Hypoxia: Methods and Protocols

Preface
Contents
Contributors
Part I: Fluorescent Reporter Methods for Hypoxia Detection
	Chapter 1: Genetically Encoded Reporters to Monitor Hypoxia
		1 Introduction
		2 Materials
			2.1 Eukaryotic Cell Culture Media, Reagents, and Equipment
			2.2 Transfection Reagents and Equipment
			2.3 Mice: Surgery, Perfusion, and Dissection: Medications, Reagents, and Equipment
			2.4 Vibratome Sectioning and Sample Mounting: Reagents and Equipment
			2.5 Microscopic Systems, Equipment, and Software
		3 Methods
			3.1 Cultivation of the Human Gli36 Tumor Cell Line
			3.2 Calcium Phosphate Transfection
			3.3 Generation of Stable Eukaryotic Cell Lines by Antibiotic and FACS Selection
				3.3.1 Counter Selection with G418
				3.3.2 FACS of Homogenously Fluorescent Cell Bulks
			3.4 In Vitro Live-Cell Oxygen Imaging and Analysis
				3.4.1 Time-Lapse Imaging of Cellular Hypoxia and Reoxygenation Using HRE-dUnaG and HRE-dUnOHR
				3.4.2 Ratiometric Sensing of Intracellular Oxygen Concentrations with CMV-dUnORS
				3.4.3 Fluorescence Lifetime-Based Cellular Oxygen Sensing with CMV-dUnOFLS
			3.5 Intracranial Tumor Injections in Mice
				3.5.1 Preparation of the Reporter Cell Lines
				3.5.2 Surgical Procedure
			3.6 Perfusion and Fixation of Intracranial Tumors In Situ for Subsequent Brain Dissection and Tumor Preparation
				3.6.1 Preparation of the Perfusates
				3.6.2 Surgical Procedure
				3.6.3 Brain Dissection
			3.7 Preparing Vibratome Sections of the Murine Brain
				3.7.1 Vibratome Sectioning
				3.7.2 Sample Mounting
			3.8 Ex Vivo Visualization of Intratumoral Hypoxia and Oxygen Gradients
		4 Notes
		References
	Chapter 2: Detection of Hypoxia in 2D and 3D Cell Culture Systems Using Genetically Encoded Fluorescent Hypoxia Sensors
		1 Introduction
		2 Materials
			2.1 Lentivirus Production in HEK293T Cells Using a Second-Generation Lentiviral System and Transduction of hAD-MSCs
			2.2 Thawing, Subcultivation, and Harvesting of Hypoxia Reporter hAD-MSCs
			2.3 Encapsulation of Hypoxia Reporter hAD-MSCs in Gelatine-Methacryloyl (GelMA) Hydrogels
			2.4 Enzymatic Digestion of GelMA Hydrogels
			2.5 Evaluation of Hypoxia Reporter Response in 2D and 3D Cultivation via Fluorescence Microscopy and Flow Cytometry
		3 Methods
			3.1 Lentivirus Production in HEK293T Cells Using a Second-Generation Lentiviral System
			3.2 Thawing of hAD-MSCs
			3.3 hAD-MSCs Transduction
			3.4 Subcultivation and Harvesting of Hypoxia Reporter hAD-MSCs
			3.5 Evaluation of Hypoxia Reporter Response in 2D Cultivation
			3.6 Evaluation of Hypoxia Reporter Response in 3D Cultivation in Gelatine-Methacryloyl Hydrogels
		4 Notes
		References
	Chapter 3: Mapping the Fate of Hypoxic Cells Using an Irreversible Fluorescent Switch
		1 Introduction
		2 Materials
			2.1 Plasmid DNA Purification
			2.2 Lentiviral Delivery
			2.3 Cell Culture
			2.4 Cell Clonal Selection
			2.5 Flow Cytometry and Fluorescence-Activated Cell Sorting (FACS)
		3 Methods
			3.1 Plasmid DNA Purification
			3.2 Lentiviral Transfection
			3.3 Generating a Stable CMV-loxp-DsRed-loxp-eGFP Expressing Cell Line
				3.3.1 Lentiviral Transduction
				3.3.2 Zeocin Selection
				3.3.3 FACS Selection
			3.4 Generating Cells that Express the Dual Lentiviral Hypoxia Fate-Mapping System
				3.4.1 Lentiviral Transduction
				3.4.2 Single-Cell Cloning
				3.4.3 Fluorescent Screening
			3.5 Validation
				3.5.1 Live-Cell Imaging
				3.5.2 Flow Cytometry
		4 Notes
		References
	Chapter 4: Application of a Specific and Sensitive NQO1 Turn-On Near-Infrared Fluorescence Probe for Live Cancer Cell and Xeno...
		1 Introduction
		2 Materials
		3 Methods
			3.1 Confocal Fluorescence Microscopic Imaging in Live Cells
			3.2 Flow Cytometric Quantitation of NQO1 Cleavage of NIR-ASM
			3.3 Imaging of NQO1 Activity/Content in Tumor Xenografts in Live Mice
		4 Notes
		References
Part II: Non-fluorescent Reporter Methods for Hypoxia Detection
	Chapter 5: A Luciferase Reporter Assay to Detect Cellular Hypoxia In Vitro
		1 Introduction
		2 Materials
			2.1 Living Materials
			2.2 Reagents and Labware
			2.3 Instrumentation
		3 Methods
			3.1 Cloning of Firefly Luciferase Reporter Vectors Containing Various Numbers of HREs (pGL4.26-2nHRE)
				3.1.1 Prepare the 2HRE Insert
				3.1.2 Assemble the 2HRE Firefly Luciferase Reporter Vector (Fig. 2)
				3.1.3 Purify the Reporter Plasmid and Confirm the Presence and Orientation of the Insert
			3.2 Cloning of Renilla Luciferase Reporter Vector (pGL4.R[hRluc/minP/Hygr]) from pGL4.26[Luc2/minP/Hygr] and pGL4.70[hRluc] (F...
				3.2.1 Prepare Linearized pGL4.26 Without the Luc2  Gene
				3.2.2 Prepare the Renilla luciferase to Insert
				3.2.3 Generate pGL4.R[hRluc/minP/Hygr] Renilla luciferase Reporter Vector
			3.3 Cloning of Firefly and Renilla Luciferase Reporter Vectors at Both Ends of a Various Number of HREs (pGL4.Dual[Luc2, hRluc...
				3.3.1 Prepare the Insert Containing the Minimal Promoter, Renilla luciferase Gene, and Poly A Sequence
				3.3.2 Prepare the Linearized pGL4.26-2nHRE- Vector
				3.3.3 Generate pGL4.Dual[Luc2, hRluc]-2nHRE- Vector (See Note 6)
			3.4 In Vitro Evaluation of the Genetic Response of Multiple Hypoxia Response Elements to Cellular Hypoxia Using a Luciferase A...
		4 Notes
		References
	Chapter 6: Oxygen Assessment in Tumors In Vivo Using Phosphorescence Lifetime Imaging Microscopy
		1 Introduction
		2 Materials
		3 Methods
			3.1 Generating a Tumor Xenograft
			3.2 Preparation of Mice for PLIM
			3.3 PLIM Acquisition
			3.4 PLIM Analysis
		4 Notes
		References
	Chapter 7: Radionuclide Reporter Imaging to Visualize Tumor Hypoxia Ex Vivo and In Vivo
		1 Introduction
		2 Materials
			2.1 Living Materials
			2.2 Reagents and Labware
			2.3 Instrumentation
		3 Methods
			3.1 Cloning of hNIS Reporter Vector Containing Multiple Copies of HRE (pGL4-12HRE-hNIS) (See Note 1 and Fig. 2)
			3.2 Establishment of Stable Cell Lines Expressing pGL4-12HRE-hNIS
			3.3 Preparation of a Mouse Subcutaneous Xenograft Model
			3.4 Ex Vivo Evaluation of the Genetic Response of Multiple Hypoxia Response Elements to Tumor Hypoxia Using Digital Autoradiog...
			3.5 In Vivo Evaluation of the Genetic Response of Multiple Hypoxia Response Elements to Tumor Hypoxia Using SPECT or PET
		4 Notes
		References
	Chapter 8: Measuring Pericellular Oxygen Tension for In Vitro Cell Culture
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparing Sensors
			3.2 Connecting Sensors to Computer
			3.3 Calibrating Sensors
			3.4 Cell Culture and Data Analysis
		4 Notes
		References
	Chapter 9: A Review of Hypoxia Imaging Using 18F-Fluoromisonidazole Positron Emission Tomography
		1 Introduction
		2 Materials and Methods
			2.1 Synthesis of FMISO
			2.2 Patient Preparation
			2.3 Tracer Administration
			2.4 Wait  Time
			2.5 Image Acquisition
			2.6 Image Reconstruction
			2.7 Image Analysis
			2.8 Other Tracers for Hypoxia Imaging
		References
	Chapter 10: Photoacoustic Lifetime Imaging of Hypoxia
		1 Introduction
			1.1 Principles of Oxygen Sensing by Photoacoustic Lifetime Imaging (PALI)
			1.2 PALI System
		2 Materials
			2.1 Multimodal Imaging System
			2.2 Small Animal Imaging Platform
			2.3 Reagents
		3 Methods
			3.1 Preparation
			3.2 In Vivo Imaging
			3.3 Signal Processing
			3.4 Image Presentation
		4 Notes
		References
	Chapter 11: Staining Hypoxic Areas of Frozen and FFPE Tissue Sections with Hypoxyprobe
		1 Introduction
		2 Materials
			2.1 Pimonidazole HCl Injection
			2.2 Frozen Tissue Processing
			2.3 FFPE Tissue Processing
			2.4 General Staining Requirements
				2.4.1 IF-Specific Requirements
				2.4.2 IHC-Specific Requirements
				2.4.3 Additional Requirements for Staining Frozen Tissues
				2.4.4 Additional Requirements for Staining FFPE Tissues
			2.5 Antibodies
		3 Methods
			3.1 Pimonidazole HCl Injection
			3.2 Tissue Processing
				3.2.1 Frozen Tissue Processing
				3.2.2 FFPE Tissue Processing
			3.3 IF Staining on Frozen Tissues
			3.4 Staining FFPE Tissues
				3.4.1 IF on FFPE Tissues
				3.4.2 IHC on FFPE Tissues
			3.5 Imaging
		4 Notes
		References
Part III: Methods to Detect Hypoxia Inducible Factors and their Downstream Targets
	Chapter 12: Multiplex Immunofluorescence Staining Protocol for the Dual Imaging of Hypoxia-Inducible Factors 1 and 2 on Formal...
		1 Introduction
		2 Materials
			2.1 Deparaffinization and Hydration
			2.2 Antigen Unmasking and Antibody Stripping
			2.3 Peroxidase Quenching
			2.4 Immunofluorescence Staining
			2.5 Tyramide Amplification
			2.6 Antibodies
			2.7 Others
		3 Methods
			3.1 Deparaffinization and Serial Hydration
			3.2 Antigen Unmasking
			3.3 Peroxidase Quenching
			3.4 Immunofluorescence Staining
			3.5 Tyramide Amplification
			3.6 Antibody Stripping
			3.7 Multiplexing
			3.8 DAPI Staining
			3.9 Mounting and Storage
			3.10 Imaging
		4 Notes
		References
	Chapter 13: Detecting Hypoxia-Inducible Factor Levels and Activity
		1 Introduction
		2 Materials
			2.1 SDS-PAGE
			2.2 Western Blot
			2.3 Reporter Assay
			2.4 RNA Isolation
			2.5 qRT-PCR
		3 Methods
			3.1 SDS-PAGE
			3.2 Western Blot
			3.3 Reporter Assay
			3.4 RNA Isolation
			3.5 qRT-PCR
		4 Notes
		References
Part IV: Methods to Detect Hypoxia-Induced Changes in Cell Metabolism
	Chapter 14: Metabolomic Investigations into Hypoxia-Mediated Metabolic Reprogramming of Pancreatic Cancer Cells
		1 Introduction
		2 Materials
			2.1 Cell Culture Reagents
			2.2 LC-MS/MS Reagent
			2.3 Western Blot Analysis
			2.4 Equipment
			2.5 Software
		3 Methods
			3.1 Mobile Phase Preparation
			3.2 Cell Culture and Hypoxia
			3.3 Metabolites Extraction
			3.4 Liquid Chromatography-Coupled Tandem Mass Spectrometry
			3.5 Data Processing
			3.6 Selecting the Metabolite Peaks for Comparative Analysis
			3.7 Data Formatting for Fold Change Analysis
			3.8 Data Analysis Using MetaboAnalyst and Data Interpretation
			3.9 Western Blotting
		4 Notes
		References
	Chapter 15: Measurement of Metabolic Alteration in Immune Cells Under Hypoxia
		Abbreviations
		1 Introduction
		2 Material
			2.1 Cell Culture Reagents
			2.2 Animal Necropsy and Bone Marrow Cell Isolation
			2.3 MDSC Purification
			2.4 Flow Cytometer Sample Preparation, Staining, and Fixing
			2.5 RNA Isolation Reagents
			2.6 cDNA Synthesis and Real-Time PCR Reagents
			2.7 Seahorse Assay
			2.8 Equipment
		3 Methods
			3.1 Harvesting Bone Marrow Cells from Mice and MDSC Differentiation
			3.2 MDSC Purification Using Kit As Per Manufacturer´s Protocol
			3.3 Cell Sample Preparation for FACS Analysis
			3.4 Hypoxic Culture
			3.5 RNA Isolation
			3.6 cDNA Synthesis
			3.7 Real-Time PCR and Data Analysis
			3.8 Seahorse Assay to Measure Oxygen Consumption  Rate
		4 Notes
		References
Part V: Methods to Simulate Hypoxia, Evaluate Oxygen Consumption Rates, and Measure FIH Activity Levels
	Chapter 16: Evaluation of Oxygen Consumption Rates In Situ
		1 Introduction
		2 Materials
			2.1 Method 1 OCR Measurements Using an Electrode
			2.2 Approach 2 Via Seahorse Instrument
		3 Methods
			3.1 Method 1: Measuring Oxygen Consumption Rates Via an Electrode
				3.1.1 Calibrating the Oxygen Electrodes (See Fig. 1)
				3.1.2 Counting Cardiomyocytes (About 10 min)
				3.1.3 Measuring Mitochondrial Respiration
				3.1.4 Cleaning the Oxygen Electrode Chambers (About 35 min)
			3.2 Method 2: Measuring Oxygen Consumption Rates Via a Seahorse Kit (Mitochondrial Stress Test)
				3.2.1 Seed Cells in XF96 Cell Culture Microplates (See Fig. 4)
				3.2.2 Hydrate a Seahorse XFe96 Sensory Cartridge
				3.2.3 Prepare to Run the Seahorse Assay
				3.2.4 Calibration and Running the Seahorse Assay
		4 Notes
		References
	Chapter 17: Cleanroom-Free Microfluidic Device for Natural Induction of Hypoxia in 2D and 3D Tumor Models
		1 Introduction
		2 Materials
			2.1 Master Mold Design and Fabrication
			2.2 Microfluidic Device Fabrication and Assembly
			2.3 Establishing 2D and 3D Tumor Models
			2.4 Confirmation of Hypoxia Gradient in Tumor Models
			2.5 Microscopy
		3 Methods
			3.1 Microfluidic Device Design and Fabrication
				3.1.1 Polycarbonate (PC) Master Mold Fabrication and PDMS Replica Molding
				3.1.2 Polycarbonate (PC) Film: PDMS Assembly for Hypoxia Microfluidic Device
			3.2 Establishing a 2D Tumor Model
			3.3 Establishing a 3D Tumor Model
			3.4 Confirming the Hypoxia Gradient in 2D and 3D Tumor Models
				3.4.1 Live Cell Staining of 2D Tumor Models
				3.4.2 Immunostaining of 2D and 3D Tumor Models
		4 Notes
		References
	Chapter 18: Oxomer- and Reporter Gene-Based Analysis of FIH Activity in Cells
		1 Introduction
		2 Materials
			2.1 General Materials
			2.2 Oxomer-Dependent Analysis of FIH Enzymatic Activity
				2.2.1 Cell Culture
				2.2.2 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
				2.2.3 Immunoblotting
			2.3 HIF-Dependent Firefly Luciferase-Based Reporter Gene Assay
		3 Methods
			3.1 Oxomer-Dependent Analysis of FIH Enzymatic Activity
				3.1.1 SDS-PAGE
				3.1.2 Immunoblotting
			3.2 HIF-Dependent Firefly Luciferase-Based Reporter Gene Assay to Analyze FIH Enzymatic Activity
		4 Notes
		References
Index