Preface
Contents
Contributors
Part I: Introduction and Review
Chapter 1: Cell Cycle Progression and Synchronization: An Overview
1 Introduction
2 Early History of Cell Cycle Discovery
3 Cell Cycle Progression Through Various Phases
3.1 G1 Phase
3.2 S Phase
3.3 G2 Phase
3.4 Mitosis and Cytokinesis
4 Cell Cycle Synchronization
4.1 Cell Synchronization Without Using Chemical Inhibitors
4.1.1 Serum Starvation
4.1.2 Contact Inhibition
4.1.3 Mitotic Shake-off
4.1.4 Flow Cytometry and Cell Sorting After DNA Staining
4.1.5 Centrifugal Elutriation
4.2 Cell Synchronization by Using Chemical Inhibitors
4.2.1 Synchronization of Cell Cycle to G1 Phase by Lovastatin
4.2.2 Synchronization of Cell Cycle to Early S Phase by Double Thymidine Block
4.2.3 Synchronization of Cell Cycle to G2 Phase by Inhibiting CDK1
4.2.4 Synchronization of Cell Cycle to Mitosis by Nocodazole, MG132, and Blebbistatin
References
Chapter 2: The Trypanosomatids Cell Cycle: A Brief Report
1 Introduction
2 G0 State and G1 Phase
3 S Phase
4 G2 Phase
5 Mitosis and Cytokinesis Phases
6 Cross-Talk Between Telomeres and the Cell Cycle
7 Cell Cycle Synchronization
8 Final Thoughts
References
Chapter 3: Cell Cycle-Related Clinical Applications
1 Introduction
2 The Regulation of Cell Cycle
3 Clinical Applications of Cell Cycle
3.1 Cancer Therapy
3.2 Cancer Biomarkers
3.3 Regulation of Stem Cells
3.4 Aging and Senescence
3.5 Other Clinical Applications
4 Future Directions
References
Chapter 4: Flow Cytometry and Cell Cycle Analysis: An Overview
1 Introduction
2 DNA Content Dyes
2.1 Propidium Iodide (PI)
2.2 4-6-Diamidino-2-Phenylindole (DAPI)
2.3 Hoescht
2.4 DRAQ5
2.5 Vybrant DyeCycle
2.6 SYTOX Dyes
2.7 FxCycle
3 Going Beyond Single-Parameter DNA Content Analysis
3.1 Uptake of Thymidine Analogs
3.2 Protein Markers of Cell Cycle Progression
4 Additional Modes of Flow Cytometric Analysis
4.1 Flow Cytometry with Acoustic Focusing
4.2 Imaging Flow Cytometry
4.3 Mass Cytometry
5 Additional Suggestions for Acquisition and Analysis of Cell Cycle Samples
5.1 Titrate Your Reagents
5.2 Fixatives, Permeabilization, and Multiplexing
5.3 Run on Low Speed
5.4 Acquire Enough Events
5.5 Use Modeling Software for Analysis
6 Discussion and Conclusions
References
Part II: Methods for Cell Cycle Synchronization of Mammalian Cells
Chapter 5: Synchronization of Cultured Cells to G1, S, G2, and M Phases by Double Thymidine Block
1 Introduction
2 Materials
2.1 Solutions, Reagents, and Chemicals
2.2 Equipment
2.3 Cell Lines
3 Methods
3.1 S Phase Synchronization: Double Thymidine Block
3.2 Synchronization of Cells to G2, M, and G1 Cells by Releasing Cells from Double Thymidine Block
3.3 Validation of Synchronization by Western Blot
3.3.1 Protein Isolation
3.3.2 Protein Quantification
3.3.3 Western Blot
3.4 Validation of Synchronization by Flow Cytometry
3.5 Validation of Synchronization by Microscopy
4 Notes
References
Chapter 6: Cell Synchronization Techniques for Studying Mitosis
1 Introduction
1.1 Enrichment of Mitotic Cells by Thymidine Block Followed by MG132 Treatment
1.2 Enrichment of Mitotic Cells Using Reversible Chemical Inhibitors
2 Materials
2.1 Cell Culture
2.2 Lysates
2.3 Western Blotting
2.4 Primary Antibodies
2.5 Secondary Antibodies
2.6 Spinning Disk Confocal Imaging
2.7 Small Molecule Inhibitors
3 Methods
3.1 Nocodazole Block
3.1.1 Western Blot
3.1.2 Imaging
3.2 STLC Block
3.2.1 Western Blot
3.2.2 Imaging
3.3 Single Thymidine Block + MG132
3.3.1 Double Thymidine Block
3.3.2 Western Blot
3.3.3 Imaging
4 Notes
4.1 Cell Culture
4.2 Western Blot
4.3 Imaging
References
Chapter 7: Synchronization of HeLa Cells to Various Interphases Including G1, S, and G2 Phases
1 Introduction
2 Materials
2.1 Solutions, Reagents, and Chemicals
2.2 Equipment
2.3 Cell Lines
3 Methods
3.1 G0/G1 Synchronization
3.2 S phase Synchronization: Double Thymidine Block
3.3 Late G2 Synchronization: Roscovitine
3.4 Validation of Synchronization by Western Blot
3.4.1 Protein Isolation
3.4.2 Protein Quantification
3.4.3 Western Blot
3.5 Validation of Synchronization by Flow Cytometry
4 Notes
References
Chapter 8: Synchronization of HeLa Cells to Mitotic Subphases
1 Introduction
2 Materials
2.1 Solutions, Reagents, and Chemicals
2.2 Equipment
2.3 Cell Lines
3 Methods
3.1 Synchronization of Cells to Prometaphase by Nocodazole
3.2 Synchronization of Cells to Metaphase by MG132
3.3 Anaphase/Telophase Synchronization: Blebbistatin
3.4 Validation of Synchronization by Western Blot
3.4.1 Protein Isolation
3.4.2 Protein Quantification
3.4.3 Western Blot
3.5 Validation of Synchronization by Fluorescence Microscopy
4 Notes
References
Chapter 9: Cell Cycle Synchronization of Primary and Cultured Articular Chondrocytes
1 Introduction
2 Materials
2.1 Tissue Harvest
2.2 Cell Isolation and Synchronization
2.3 Cell Monolayer Culture
2.4 Cell Recovery and Flow Cytometry
3 Methods
3.1 Synchronization of Cultured Articular Chondrocytes in Monolayer
3.2 Simultaneous Isolation and Synchronization of Primary Articular Chondrocytes
3.3 Flow Cytometry
4 Notes
References
Part III: Methods for Cell Cycle Synchronization of Unicellular Organisms
Chapter 10: Synchronization of Leishmania amazonensis Cell Cycle Using Hydroxyurea
1 Introduction
2 Materials
2.1 Cell Culture and Counting
2.2 Hydroxyurea Treatment
2.3 Sample Preparation for Flow Cytometry
2.4 Flow Cytometry Data Collection and Analysis
3 Methods
3.1 Cell Culture: Starting, Counting, and Passage
3.1.1 L. amazonensis Procyclic Promastigote Axenic Culture
3.1.2 Counting and Maintaining the Cells in Culture
3.2 Growth Curve
3.3 Cell Synchronization
3.4 DNA Content Analysis
3.4.1 Preparation of Cells to Flow Cytometer Analysis
3.4.2 Data Analysis in FlowJo
4 Notes
References
Chapter 11: Synchronization of Trypanosoma brucei by Counter-Flow Centrifugal Elutriation
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Centrifugal Counter-Flow Elutriation
2.3 Flow Cytometry for Cell Cycle Analysis
2.4 DAPI-Staining for Cell Cycle Analysis
3 Methods
3.1 Cell Culture
3.2 Centrifugal Counter-Flow Elutriation
3.3 Flow Cytometry for Cell Cycle Analysis
3.4 DAPI-Staining for Cell Cycle Analysis by Microscopy
4 Notes
References
Chapter 12: Synchronization of Saccharomyces cerevisiae Cells for Analysis of Progression Through the Cell Cycle
1 Introduction
2 Materials
2.1 Yeast Media
3 Methods
3.1 Growth of S. cerevisiae
3.1.1 Monitoring Cell Density
3.1.2 Counting Cells with Hemocytometer
3.2 Monitoring Synchrony
3.2.1 Determining the Percent of Budded Cells
3.2.2 Analysis of DNA Content by Flow Cytometry
3.2.3 Analysis of Nuclear Morphology by DAPI (4โฒ,6-Diamidino-2-Phenylindole) Staining
3.3 Synchronization of S. cerevisiae Cell Cultures
3.4 Induced Synchronization Block and Release Protocols
3.4.1 Pheromone-Mediated Arrest and Release
3.4.2 Hydroxyurea-Induced S-Phase Arrest and Release
3.4.3 Nocodazole-Induced G2/M-Phase Arrest and Release
3.4.4 G1-Phase Arrest Induced by Cyclin Depletion
3.4.5 Metaphase Arrest Achieved by Depletion of Cdc20
3.4.6 Specific Cell Cycle Arrest Induced by Inactivation of Thermosensitive cdc Mutants
3.5 Selection Synchrony
3.5.1 Centrifugal Elutriation
3.5.2 Selection of G1-Phase Cells by Centrifugal Elutriation
4 Notes
References
Chapter 13: Cell Cycle Synchrony Methods for Fission Yeast, Schizosaccharomyces pombe
1 Introduction
2 Materials
2.1 Fission Yeast Strains
2.2 Yeast Extract with Supplements Medium (YES)
2.3 Edinborough Minimal Medium (EMM)
2.3.1 50x EMM Salts Recipe
2.3.2 1000x EMM Vitamins Recipe
2.3.3 EMM Minerals Recipe
2.4 Hydroxyurea (HU) Stock Solution
2.5 Orbital Shaking Water Bath
2.6 Cooling Pan
2.7 Thermometer
2.8 70% Ethanol
2.9 Light Microscope
2.10 Vacuum Filtration System
3 Methods
3.1 Yeast Culture Growth
3.2 Culture Harvest and Septation Index Calculation
3.3 Drug-Induced Synchrony: Hydroxyurea (HU) Block and Release
3.4 Environmental Control of Synchrony: Nitrogen Block and Release
3.5 Conditional Allele Arrest: Temperature-Sensitive Cell Cycle Mutants
4 Notes
References
Part IV: Methods to Assess Cell Cycle Progression
Chapter 14: Analysis of Cell Cycle by Flow Cytometry
1 Introduction
2 Materials
2.1 Cell Culturing
2.2 Staining of DNA with Propidium Iodide (PI)
2.3 DNA Contents Analysis
3 Methods
3.1 Cell Culturing and Treatment of the Cells
3.2 Staining of DNA with PI
3.3 DNA Contents Analysis
3.3.1 DNA Contents Analysis by BD FACS Canto II Machine
3.3.2 Analysis of Flow Cytometry Data by FlowJo Software
4 Notes
References
Chapter 15: Analysis of Cell Proliferation by Three-Dimensional Culture
1 Introduction
2 Materials
3 Methods
3.1 3D Culture of Breast Cancer Cells in 8-Well Chamber Slides
3.2 Fix Breast Cancer Cell Spheroids in 3D Culture for Immunofluorescence Staining
3.3 Extract Protein from Breast Cancer Cells in 3D Culture
3.4 Extract RNA from Breast Cancer Cells in 3D Culture
4 Notes
References
Chapter 16: BrdU Incorporation Assay to Analyze the Entry into S Phase
1 Introduction
2 Materials
2.1 General Lab Supplies
2.2 Reagents
3 Methods
3.1 In Vitro Experiment
3.1.1 Slide Preparation (All of the Operations Are Performed Inside the BSC)
3.1.2 In Vitro Adherent Cells Labeling
3.1.3 Fixation and Permeabilization (DNA Hydrolysis)
3.1.4 Staining [Standard ICC/IF Staining]
3.2 In Vivo Experiment
3.2.1 In Vivo Labeling
3.2.2 Tissue Slices from Frozen Section Labeling
3.2.3 Fixation and Permeabilization (DNA Hydrolysis)
3.2.4 Blocking [Can Also Be Combined with Permeabilization Depending on Preference]
3.2.5 Immunostaining
3.3 Flow Cytometry [Alternative Method]
3.3.1 Condition and Preparation
3.3.2 (Optional Step) Count Cell Number and Viability
3.3.3 Procedure
4 Notes
5 Limitations
References
Chapter 17: Assessment of Growth Plate Chondrocytes Proliferative Activity in Embryonic Endochondral Ossification via Ki-67 Im...
1 Introduction
2 Materials
3 Methods
3.1 Deparaffinize and Rehydrating
3.2 Antigen Retrieval
3.3 Permeabilization
3.4 Blocking
3.5 Immunostaining: Primary Antibody
3.6 Secondary Antibody
3.7 Multicolor Detections (Optional)
3.8 Data Analysis
4 Notes
References
Chapter 18: Detecting Cell Cycle Stage and Progression in Fission Yeast, Schizosaccharomyces pombe
1 Introduction
2 Materials and Equipment
2.1 Fixed Cell Samples
2.2 Flow Cytometry Staining Tubes
2.3 50 mM Sodium Citrate Buffer
2.4 Ribonuclease A (RNaseA) Stock Solution
2.5 1x RNaseA Solution
2.6 1x SYTOX Green Solution
2.7 Sonicator
2.8 Mesh Filter
2.9 10x Aniline Blue Stock Solution
2.10 Aniline Blue Working Solution
2.11 Mounting Medium with DAPI
3 Methods
3.1 Flow Cytometry
3.2 Nucleus and Septum Staining of Cell Populations
3.3 Assessing Cell Cycle Synchrony and Effect Using Nucleus and Septum Stain
4 Notes
References
Index