دانلود کتاب Bioluminescence: Methods and Protocols, Volume 1

فهرست مطالب :

Preface
Contents
Contributors
Part I: Establishment of Luciferins and Luciferases
Chapter 1: Gene Cloning and Functional Analysis of the Luciferase from Luminous Syllids of the Genus Odontosyllis
1 Introduction
2 Materials
2.1 Labware and Reagents for Sampling and Manipulating Worms
2.2 RNA Experiments
2.3 Protein Experiments
2.4 Recombinant Protein Expression Experiments
2.5 Measurement of BL Intensity In Vitro
3 Methods
3.1 Sampling of the Japanese Syllid Worms
3.2 Collection of Luminous Mucus
3.3 Luciferin-Luciferase (LL) Reaction
3.4 RNA Extraction from the Worm
3.5 RNA-Sequencing (RNA-Seq) Analysis
3.6 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
3.7 De Novo Mass Spectroscopy (MS) Analysis of Protein
3.8 Homology Search of the RNA-Seq Data to the Amino Acid Sequences
3.9 cDNA Cloning and Construction of Expression Vector
3.10 Recombinant Protein Expression Using Mammalian Cells (See Note 22)
3.11 Measurement of BL In Vitro
4 Notes
References
Chapter 2: Synthetic Coelenterazine Derivatives and Their Application for Bioluminescence Imaging
1 Introduction
2 Materials
2.1 Reagents for the Synthesis of CTZ Derivatives
2.2 Other Reagents and Labware
2.3 Instrumentation
2.4 Softwares
3 Methods
3.1 General Procedure for Synthesis (See Fig. 1)
3.1.1 Synthesis of 3-Benzyl-5-bromo-2-amino-pyrazine (1-1)
3.1.2 General Procedure for Preparations of Compound 2 Series
3.1.3 Synthesis of 3-Phenyl-1,1-diethoxyacetone (3)
3.1.4 General Procedure for Preparation of Derivative A Series
3.1.5 General Procedure for Preparation of Compound 4 Series
3.1.6 General Procedure for Preparation of Derivative B Series
3.1.7 Synthesis of 2-Amino-3-benzyl-5-((trimethylsilyl)ethynyl)-pyrazine (Compound 5)
3.1.8 Synthesis of 2-Amino-3-benzyl-5-ethynyl-pyrazine (Compound 6)
3.1.9 Synthesis of 2-Azidoethanol (Compound 7-1)
3.1.10 Synthesis of 3-Azidopropyl (Compound 7-2)
3.1.11 Synthesis of 1-Azido-2-fluoroethane (Compound 7-3)
3.1.12 General Procedure for Preparation of Compound 8 Series
3.1.13 General Procedure for Preparation of Derivative T Series
3.2 BL Assay of CTZ Derivatives
3.2.1 General Procedure for BL Assay
3.2.2 BL Intensities with RLuc
3.2.3 BL Spectra with RLuc
3.2.4 Kinetic Profiles of BL with RLuc
3.2.5 BL Intensities with GLuc
3.2.6 BL Spectra with GLuc
3.2.7 Kinetic Profiles of BL with GLuc
3.2.8 BL Imaging In Cellulo
3.2.9 BL Imaging In Vivo
3.3 General Procedure for Chemiluminescent Assay
3.4 General Procedure for Fluorescence Assay
4 Notes
References
Chapter 3: Visible Light Bioluminescence Imaging Platform for Animal Cell Imaging
1 Introduction
2 Materials
2.1 Reagents for Synthesis
2.2 Reagents for Cell Assays and Labware
2.3 Device and Instrumentation
2.4 Softwares
3 Methods
3.1 Synthesis of CTZ Analogues 1a-d and 2a-d (See Fig. 1)
3.1.1 Synthesis of Compounds 6a-6c
3.1.2 Synthesis of Compounds 7a-7d and 8a-8d
3.1.3 Synthesis of Compounds 1a-1d and 2a-2d
3.2 Synthesis of CTZ Analogues 3a-d (See Fig. 2)
3.2.1 Synthesis of Compound 12a
3.2.2 Synthesis of Compounds 12b-12d
3.2.3 Synthesis of Compounds 3a-3d
3.3 Determination of BL Spectra of the CTZ Analogues (See Fig. 3)
3.4 Determination of the Luciferase Specificity of the CTZ Variants (See Fig. 4)
3.5 Determination of the Kinetic Parameters of the BL According to the CTZ Analogues Lineweaver-Burk (See Table 1)
4 Notes
References
Chapter 4: Biosynthesis-Inspired Deracemizative Production of D-Luciferin In Vitro by Combining Luciferase and Thioesterase
1 Introduction
2 Materials
2.1 Instrumentation
2.2 Reagents and Labware
2.3 Buffers and Solutions
3 Methods
3.1 Chirality Analysis of Firefly Luciferin
3.2 Luciferin Racemization In Vitro
3.3 Enzymatic Deracemizative Reaction of D-Luciferin from L-Luciferin
3.4 BL Intensity Measurement During the Enzymatic Deracemizative Reaction
4 Notes
References
Chapter 5: Production of Metridia Luciferase in Native Form by Oxidative Refolding from E. coli Inclusion Bodies
1 Introduction
2 Materials
2.1 Production and Washing of Inclusion Bodies
2.2 Solubilization and Refolding
2.3 Chromatography Protein Purification
2.4 BL Assay
2.5 Equipment
3 Methods
3.1 Production of Inclusion Bodies
3.2 Washing of Inclusion Bodies
3.3 Solubilization
3.4 Refolding and Recovery
3.5 Chromatographic Purification
3.5.1 Desalting of the Concentrated Refolding Mixture
3.5.2 Anion-Exchange Chromatography on HiTrap Q HP Column
3.5.3 Size-Exclusion Chromatography
3.6 BL Assay
4 Notes
References
Chapter 6: Production of Copepod Luciferases via Baculovirus Expression System
1 Introduction
2 Materials
2.1 Cell Strains
2.2 Bacmid DNA Preparation
2.3 Maintenance and Transfection of Insect Cells
2.4 Protein Purification and BL Measurements
2.5 Equipment
3 Methods
3.1 Bacmid DNA Preparation
3.2 Transfection of Sf9, Production of Viral Stocks for the Expression of Recombinant Luciferases
3.3 Harvesting of Culture Medium, Ammonium Sulfate Protein Precipitation
3.4 Purification of CopLuc
3.4.1 First Immobilized Metal Affinity Chromatography (IMAC)
3.4.2 Second Metal Affinity Chromatography
3.4.3 Size-Exclusion Chromatography
3.5 BL Activity Measurements
4 Notes
References
Chapter 7: Molecular Tension Probe for In Vitro Bioassays
1 Introduction
2 Materials
2.1 Reagents and Labware
2.2 Living Materials
2.3 Instrumentation
2.4 Software
3 Methods
3.1 Preparation of the DNA Constructs Encoding a Molecular Tension Probe (Fig. 1)
3.2 Purification of an ALuc Tension Probe, FRB-A23-FKBP (Fig. 2)
3.3 Determination of pH-Driven BL Intensities of FRB-A23-FKBP (Fig. 3)
3.4 Multivalent Cation-Driven BL Intensities of FRB-A23-FKBP (Fig. 4)
3.5 Determine the Kinetic Constants of FRB-A23-FKBP (Fig. 5 and Table 1)
4 Notes
References
Part II: Basic In Vitro Applications
Chapter 8: Optimized Loop-Mediated Isothermal Amplification (LAMP) Allows Single Copy Detection Using Bioluminescent Assay in ...
1 Introduction
2 Materials
2.1 Reagents for LAMP
2.2 Reagents for BART
2.3 Consumables and Specialist Equipment
2.4 Consumables for Genome Extraction from Maize
3 Methods
3.1 Preparation of LAMP Primers
3.2 Preparation of Maize Samples
3.3 Preparation of LAMP-BART Mix
3.4 LAMP-BART Assay
3.5 Optimization of LAMP-BART Assay for GM Detection
4 Notes
References
Chapter 9: A Simple and Rapid Bioluminescence-Based Functional Assay of Organic Anion Transporter 1 as a D-Luciferin Transport...
1 Introduction
2 Materials
2.1 Reagents and Labware
2.2 Instrumentation
2.3 Software
3 Methods
3.1 Preparation of Cells and Test Solution
3.2 Uptake Study
3.3 Acquisition of Bioluminescence (BL) Image
3.4 Data Analysis of BL Image
4 Notes
References
Chapter 10: A Simple Bioluminescent Assay for the Screening of Cytotoxic Molecules Against the Intracellular Form of Leishmani...
1 Introduction
2 Materials
2.1 General Reagents (See Note 1)
2.2 Parasites
2.3 Macrophages
2.4 Culture Media
2.5 Reagent Solutions to Be Prepared
2.6 Equipments
2.7 Consumables
3 Methods
3.1 Parasite Culture
3.1.1 Stock Culture
3.1.2 Pre-inoculum Culture (See Comments About In Vitro Metacyclogenesis in Note 7)
3.2 Macrophage Culture
3.3 Screening Assay (See Scheme of Assay´s Timeline in Fig. 1)
3.3.1 Infection of Macrophages (Tue)
3.3.2 Compound Screening (Wed)
3.4 Assay Readouts (from Thu up to 1 Week, See Note 19)
3.4.1 Determination of FL Intensity
3.4.2 Determination of BL Signal
3.4.3 Microscopy
3.5 Assay Validation and Data Analysis
3.5.1 Assay Validation
3.5.2 Parasite Viability for Test Compounds
3.5.3 Microscopy-Based Analysis
4 Notes
References
Chapter 11: A Simple, Robust, and Affordable Bioluminescent Assay for Drug Screening Against Infective African Trypanosomes
1 Introduction
2 Materials
2.1 General Reagents
2.2 Parasites
2.3 Reagent Solutions to Be Prepared
2.4 Culture Medium
2.5 Equipment
2.6 Consumables
3 Methods
3.1 Culture Synchronization
3.2 Screening Assay (Wed, See Fig. 1 for the Overall Timeline of the Assay)
3.2.1 Compound´s Preparation
Positive Controls
Test Compounds
3.2.2 Compounds´ Platting
3.2.3 Parasites´ Platting
3.3 Assay Readout (Thu) (See Note 11)
3.4 Data Analysis and Assay Validation
4 Notes
References
Chapter 12: Imaging of Autonomous Bioluminescence Emission From Single Mammalian Cells
1 Introduction
2 Materials
2.1 Microscope
2.2 Reagents and Labware
3 Methods
3.1 Construction of the Microscope
3.2 Sample Preparation
3.3 Imaging
3.4 Data Post-processing
4 Notes
References
Chapter 13: Rapid Single-Cell Detection of Beer-Contaminating Lactic Acid Bacteria Using Bioluminescence/Rapid Microbe Detecti...
1 Introduction
2 Materials
2.1 Test Strain
2.2 Cultivation Medium and Device
2.3 Apparatus for Beer Filtration
2.4 Reagent and Equipment for Standard ATP Sheet
2.5 Reagents and Equipment for RMD Method
2.6 Equipment for Agar Plate Method
2.7 Reagent for Accuracy Detection Test
3 Methods
3.1 Beer Filtration
3.2 RMD Method
3.2.1 Preparation of High-Sensitivity Reagent
3.2.2 Preparation of the Standard ATP Sheet for Sensitivity Analysis
3.2.3 System Start-Up
3.2.4 Operation of Sensitivity Analysis
3.2.5 Sample Measurement
3.3 Agar Plate Method
3.4 Accuracy Detection Test
4 Notes
References
Chapter 14: Bioluminescence of Aliivibrio Fischeri in Artificial Seawater and Its Application in Fungicide Sensing
1 Introduction
2 Materials
2.1 Reagents and Labware
2.2 Instrumentation
3 Methods
3.1 Preparation of Luminescent Bacterial Glycerol Stock
3.2 The ASW-Driven Variance in the Cell Growth and the Corresponding BL Intensity over Time
3.3 Effect of ASW Components on BL
3.4 Effect of K+ on A. fischeri BL
3.5 Effect of HCO3- on A. fischeri BL
3.6 Effect of Inorganic Sulfur on A. fischeri BL
3.7 Effect of Sulfur-Containing Amino Acids and Serine on A. fischeri BL
3.8 Effect of the Fungicides (OPPNa and IMZ) on A. fischeri BL
4 Notes
References
Chapter 15: A Bioluminescence Reporter Assay for Retinoic Acid Control of Translation of the GluR1 Subunit of the AMPA Glutama...
1 Introduction
2 Materials
2.1 Reagents and Labware
2.2 Instrumentation
3 Methods
3.1 Construction of GluR1 Assay Plasmid (pTK1)
3.2 GluR1 Translation Dual Luciferase Assay
4 Notes
References
Chapter 16: Design of an Intron-Retained Bioluminescence Reporter and its Application in Imaging of Pre-mRNA Splicing in Livin...
1 Introduction
2 Materials
2.1 Reagents
2.2 Instrumentation
2.3 Software
3 Methods
3.1 Plasmid Construction Scheme
3.2 Experimental Manipulation of Cells
3.2.1 Plasmid Transfection
3.2.2 RT-PCR
3.2.3 Polymerase Chain Reaction (PCR)
3.2.4 Agarose Gel Electrophoresis
3.3 BL Assays
3.4 Specific Experimental Operations as Below
3.4.1 Validation of the Reporter
3.4.2 Responses of the Reporter to Splicing Inhibitor
3.4.3 Responses of the Reporter to Dosage in Splicing Inhibitor
3.4.4 Measuring Splicing Activity Responded to Pladienolide B
3.4.5 In Vivo Imaging of the Reporter
3.5 Data Processing
4 Notes
References
Chapter 17: Generation of Bi-Reporter-Expressing Tri-Segmented Arenavirus
1 Introduction
2 Materials
2.1 Reagents
2.2 Plasmids
2.3 Cells
2.4 Labware
2.5 Instrumentation
3 Methods (See Note 4)
3.1 Generation of r3LCMV
3.2 Confirmation of Viral Rescue and Titration of r3LCMV
3.3 Amplification and Generation of a r3LCMV Stock
3.4 In Vitro Characterization of r3LCMV
4 Notes
References
Chapter 18: Bioluminescent and Fluorescent Reporter-Expressing Recombinant SARS-CoV-2
1 Introduction
2 Materials
2.1 Reagents and Labware
2.2 Cells
2.3 Virus
2.4 Software
2.5 Instrumentation
3 Methods
3.1 Generation of rSARS-CoV-2 (See Fig. 2b)
3.2 Viral Titration by Plaque Assay
3.3 Viral Growth Kinetics
4 Notes
References
Chapter 19: Generation, Characterization, and Applications of Influenza A Reporter Viruses
1 Introduction
2 Materials
2.1 Reagents and Labware
2.2 Solutions
2.3 Cell Lines
2.4 Viruses
2.5 Plasmids
2.6 Software
2.7 Instrumentation
3 Methods
3.1 Generation of PR8-mCherry (See Fig. 4)
3.2 Characterization of PR8-mCherry by Immunofluorescence (See Fig. 5a)
3.3 Multicycle Growth Kinetics (See Fig. 5b, c)
3.4 Plaque Assay to Determine Viral Titers, Plaque Phenotype, and Genetic Stability (See Fig. 5d)
3.5 A FL-Based Assay to Evaluate Antivirals (See Fig. 6a) or Neutralizing Antibodies (NAbs) (See Fig. 6b)
4 Notes
References
Part III: Basic In Vivo Applications
Chapter 20: Optimized Aequorin Reconstitution Protocol to Visualize Calcium Ion Transients in the Heart of Transgenic Zebrafis...
1 Introduction
2 Materials
2.1 Reagents and Labware
2.2 Instrumentation
2.3 Image Processing Software
2.4 Animal
3 Methods
3.1 Collection of Tg(myl7:GA) Zebrafish Eggs
3.2 Abrogate Ca2+ Transients in the Heart with Nifedipine
3.3 CTZ Reconstitution Period
3.4 Recovery Period in E3 Medium
3.5 Mounting the Embryos in Agarose for BLI
3.6 BLI
3.7 Image and Data Processing in ImageJ
4 Notes
References
Chapter 21: Quantification and Imaging of Exosomes via Luciferase-Fused Exosome Marker Proteins: ExoLuc System
1 Introduction
2 Materials
2.1 Reagents and Labware
2.2 Instrumentation
3 Methods
3.1 Establishment of ExoLuc Cells
3.1.1 Preparation of Expression Virus
3.1.2 Generation of ExoLuc Cells
3.2 NanoLuc Luciferase Assay
3.3 Exosome Isolation
3.4 Cellular Uptake Assay
3.4.1 Direct Addition System
3.4.2 Co-culture System
3.5 Exosome Biodistribution
4 Notes
References
Chapter 22: Bioluminescent Tracking of Human Induced Pluripotent Stem Cells In Vitro and In Vivo
1 Introduction
2 Materials
2.1 Reagents and Labware
2.2 Instrumentation
3 Methods
3.1 Routine Culture of iC9-iPSCs and Transduction
3.1.1 Thawing iC9-iPSCs
3.1.2 Split iC9-iPSCs
3.1.3 Lentiviral Transduction of Luc in iC9-iPSCs
3.2 Monitoring and Clearance of Luc Expressing iC9-iPSCs In Vitro
3.3 Monitoring and Clearance of Luc Expressing iC9-iPSCs In Vivo
3.3.1 Inoculation of iC9-iPSCs into NOD-SCID Mice
3.3.2 Luc Tracking of Teratoma Formation from iC9-iPSCs
3.3.3 Injection of CID for Removal of iC9-iPSCs-Derived Teratoma
4 Notes
References
Chapter 23: Non-invasive In Vivo Tracking of Mammalian Cells Stably Expressing Firefly Luciferase
1 Introduction
2 Critical Parameters
2.1 Materials
2.2 Instrumentation
3 Methods
3.1 Retrovirus Construction
3.2 Establishment of Stable Cell Lines
3.3 Luciferase Assay
3.4 In Vivo BLI
4 Notes
References
Chapter 24: Bioluminescence Imaging for Evaluation of Antitumor Effect In Vitro and In Vivo in Mice Xenografted Tumor Models
1 Introduction
2 Materials
2.1 Reagents and Labware
2.2 Cell Culture
2.3 Instrumentation
2.4 Animals
3 Methods
3.1 Transfection Firefly Luciferase (FLuc) to Cancer Cell Lines
3.2 Evaluating BLI In Vitro 2D Cell Culture
3.3 Evaluating BL In Vitro 3D Cell Culture, Spheroid
3.4 Creating In Vivo Mouse Subcutaneous (s.c.) Tumor Model
3.5 Creating In Vivo Peritoneal Disseminated Mouse Tumor Model
3.6 Creating In Vivo Pleural Disseminated Mouse Tumor Model
3.7 Creating In Vivo Lung Metastasis Mouse Tumor Model
3.8 In Vivo BLI
4 Notes
References
Chapter 25: Detection of Spontaneous Bone Metastases of Solid Human Tumor Xenografts in Mice
1 Introduction
2 Materials
2.1 Reagents and Labware
2.2 Instrumentation
3 Methods
3.1 Spontaneous Bone Metastasis Xenograft Model and In Vivo and Ex Vivo BLI
3.2 Preparing Bones for Morphologic Analyses of the Spontaneous Bone Metastases by Histological and Immunohistochemical Staini...
3.3 Hematoxylin-Eosin Staining for Analyzing Bone Metastasis Morphology (See Note 12)
3.4 Immunohistochemical Staining with Anti-Luciferase for Detection of Single Disseminated Tumor Cells in the Bone Marrow (See...
4 Notes
References
Chapter 26: In Vivo Imaging Analysis of an Inner Ear Drug Delivery in Mice: Comparison of Inner Ear Drug Concentrations Over T...
1 Introduction
2 Materials
2.1 Animals
2.2 Reagents and Labware
2.3 Instrumentation
2.4 Software
3 Methods
3.1 Injection Methods: Intravenous (I.V.) or Transtympanic or Intraperitoneal (I.P.) Injections
3.2 BLI System
4 Notes
References
Chapter 27: Protocols for the Evaluation of a Lymphatic Drug Delivery System Combined with Bioluminescence to Treat Metastatic...
1 Introduction
2 Materials
2.1 Mouse Model Inoculated with Luminescent Tumor Cells
2.2 Anesthetic Solution
2.3 Tumor Cell Inoculation
2.4 Operation Desk for the Inoculation of the Luciferase-Expressing Cell
2.5 Preparation of D-Luciferin
2.6 In Vivo BL Imaging
2.7 Ex Vivo BL Imaging
2.8 Devices
3 Methods (See Note 2)
3.1 Preparation of Animals
3.1.1 Intranodal Inoculation of Tumor Cells
3.2 D-Luciferin Aliquot Preparation and Storage
3.2.1 D-Luciferin Stock Solution Preparation
3.2.2 Preparation of D-Luciferin Solution for In Vivo Use
3.2.3 D-Luciferin Ex Vivo Solution Preparation
3.3 In Vivo BL Imaging
3.3.1 In Vivo BL Imaging of the Whole Body
3.3.2 In Vivo BL Imaging of the Region of Interest (ROI) (See Note 13, Fig. 6d)
3.4 Ex Vivo BL Imaging of the Organs of Interest (See Note 14)
4 Notes
References
Chapter 28: In Vivo Bioluminescent Imaging of Rabies Virus Infection and Evaluation of Antiviral Drug
1 Introduction
2 Materials
2.1 Reagents and Labware
2.2 Instrumentation
3 Methods
3.1 Virus Inoculation and Antiviral Treatment
3.2 In Vivo BLI
4 Notes
References
Chapter 29: Imaging Infection by Vector-Borne Protozoan Parasites Using Whole-Mouse Bioluminescence
1 Introduction
2 Materials
2.1 Parasites
2.2 Mosquitoes
2.3 Mice
2.4 Reagents and Labware
2.5 Equipment
2.6 Software
3 Methods
3.1 Homing and Infectivity of P. berghei Sporozoites
3.1.1 Collection of P. berghei Sporozoites from the Salivary Glands of Mosquitoes
3.1.2 Infection of Mice with P. berghei Sporozoites
3.1.3 Image Acquisition
3.2 Drug Testing in L. infantum-Infected Mice
3.2.1 In Vitro Culture of L. infantum Axenic Amastigotes
3.2.2 Infection of Mice with L. infantum Axenic Amastigotes and Drug Treatment
3.2.3 Image Acquisition
3.3 Study of Host Susceptibility to T. b. brucei Infection
3.3.1 Infection of Mice with T. b. brucei GVR35 Bloodstream Forms
3.3.2 Image Acquisition
3.4 Image Analysis
4 Notes
References
Chapter 30: Longitudinal Tracing of Lyssavirus Infection in Mice via In Vivo Bioluminescence Imaging
1 Introduction
2 Materials
2.1 Reagents and Labware
2.2 Instrumentation
2.3 Software
3 Methods
3.1 Generation of Recombinant Lyssavirus Expressing Luciferase
3.1.1 Cloning FLuc into a Recombinant Lyssavirus Genome
3.1.2 Transfecting HEK293T Cells to Generate Recombinant Reporter Virus
3.1.3 Amplification and Purification of Recombinant Reporter Virus
3.1.4 Determining Viral Titer
3.2 In Vivo BLI
3.2.1 Lyssavirus Challenge in Mice
3.2.2 Imaging Mice
3.2.3 BLI Analysis
3.3 Validation of Luminescence Data
3.3.1 Preparation of Tissue Samples for Quantitative-PCR Analysis
3.3.2 Preparation of Tissue Sample for Microscopy
4 Notes
References
Part IV: Multiplex Imaging Platforms
Chapter 31: Dual-Luciferase-Based Fast and Sensitive Detection of Malaria Hypnozoites for the Discovery of Anti-relapse Compou...
1 Introduction
2 Materials
2.1 Dual Reporter Line
2.2 Animals
2.3 Mosquitoes
2.4 Reagents and Labware
2.5 Instrumentation
2.6 Software
3 Methods
3.1 Harvesting Transgenic P. cynomolgi Sporozoites
3.1.1 Thawing a Cryopreserved Stock of Blood Stage Parasites (see Note 4)
3.1.2 Mosquito Feeding and Oocyst Counting
3.1.3 Sporozoite Isolation
3.2 BL-Based Analysis and Quantification of Liver Stages, Including Hypnozoites (See Fig. 1)
3.2.1 Macaque Hepatocyte Culture
3.2.2 Infection of Hepatocyte Cultures with Sporozoites
3.2.3 BL-Based Quantification of In Vitro Liver Stage Development, Including Hypnozoites
4 Notes
References
Chapter 32: Synthetic Assembly DNA Cloning of Multiplex Hextuple Luciferase Reporter Plasmids
1 Introduction
2 Materials
2.1 Living Subjects
2.2 Reagents and Labware
2.3 Instrumentation
2.4 Software
3 Methods
3.1 Generating Vectors Containing Transcription Factor-Binding Motifs
3.2 Generating Vectors Containing Single Luciferase Reporter Units
3.3 Generating Multiplex Hextuple Luciferase Reporter Vectors
3.4 Time Considerations for the Entire Procedure
4 Notes
References
Chapter 33: Multiplex Hextuple Luciferase Assaying
1 Introduction
2 Materials
2.1 Living Subjects
2.2 Reagents and Labware
2.3 Instrumentation
2.4 Software
3 Methods
3.1 Preparative Cell Culture Work for Multiplex Hextuple Luciferase Assaying
3.2 RNAi Perturbations and Transfection of the Multiplex Hextuple Luciferase Reporter Plasmid
3.3 Multiplex Hextuple Luciferase Assaying
3.4 Time Considerations for the Entire Procedure
4 Notes
References
Chapter 34: Molecular Imaging of Tumor Progression and Angiogenesis by Dual Bioluminescence
1 Introduction
2 Materials
2.1 Cell Line and Mouse
2.2 Reagents and Labware
2.3 Instrumentation
3 Methods
3.1 4T1 Cell Culture
3.2 LV-RLuc-RFP (RR) and LV-RLuc-RFP-HSV-Ttk (RRT) Lentiviral Packaging and Production
3.3 LV-RR and LV-RRT Lentiviral Transduction for Gene Expression in 4T1 Cells
3.4 Drug Screening and Identification of 4T1-RR and 4T1-RRT Cell Lines
3.5 Establishment of Subcutaneous Tumor Mouse Model
3.6 Dual BLI of Tumor Progression (RLuc) and Tumor Angiogenesis (FLuc)
4 Notes
References
Index