysis. In a methodical manner, individual test methods are described step-by-step, along with equipment, suggested suppliers, recipes for reagents, and evaluation criteria.

An invaluable resource in the lab, this book will help to troubleshoot any assay problems you may encounter to optimise quality and efficiency in your genetic toxicology tests. Genetic Toxicology Testing: A Laboratory Manual is an essential reference for those new to the genetic toxicology laboratory, or anyone involved in setting up their own.


  • Offers practical and consistent guidance on the most commonly-performed tests and procedures in a genetic toxicology lab
  • Describes standard genetic toxicology assays, their methodology, reagents, suppliers, and analysis of their results
  • Includes guidance on general approaches: formulation for in vitro assays, study monitoring, and Good Laboratory Practice (GLP)
  • Serves as an essential reference for those new to the genetic toxicology laboratory, or anyone involved in setting up their own lab
  • Englisch
  • San Diego
  • |
  • USA
Elsevier Science
  • 6,95 MB
978-0-12-801006-8 (9780128010068)
0128010061 (0128010061)
weitere Ausgaben werden ermittelt
  • Front Cover
  • Genetic Toxicology Testing
  • Copyright Page
  • Contents
  • List of Contributors
  • Foreword
  • Preface
  • References
  • 1 A Practical Guide to Genetic Toxicology Testing
  • 1.1 Introduction
  • References
  • 2 General Recommendations
  • 2.1 Establishing a New Assay
  • 2.1.1 Method Set-Up
  • 2.1.2 Method Validation
  • 2.2 Spreadsheets and Manipulation of Results
  • 2.3 Laboratory Historical Control Databases
  • 2.3.1 Vehicle/Negative Control Database
  • 2.3.2 Positive Control Database
  • 2.3.3 Spreadsheet Calculations
  • 2.4 Use of Computer Systems
  • 2.5 Study Design
  • 2.6 Evaluation Criteria
  • 2.6.1 Valid Assay
  • 2.6.2 Criteria for Interpretation of Results
  • 2.6.3 Statistical Analysis
  • 2.7 Organization of SOPs
  • 2.8 Planning a Study
  • 2.9 Preparing a Protocol Complying with GLP
  • 2.9.1 Standardized Boilerplate Protocols
  • 2.10 Collecting Results
  • 2.10.1 Data Tabulation
  • 2.10.2 Presentation of Report Tables
  • 2.11 Reports
  • 2.12 Training
  • 2.13 Improving Quality and Efficiency
  • 2.13.1 Improving Quality
  • 2.13.2 Minimizing the Potential Problems on Studies
  • 2.13.3 Reducing the Need for Repetition of Parts of the Study
  • 2.13.4 Accommodating Repeat or Supplementary Tests
  • 2.13.5 Running More Studies in a Given Timeframe
  • 2.13.6 Timeframe for Routine Studies
  • 2.13.7 Reducing the Effort Needed to Prepare Protocols, Tables, and Reports
  • 2.13.8 Reducing the Effort Needed to Perform the Study
  • 2.13.9 Reducing Costs or Labor Requirement
  • 2.14 QA
  • 2.15 Qualifying a Contract Laboratory
  • 2.16 Responsibilities of the Study Monitor
  • References
  • 3 Formulation of Test Articles
  • 3.1 Introduction
  • 3.1.1 Safety
  • 3.1.2 Selecting an Appropriate Formulation
  • 3.2 Formulation Laboratories
  • 3.2.1 Designing and Equipping a Genetic Toxicology Formulation Area
  • 3.2.1.1 Hoods
  • 3.2.1.2 Storage equipment
  • 3.2.2 Personal Protective Clothing
  • 3.2.2.1 Standard
  • 3.2.2.2 Capital equipment
  • 3.2.2.3 Consumables
  • 3.3 Safety Data Sheets
  • 3.4 Receipt of the Test Article
  • 3.5 Formulation Types and Planning
  • 3.6 Solubility and In Vitro Compatibility Testing
  • 3.6.1 Introduction
  • 3.6.2 Choice of Solvent
  • 3.6.3 Solubility Testing
  • 3.6.4 Calculations and Checking: Small-Volume (In Vitro) Assays
  • 3.6.5 Compatibility of Formulation with Culture Medium
  • 3.7 Formulation of Dose Solutions
  • 3.8 Formulation of Bulk Formulations
  • 3.9 Formulation of Suspensions
  • 3.9.1 Aqueous Suspending Agents
  • 3.9.2 Large Volume Suspensions
  • 3.9.3 Small Volume Suspensions
  • 3.10 Chemical Analysis and Stability
  • References
  • 4 The Bacterial Reverse Mutation Test
  • 4.1 Introduction
  • 4.2 History
  • 4.3 Fundamentals
  • 4.4 Equipment
  • 4.5 Consumables
  • 4.6 Reagents and Recipes
  • 4.6.1 Ampicillin 2 µg/disc
  • 4.6.2 Biotin 0.37 mg/mL
  • 4.6.3 Crystal Violet 5 µg/disc
  • 4.6.4 Glucose 0.4 g/mL
  • 4.6.5 G6P 1M: Glucose-6-Phosphate
  • 4.6.6 HBT: 500 µM Histidine, 500µM Biotin, 500 µM Tryptophan Solution
  • 4.6.7 Histidine HCl.H2O 5 mg/mL
  • 4.6.8 KMg
  • 4.6.9 MGA Plates
  • 4.6.10 Minimal Glucose Master (MGM, MGMA and MGMAT) Plates
  • 4.6.11 NADP 0.1 M
  • 4.6.12 Nutrient Agar Plates
  • 4.6.13 Nutrient Broth
  • 4.6.14 Phosphate Buffer 0.2 M pH 7.4
  • 4.6.15 Positive Control and Diagnostic Mutagen Solutions
  • 4.6.16 S9 Fraction
  • 4.6.17 S9 Mix
  • 4.6.18 Tetracycline 1 µg/disc
  • 4.6.19 Top Agar Incomplete: TAI
  • 4.6.20 Top Agar Complete: TAC
  • 4.6.21 Tryptophan 5 mg/mL
  • 4.6.22 VB Salts 50×: Vogel-Bonner Salts
  • 4.7 Suggested Phases in Development of the Test
  • 4.8 The Bacterial Strains
  • 4.8.1 Genotypes of Routinely Used Strains
  • 4.8.2 Obtaining the Tester Strains
  • 4.8.3 Receipt of Bacterial Strains
  • 4.8.4 Phenotyping of New Isolates
  • 4.8.5 Freezing of Selected Isolates
  • 4.8.6 Diagnostic Mutagen Test
  • 4.9 Routine Testing
  • 4.9.1 Designing a Study
  • 4.9.1.1 Metabolic activation system
  • 4.9.2 Test Article Considerations
  • 4.9.2.1 Solvent selection
  • 4.9.2.2 Dose volumes
  • 4.9.2.3 Dose levels
  • 4.9.3 Positive Controls
  • 4.10 Standard Test Procedures
  • 4.10.1 Plate Incorporation Method
  • 4.10.2 Preincubation Method
  • 4.10.3 Standard Study Design
  • 4.10.4 Examination of the Plates
  • 4.10.5 Interpretation of Results
  • 4.10.5.1 Evaluation of toxicity
  • 4.10.5.2 Validity of the study
  • 4.10.5.3 Criteria for negative/positive/equivocal outcome
  • 4.10.5.4 Unexpected and borderline results
  • 4.10.6 Presentation of Results
  • 4.10.7 Testing of Volatile and Gaseous Compounds
  • 4.11 Screening Tests
  • 4.11.1 Simplified Test Systems
  • 4.11.2 Screening Tests Using Standard Tester Strains
  • 4.11.3 Reduced Format Tests Using Standard Tester Strains
  • 4.12 Appendix 1: Growing and Monitoring Suspension Cultures
  • References
  • 5 The Mouse Lymphoma TK Assay
  • 5.1 Introduction
  • 5.2 History
  • 5.3 Provenance of the Cells
  • 5.4 Spontaneous Mutation Frequency
  • 5.5 Materials
  • 5.5.1 Safety
  • 5.5.2 Growth Medium
  • 5.5.3 Cell Culture
  • 5.5.4 Metabolic Activation
  • 5.5.5 Test Item
  • 5.5.6 Vehicle
  • 5.5.7 Positive Controls
  • 5.6 Study Design
  • 5.6.1 General Test Conditions
  • 5.6.2 Preliminary Toxicity Test
  • 5.6.3 Main Mutation Test
  • 5.6.3.1 Posttreatment procedures
  • 5.6.3.2 Expression period
  • 5.6.3.2.1 Expression period day 1
  • 5.6.3.2.2 Expression period day 2
  • 5.6.3.3 Viability assessment and mutant selection (microtiter version)
  • 5.6.3.3.1 Colony counting (microtiter version)
  • 5.6.3.4 Viability assessment and mutant selection (agar version)
  • 5.6.3.4.1 Colony counting (agar version)
  • 5.6.3.5 Analysis of results
  • 5.6.3.5.1 Relative suspension growth
  • 5.6.3.5.2 Toxicity assessment
  • 5.6.3.5.3 MF assessment (microtiter version)
  • 5.6.3.5.4 MF assessment (agar version)
  • 5.6.3.6 Acceptance criteria
  • 5.7 Evaluation Criteria
  • 5.8 Predictivity of the MLA
  • References
  • 6 The In Vitro Micronucleus Assay
  • 6.1 Introduction
  • 6.2 Practical Considerations
  • 6.2.1 Regulatory Guidelines
  • 6.2.2 Good Laboratory Practice (GLP)
  • 6.2.3 Cell Types
  • 6.2.4 Laboratory Proficiency
  • 6.2.5 Controls
  • 6.2.6 Metabolic Activation
  • 6.2.7 S9 Rat Liver Homogenate
  • 6.2.8 Experimental Design
  • 6.2.9 Cytotoxicity Measures
  • 6.2.9.1 Methods used to determine cytotoxicity in the absence of cytochalasin B [16] (reproduced with permission of the author)
  • 6.2.9.2 Method used to determine cytotoxicity in the presence of cytochalasin B
  • 6.2.10 Historical Controls
  • 6.3 Methods
  • 6.3.1 Mononuclear Assay
  • 6.3.2 Binuclear Assay
  • 6.3.3 Centromeric Labeling
  • 6.3.4 Nondisjunction Assay
  • 6.4 Materials
  • 6.4.1 Mononuclear Assay
  • 6.4.2 Binuclear Assay
  • 6.4.3 Centromeric Labeling
  • 6.4.4 Nondisjunction Assay
  • 6.5 Protocols
  • 6.5.1 S9 Mix
  • 6.5.2 Mononuclear Assay
  • 6.5.2.1 Treatment schedules
  • 6.5.2.2 Cell culture and treatment
  • 6.5.2.3 Slide preparation
  • 6.5.2.4 Coding of slides
  • 6.5.2.5 Analysis of slides (microscope)
  • 6.5.2.6 Analysis of slides (semiautomated scoring)
  • Criteria for evaluation
  • 6.5.3 Binuclear Assay
  • 6.5.3.1 Treatment schedules
  • 6.5.3.2 Human peripheral blood lymphocytes
  • 6.5.3.3 Donors
  • 6.5.3.4 Lymphocyte culture
  • 6.5.3.5 Staining and analysis
  • 6.5.3.6 Coding of slides
  • 6.5.3.7 Analysis of slides
  • 6.5.3.8 Evaluation of results (acceptance criteria and statistics)
  • 6.5.3.9 Criteria for a valid assay
  • 6.5.3.10 Evaluation of data
  • 6.5.4 Centromeric Labeling
  • 6.5.4.1 FISH using a programmable hotplate such as HYBriteT or ThermobriteT
  • 6.5.4.2 Alterative protocol for FISH
  • 6.5.4.3 Slide checking
  • 6.5.4.4 Slide scoring
  • 6.5.5 Nondisjunction Assay
  • 6.5.5.1 FISH method
  • 6.5.5.2 Slide checking
  • 6.5.5.3 Slide scoring
  • 6.6 Flow Cytometric Method
  • 6.6.1 Equipment
  • 6.6.2 Consumables
  • 6.6.3 Reagents and recipes
  • 6.6.4 Suspension Cell Protocol
  • 6.6.5 Attachment Cell Protocol
  • 6.6.6 Flow Cytometric Data Acquisition
  • 6.6.7 Flow Cytometric Data Analysis
  • 6.6.7.1 Independent cytotoxicity assessment
  • 6.6.7.2 Criteria for a valid assay
  • 6.6.7.3 Criteria for positive/negative outcomes
  • 6.6.8 Creating an Analysis Template
  • 6.6.9 Example Plate Layout
  • 6.6.10 Example Results Table
  • 6.6.11 Advice for Test Article Exposure
  • 6.6.11.1 Suspension cells
  • 6.6.11.2 Attachment cells
  • 6.6.11.3 Metabolic activation
  • 6.6.11.4 Positive controls
  • 6.6.12 Use of Multichannel Aspirator with Bridge
  • 6.6.13 Plate Placement During Nucleic Acid Dye B Photoactivation
  • 6.6.14 Updates and Future Work
  • References
  • 7 The In Vitro Chromosome Aberration Test
  • 7.1 Introduction
  • 7.2 History
  • 7.3 Fundamentals
  • 7.4 Equipment
  • 7.5 Consumables and Reagents
  • 7.6 Reagents and Recipes
  • 7.6.1 Colcemid 10 µg/mL in PBS
  • 7.6.2 Fix
  • 7.6.3 F-12 Complete
  • 7.6.4 Freezing Medium 10% (CHO Cells)
  • 7.6.5 Hypotonic Solution (0.075 M KCl)
  • 7.6.6 Heparin Sodium 1000 U/mL
  • 7.6.7 G6P 1 M: Glucose-6-Phosphate
  • 7.6.8 KMg
  • 7.6.9 NADP 0.1 M
  • 7.6.10 PHA M Form (Phytohemagglutinin)
  • 7.6.11 Phosphate Buffer 0.2 M, pH 7.4
  • 7.6.12 Positive Control Solutions
  • 7.6.13 RPMI Complete
  • 7.6.14 S9 Fraction
  • 7.6.15 S9 Mix
  • 7.7 Phases in Development of the Test
  • 7.8 Cell Characterization
  • 7.8.1 Modal Chromosome Number
  • 7.8.2 Mycoplasma
  • 7.8.3 Cell-Cycle Time
  • 7.9 Routine Testing
  • 7.9.1 General Considerations
  • 7.9.2 Dose Regimens
  • 7.9.3 Metabolic Activation System
  • 7.9.4 Test Substance Considerations
  • 7.9.5 Vehicle Selection and Dose Volume
  • 7.9.6 Dose Level Selection
  • 7.9.7 Positive Controls
  • 7.10 Standard Test Procedures
  • 7.10.1 Experimental Design Spreadsheet
  • 7.10.1.1 CHO cells: routine maintenance
  • 7.10.2 CHO Cells: Test Procedures
  • 7.10.3 HPBL Test Procedures
  • 7.10.4 Slide Staining: All Cell Types
  • 7.10.5 Selection of Slides for Detailed Examination
  • 7.10.5.1 Cell lines
  • 7.10.5.2 HPBL selection of slides for provisional detailed examination
  • 7.10.6 Slide Coding
  • 7.10.7 Preliminary Slide Reading
  • 7.10.8 Slide Scoring
  • 7.10.8.1 Basics
  • 7.10.8.2 Understanding the normal karyotype
  • 7.10.8.3 Routine scoring
  • 7.10.8.4 Classification
  • 7.11 Interpretation of Results
  • 7.11.1 Evaluation of Toxicity
  • 7.11.2 Validity of the Study
  • 7.11.3 Criteria for Negative/Positive/Equivocal Outcome
  • 7.11.4 Interpretation of Numerical Aberrations
  • 7.11.5 Unexpected and Borderline Results
  • 7.11.6 Follow-up In Vivo Testing
  • 7.11.7 Reporting
  • 7.11.7.1 Results tables
  • 7.11.8 Historical Control Results
  • 7.11.9 Testing of Volatile and Gaseous Compounds
  • 7.12 Screening Versions of the Test
  • 7.13 Automation
  • References
  • 8 The In Vivo Rodent Micronucleus Assay
  • 8.1 Introduction
  • 8.2 History
  • 8.3 Fundamentals
  • 8.4 Test Substance Considerations
  • 8.5 Study Design
  • 8.5.1 Animal Source, Housing, Maintenance, and Identification
  • 8.5.2 Animal Species, Strain, Age, and Sex
  • 8.5.3 Historical Negative/Vehicle and Positive Control Data
  • 8.5.4 Number and Size of Treatment Groups
  • 8.5.5 Acute and Repeat-Dose Schedules
  • 8.5.6 Dose Level Selection
  • 8.5.7 Dose Range-Finding Experiment
  • 8.5.8 Dose Administration
  • 8.5.9 Dose Volume
  • 8.6 Manual Methods
  • 8.6.1 Equipment and Consumables
  • 8.6.2 Reagent Preparation
  • 8.6.2.1 Fetal calf serum
  • 8.6.2.2 Acridine orange
  • 8.6.2.3 Giemsa stain-Gurr's improved R66
  • 8.6.3 Sample Preparation
  • 8.6.4 Microscopic Methods
  • 8.6.4.1 Blood smears
  • 8.6.4.2 Mouse bone marrow smears
  • 8.6.4.3 Rat bone marrow smears
  • 8.6.4.4 Smear fixation and staining
  • 8.6.4.5 Giemsa staining
  • 8.6.4.6 Acridine orange staining
  • 8.6.4.7 Supravital staining of blood
  • 8.6.4.8 Microscopic evaluation
  • 8.6.4.9 Identification of micronuclei
  • 8.6.5 Records
  • 8.6.6 Identification of Aneugenic Agents
  • 8.6.7 Centromeric Staining Using FISH
  • 8.6.7.1 Materials
  • 8.6.7.2 FISH method using programmable hotplate
  • 8.6.7.3 Alterative protocol for FISH
  • 8.6.7.4 FISH: slide checking
  • 8.6.7.5 FISH: slide scoring
  • 8.6.8 Kinetochore Labeling
  • 8.6.8.1 Materials
  • 8.6.8.2 Methods
  • Phase 1
  • Phase 2
  • Slide Checking
  • Slide Coding
  • Analysis of Slides
  • Assessment of results
  • 8.7 Automated Analysis and Flow Cytometry
  • 8.7.1 Individual Reagents
  • 8.7.2 Solution and Material Preparation
  • Fixative tubes (must be prepared at least 1 day prior to blood collection)
  • Washing solution for samples in LTSS
  • Anticoagulant/diluent vials (prepare prior to blood collection)
  • Labeling solution I
  • Labeling solution II
  • DNA staining solution
  • 8.7.3 Flow Cytometry Method
  • 8.7.4 Blood Collection
  • 8.7.5 Bone Marrow Collection and Processing
  • 8.7.5.1 Prepare cellulose columns
  • 8.7.6 Collect and Fractionate Bone Marrow Samples
  • 8.7.7 Fixation of Blood and Bone Marrow Samples
  • 8.7.8 Storage of Fixed Samples
  • 8.7.8.1 Wash samples out of fixative
  • 8.7.9 Transfer Samples to LTSS
  • 8.7.9.1 Wash samples out of LTSS
  • 8.7.10 Label Washed Samples for Flow Analysis
  • 8.7.11 Flow Cytometric Analysis
  • 8.7.11.1 Flow cytometer calibration with biological standards
  • 8.7.12 Analysis of Experimental Samples
  • 8.7.13 Template Preparation for Flow Cytometric Analyses
  • 8.8 Study Validity
  • 8.9 Interpretation of Results and Statistical Analysis
  • 8.9.1 MIE Values
  • 8.9.2 Proportion of Immature Erythrocytes
  • 8.10 Nongenotoxic Mechanisms of Induction of Micronucleated Erythrocytes
  • 8.10.1 Causes
  • 8.10.2 Avoiding and Recognizing Irrelevant Positives
  • 8.11 Limitations of the Rodent Erythrocyte Micronucleus Test
  • References
  • 9 The Rodent Bone Marrow Chromosomal Aberration Test
  • 9.1 Introduction
  • 9.2 History
  • 9.3 Related Methods
  • 9.4 Study Design and Performance
  • 9.4.1 Vehicle/Negative Control and Formulation
  • 9.4.2 Positive Control Group
  • 9.4.3 Sex and Group Size
  • 9.4.4 Number of Groups
  • 9.4.5 Evidence of Target Organ Exposure
  • 9.4.6 Dose Selection
  • 9.4.7 Dose Administration
  • 9.4.8 Treatment Schedule
  • 9.4.9 Animal Observations
  • 9.4.10 Animal Euthanasia
  • 9.5 Terminal Procedures
  • 9.5.1 Equipment
  • 9.5.2 Consumables
  • 9.5.3 Advance Preparation
  • 9.5.4 Bone Marrow Collection
  • 9.5.5 Slide Preparation
  • 9.5.6 Slide Staining
  • 9.5.7 Slide Examination
  • 9.5.8 Calculations and Reporting of Results
  • 9.5.9 Acceptability of the Study
  • 9.5.10 Evaluation and Interpretation of Results
  • 9.5.11 Test Report
  • 9.5.12 Integration into Other Studies
  • 9.5.13 Cytonucleus Test
  • Appendix
  • References
  • 10 The In Vivo Comet Assay Test
  • 10.1 Introduction
  • 10.2 The In Vivo Comet Assay in Regulatory Safety Testing
  • 10.2.1 Follow-Up of Positive In Vitro Standard Battery Tests
  • 10.2.2 Follow-Up of Negative In Vitro Standard Battery Tests
  • 10.3 Fundamentals
  • 10.4 Equipment and Nondisposable Supplies
  • 10.5 Consumables
  • 10.6 Reagents and Solutions
  • 10.6.1 Reagents
  • 10.6.2 Solutions
  • 10.6.2.1 Mincing Buffer (500 mL final volume)
  • 10.6.2.2 1% Normal Melting Agarose (200 mL final volume)
  • 10.6.2.3 0.5% Low Melting Point Agarose (200 mL final volume)
  • 10.6.2.4 Lysing stock solution (900 mL final volume)
  • 10.6.2.5 Working lysing solution (40 mL final volume in Coplin jars)
  • 10.6.2.6 10 M NaOH stock (500 mL final volume)
  • 10.6.2.7 200mM EDTA stock, pH 10 (200 mL final volume)
  • 10.6.2.8 Alkaline electrophoresis buffer (1 L final volume)
  • 10.6.2.9 0.4M Tris Buffer (1 L final volume)
  • 10.7 Test System
  • 10.8 Study Design Considerations
  • 10.8.1 Test Article
  • 10.8.2 Vehicle Selection
  • 10.8.3 Positive Control
  • 10.8.4 Number of Animals
  • 10.8.5 Route of Exposure
  • 10.8.6 Treatment Schedule/Sample Time
  • 10.8.7 Dose Selection and Cytotoxicity
  • 10.8.8 Tissue Selection
  • 10.9 Standard Test Procedures
  • 10.9.1 Preliminary Procedures
  • 10.9.2 Sample Collection
  • 10.9.3 Comet Slide Preparation
  • 10.9.4 Alkaline Electrophoresis
  • 10.9.5 Slide Staining
  • 10.9.6 Image Analysis Scoring
  • 10.10 Data and Reporting
  • 10.10.1 Statistical Analysis
  • 10.10.1.1 Normality Test
  • 10.10.1.2 Pairwise comparisons
  • Parametric tests
  • Nonparametric tests
  • 10.10.1.3 Trend tests
  • Parametric test
  • Nonparametric test
  • 10.10.2 Validity of a Test
  • 10.10.3 Positive Response Criteria
  • 10.10.4 Cytotoxicity
  • 10.10.5 Reporting results
  • 10.11 Evaluating Unclear Results
  • 10.11.1 Equivocal Results
  • 10.11.2 Positive Results
  • 10.11.3 Negative Results
  • Acknowledgments
  • References
  • 11 The Pig-a Endogenous Gene Mutation Assay
  • 11.1 Introduction
  • 11.2 History
  • 11.3 Fundamentals
  • 11.4 Study Design
  • 11.4.1 Animal Species/Strain/Sex/Age
  • 11.4.2 Number of Animals Per Experimental Group
  • 11.4.3 Group Selection/Identification
  • 11.4.4 Negative and Positive Controls
  • 11.4.5 Acute or Integrated Repeat-Dose Studies
  • 11.4.5.1 Dose-range finding
  • 11.4.5.2 Top dose and dose spacing
  • 11.4.5.3 Timing of sample collection
  • 11.4.5.4 Assay configurations
  • 11.5 Equipment
  • 11.6 Consumables
  • 11.7 Reagents and Recipes
  • 11.7.1 Solution and Materials Preparation
  • 11.7.1.1 Buffered salt solution plus 2% FBS
  • 11.7.1.2 Working nucleic acid dye plus counting beads solution
  • 11.7.1.3 Working antibody solution
  • 11.7.1.4 Working anti-PE MicroBead suspension
  • 11.7.1.5 Aliquot anticoagulant solution and Lympholyte®-Mammal
  • 11.8 Method Overview
  • 11.9 Blood Collection
  • 11.10 Leukodepletion and Platelet Removal
  • 11.11 Sample Labeling
  • 11.12 Column Separation and Sample Staining
  • 11.13 Flow Cytometric Analysis: 96-Well Plate-Based Protocol
  • 11.14 Tabulating and Summarizing Results
  • 11.15 Evaluation and Interpretation of Results
  • 11.15.1 Statistics
  • 11.15.2 Criteria for a Valid Assay
  • 11.15.3 Comparison to Historical Controls
  • 11.15.4 Biological Relevance
  • 11.16 Flow Cytometric Template Preparation
  • 11.17 Example Plots
  • 11.18 Storage and Shipment of Blood Samples
  • 11.19 Aspiration of Postcolumn Samples
  • References
  • Index
  • Back Cover

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