Metabolic Reprogramming: Methods and Protocols (Methods in Molecular Biology, 2675)

Preface
Acknowledgments
Contents
Contributors
Chapter 1: In Vivo Tissue Lipid Uptake in Antisense Oligonucleotide (ASO)-Treated Mice
	1 Introduction
	2 Materials
		2.1 Animal Treatments
	3 Methods
		3.1 Animal Treatments
		3.2 Sample Preparation and Measurements
		3.3 Data Analysis
	4 Notes
	References
Chapter 2: In Vivo Hepatic Triglyceride Secretion Rate in Antisense Oligonucleotide (ASO)-Treated Mice
	1 Introduction
	2 Materials
		2.1 In Vivo Antisense Oligonucleotide (ASO) Treatment
		2.2 Triglyceride (TG) Secretion Rate
	3 Methods
		3.1 In Vivo ASO Treatment
		3.2 Triglyceride (TG) Secretion Rate
		3.3 VLDL Isolation and Characterization
		3.4 Further Processing and Use of VLDLs
	4 Notes
	References
Chapter 3: Measurement of Fatty Acid Oxidation by High-Resolution Respirometry: Special Considerations for Analysis of Skeleta...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Air/Zero Calibration of OROBOROS Oxygraph-2K
		3.2 Sample Preparation for Skeletal Muscle
		3.3 Sample Preparation for Cardiac Muscle
		3.4 Sample Preparation for Adipose Tissue
		3.5 The Making of Adipose Tissue Slices
		3.6 The Making of Adipose Tissue Homogenates
		3.7 Isolation of Mitochondria from Adipose Tissue
		3.8 Analysis of OXPHOS by High-Resolution Respirometry
		3.9 Modifications of the SUIT Protocol
	4 Notes
	References
Chapter 4: In Vivo Imaging of Bone Marrow Long-Chain Fatty Acid Uptake
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Lentiviral Transduction
		2.3 Transplantation
		2.4 In Vivo Imaging
	3 Methods
		3.1 Transfection of Packaging Cells to Produce pCDH-Luciferase-T2A-mCherry Virus
		3.2 Lentiviral Transduction of cKit Cells
		3.3 Conditioning Mice for Transplant
		3.4 FFA-Luciferase Allograft Mouse Engraftment Is Monitored by In Vivo Bioluminescent Live Animal Imaging
	4 Notes
	References
Chapter 5: Quantification and Tracing of Stable Isotope into Cysteine and Glutathione
	1 Introduction
	2 Materials
		2.1 Cell Culture and Buffers
		2.2 LC-Ms
	3 Methods
		3.1 Plating of A549 Cells for Stable Isotope Labeling
		3.2 Stable Isotope-Labeled Metabolite Labeling
		3.3 Extraction of Cells
		3.4 LC-MS Analysis
		3.5 LC-MS File Conversion with Xcalibur File Converter
		3.6 Stable Isotope-Labeling Analysis with El-MAVEN
		3.7 Calculating Intracellular Concentrations
	4 Notes
	References
Chapter 6: Targeted Quantification of Amino Acids by Dansylation
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Supplies
		2.3 Standard Reagents
		2.4 Dansyl Chloride (DNS) Derivatization Reagents
	3 Methods
		3.1 Extraction of Amino Acids from Standards and Samples
		3.2 DNS Derivatization in  Dark
		3.3 Separation by Liquid Chromatography and Compound Detection by Electrospray Ionization Mass Spectrometry
		3.4 Data Analysis
	4 Notes
	References
Chapter 7: Isolation of Mitochondria from Mouse Tissues for Functional Analysis
	1 Introduction
	2 Materials
		2.1 Mitochondrial Isolations for Mouse Tissues
		2.2 Isolation of Mitochondria from the Liver and Kidney
		2.3 Isolation of Mitochondria from the Heart
		2.4 Isolation of Mitochondria from the Brain
		2.5 Isolation of Mitochondria from Skeletal Muscle
		2.6 Isolation of Mitochondria from Brown and White Adipose Tissue
	3 Methods
		3.1 Isolation of Mitochondria from Mouse Liver
		3.2 Isolation of Mitochondria from Mouse Kidney
		3.3 Isolation of Mitochondria from Mouse Heart
		3.4 Isolation of Mitochondria from Mouse Brain
		3.5 Isolation of Mitochondria from Mouse Skeletal Muscle
		3.6 Isolation of Mitochondria from Mouse Brown Adipose Tissue
		3.7 Isolation of Mitochondria from Mouse White Adipose Tissue
		3.8 Mitochondrial Functional Assays
	4 Notes
	References
Chapter 8: Methods for Monitoring Mitochondrial Biogenesis and Turnover in Cultured Hepatocytes and Mouse Liver Using MitoTime...
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
	3 Methods
		3.1 MitoTimer Assay: Primary Mouse Hepatocyte Isolation
		3.2 MitoTimer Assay: Adenovirus Transduction in Primary Mouse Hepatocytes
		3.3 MitoTimer Assay: Image Capture
		3.4 Detection of Mitochondrial Biogenesis in Mouse Liver: Adenovirus Injection and Tissue Processing
		3.5 Detection of Mitochondrial Biogenesis in Mouse Liver: Image Capture
	4 Notes
	References
Chapter 9: Quantification of Intracellular ATP Content in Ex Vivo GC B Cells
	1 Introduction
	2 Materials
		2.1 Sample Processing
		2.2 B-Cell Purification and Maintenance (for In Vitro Experiments) (See Note 1)
		2.3 Stimulus
		2.4 Metabolic Modifiers (for In Vitro Experiments)
		2.5 B-Cell and Quinacrine Staining (See Note 2)
		2.6 Flow Cytometry
	3 Methods
		3.1 Sample Processing
		3.2 B-Cell Purification and Culture (for In Vitro Experiments) (See Note 7)
		3.3 B-Cell and Quinacrine Staining
		3.4 Flow Cytometry and Analysis
	4 Notes
	References
Chapter 10: UHPLC-HRMS-Based Analysis of S-Hydroxymethyl-Glutathione, GSH, and GSSG in Human Cells
	1 Introduction
	2 Materials
		2.1 Samples, Solutions, Buffers, Media, and Mobile Phases
		2.2 Disposable Material
		2.3 Chemical Synthesis Reagents
		2.4 Instruments
	3 Methods
		3.1 HSMGSH Synthesis
		3.2 Sample Generation and Collection
			3.2.1 Cell Growth and Formaldehyde Treatment
			3.2.2 Cell Wash and Quenching
			3.2.3 Harvest  Step
			3.2.4 Metabolite Extraction
			3.2.5 Protein Quantification and Cell Counting
		3.3 Sample Lyophilization
		3.4 Sample Reconstitution for UHPLC-HRMS Analysis
		3.5 Preparation of Pooled Quality Control Samples
		3.6 Preparation of System Suitability Sample and Mass Spectrometer Calibration
		3.7 UHPLC-HRMS-Based Method
		3.8 Determination of HSMGSH Stability by UHPLC-HRMS
		3.9 Determination of Linearity of Response and Reaction Yield by UHPLC-HRMS
		3.10 UHPLC-MS/MS Analysis
		3.11 Sample Analysis by UHPLC-HRMS
		3.12 Data Analysis
	4 Notes
	References
Chapter 11: Quantitation of Glutathione and Oxidized Glutathione Ratios from Biological Matrices Using LC-MS/MS
	1 Introduction
	2 Materials
	3 Methods
		3.1 Mass Spectrometer Optimization
		3.2 Liquid Chromatography Optimization
		3.3 Establishing Linear Ranges and Constructing Calibration Curves
		3.4 Method Validation
		3.5 Sample Preparation
		3.6 Building Sample Batches
		3.7 Data Analysis
	4 Notes
	References
Chapter 12: Real-Time Monitoring of Hydrogen Peroxide Levels in Yeast and Mammalian Cells
	1 Introduction
	2 Materials
		2.1 Yeast: Plasmid and Yeast Strain Transformation (See Note 1)
		2.2 Yeast Culture Media
		2.3 Yeast Redox Measurement
		2.4 Yeast: Calculation, Visualization, and Statistical Analysis Software
		2.5 Mammalian Cells: Plasmids
		2.6 Cultivation and Transfection of HEK293 Cells
		2.7 Mammalian Cells: H2O2 Measurement and Analysis
		2.8 Imaging Filters
		2.9 Software
	3 Methods
		3.1 Yeast Transformation and Selection
		3.2 Yeast Cell Culture and Harvesting
		3.3 Sample Preparation in 96-Well Plate with Controls
		3.4 Real-Time roGFP2 Fluorescence Measurement
		3.5 Oxidation Degree Calculation
		3.6 Mammalian Cells: Cultivation of HEK293 Cells (Day 1)
		3.7 Transfection of HEK293 Cells (Day 2)
		3.8 H2O2 Measurement in HEK293 Cells (Day 4)
		3.9 Data Analysis
	4 Notes
	References
Chapter 13: Tracer-Based Metabolic Analysis by NMR in Intact Perfused Human Liver Tissue
	1 Introduction
	2 Materials
		2.1 Media, Buffers, and Reagents for Perfusion
		2.2 Reagents for NMR Sample Preparation
		2.3 Reagents for Isolation of Primary Hepatocytes
	3 Methods
		3.1 Perfusion of Encapsulated Tissue Wedges and Sampling of Tissue
		3.2 Preparation of Tissue Extracts for NMR Spectroscopy
		3.3 NMR Data Acquisition
		3.4 NMR Data Processing
		3.5 Use of Primary Hepatocytes for NMR-Based Metabolic Experiments
	4 Notes
	References
Chapter 14: 13C Isotope Labeling and Mass Spectrometric Isotope Enrichment Analysis in Acute Brain Slices
	1 Introduction
	2 Materials
		2.1 Preparation of Acute Cerebellar Slices
		2.2 Metabolic Tracing of Slices Using U-13C6-Glucose
		2.3 LC-MS Analysis of 13C-Labeled Amino Acids and Amines
		2.4 LC-MS Analysis of 13C-Labeled Anionic Compounds
		2.5 Data Analysis of 13C-Labeled Metabolites
	3 Methods
		3.1 Preparation of Acute Cerebellar Brain Slices
		3.2 Metabolic Tracing of Slices Using U-13C6-Labeled Glucose
		3.3 LC-MS Measurement and Data Analysis of 13C-Labeled Metabolites
	4 Notes
	References
Chapter 15: Metabolite Analyses Using Nuclear Magnetic Resonance (NMR) Spectroscopy in Plasma of Patients with Prostate Cancer
	1 Introduction
	2 Materials
		2.1 Blood Collection
		2.2 NMR Sample Preparation
		2.3 NMR Experiments and Profiling
	3 Methods
		3.1 Blood Collection: Plasma
		3.2 Blood Collection: Serum
		3.3 NMR Sample Preparation
		3.4 NMR: Experimental Setup
		3.5 NMR: Creating of Shimming Parameters for Blood Sample
		3.6 Run NMR Experiment
		3.7 NMR Pre-processing
		3.8 NMR Profiling
	4 Notes
	References
Chapter 16: Screening Kinase-Dependent Phosphorylation of Key Metabolic Reprogramming Regulators
	1 Introduction
	2 Materials
		2.1 Plasmids and Purified Proteins
		2.2 Expression and Purification of Histidine-Tagged Recombinant Proteins
		2.3 Ectopic Expression and Purification of JNK1 Constitutive Active (JNK1CA) in HEK293T Cells
		2.4 Purification of Endogenous Active JNK1 in Lysates of Human Cancer Cells
		2.5 PKM2 Phosphorylation Assay
		2.6 SDS-PAGE, Coomassie Blue Staining, and Autoradiography
	3 Methods
		3.1 Expression of Histidine-Tagged Recombinant Proteins
		3.2 Purification of Histidine-Tagged Recombinant PKM2 Proteins (His-PKM2)
		3.3 Ectopic Expression of JNK1 Constitutive Active (JNK1CA) in HEK293T Cells
		3.4 Purification of Ectopically Expressed FLAG-JNK1CA
		3.5 Purification of Endogenous Active JNK1
		3.6 In Vitro Kinase Assay to Assess PKM2 Phosphorylation by JNK
		3.7 Electrophoresis, Gel Drying, and Autoradiography
	4 Notes
	References
Chapter 17: Measuring the Oxidation State and Enzymatic Activity of Glyceraldehyde Phosphate Dehydrogenase (GAPDH)
	1 Introduction
	2 Materials
		2.1 Trichloroacetic Acid (TCA) Precipitation, Lysis, and Protein Extraction
		2.2 Alkylation of Free Thiols, SDS-PAGE, and Immunoblotting
		2.3 Yeast Harvesting and Cell Lysis
		2.4 GAPDH Activity Measurement
	3 Methods
		3.1 Measuring Reduced GAPDH and Probing Reversibly Oxidized GAPDH: Cell Growth and Quenching with Trichloroacetic Acid (TCA)
		3.2 Measuring Reduced GAPDH and Probing Reversibly Oxidized GAPDH: TCA Lysis and Protein Extraction
		3.3 Measuring Reduced GAPDH
		3.4 Measuring Reversibly Oxidized GAPDH
		3.5 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)
		3.6 Immunoblotting and Quantification of % GAPDH Oxidation
		3.7 GAPDH Activity Measurement: Yeast Cell Growth and Preparation of Lysates
		3.8 GAPDH Activity Measurement: Preparation of Assay Mixture
		3.9 GAPDH Activity Measurement: Quantification of Enzymatic Activity
	4 Notes
	References
Chapter 18: Quantitation of Glutamine Synthetase 1 Activity in Drosophila melanogaster
	1 Introduction
		1.1 The Glutamine-Glutamate Cycle
		1.2 Glutamine Synthetase Activity Assays
	2 Materials
		2.1 Enzymes
		2.2 Activity Assays
	3 Methods
		3.1 Drosophila Extracts
		3.2 Colorimetric Assay of γ-Glutamylhydroxyamate
		3.3 Glutamine Synthetase Activity Assays
			3.3.1 Fixed-Time Assays
			3.3.2 Time-Course Assays
			3.3.3 Calculation of Glutamine Synthetase Activity
	4 Notes
	References
Chapter 19: Fixed Cell Immunofluorescence for Quantification of Hypoxia-Induced Changes in Histone Methylation
	1 Introduction
	2 Materials
		2.1 Cell Culture and Treatments
		2.2 Immunofluorescence
	3 Methods
		3.1 Cell Culture Preparation and Treatment
		3.2 Fixation and Permeabilization
			3.2.1 Methanol Fixation and Permeabilization
			3.2.2 PFA Fixation and Triton X-100 Permeabilization
		3.3 Blocking
		3.4 Immunostaining (See Note 14)
		3.5 Image Acquisition and Analysis
	4 Notes
	References
Chapter 20: Metabolic Reprogramming During B-Cell Differentiation
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Phenotype Analysis
	3 Methods
		3.1 Preparation of Irradiated CD40L-L Cells for Co-Culture of B Cells
		3.2 Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
		3.3 Isolation of B Cells
		3.4 Phenotypic Analysis by Flow Cytometry
		3.5 In Vitro Culture Conditions for the Generation of Resting B Cells (Day 0)
		3.6 In Vitro Culture Conditions for the Generation of Activated B Cells (Day 3)
		3.7 In Vitro Culture Conditions for the Generation of Plasmablasts (Day 6)
		3.8 In Vitro Culture Conditions for the Generation of Plasma Cells (Day 13 Onward)
	4 Notes
	References
Chapter 21: An Integrated Methodology to Quantify the Glycolytic Stress in Plasma Cell Myeloma in Response to Cytotoxic Drugs
	1 Introduction
	2 Materials
		2.1 Reagents and Materials
		2.2 Equipment
		2.3 Software
	3 Methods
		3.1 Growing and Maintenance of Myeloma Cells
		3.2 Cell Seeding, Day 1
		3.3 Setting Up the XFe96 Extracellular Flux Analyzer, Day 2
		3.4 Preparing a Sensor Cartridge before Running an XF Glycolysis Stress Test, Day 2
		3.5 Cytotoxic Treatment, Day 2
		3.6 Seeding Pre-Treated Myeloma Cells into XF96 Cell Culture Microplate, Day 3
		3.7 The Making of the Injection Solutions, Day 3
		3.8 Injection of the Solutions and Running Assay, Day 3
		3.9 Run the XF Glycolysis Stress Test, Day 3
		3.10 Data Analysis and Normalization
	4 Notes
	References
Chapter 22: Spatial Analysis of Nucleotide Metabolism: From CRISPR Knockout Cancer Cells to MALDI Imaging of Tumors
	1 Introduction
	2 Materials
		2.1 Single Cell Derived CRISPR/Cas9 Knockout Clones´ Production
		2.2 Xenograft Mouse Model Generation
		2.3 Sample Preparation for MALDI Imaging
		2.4 MALDI Imaging
	3 Methods
		3.1 Single Cell Derived CRISPR/Cas9 Knockout Clones´ Production
		3.2 Xenograft Tumor Model: Preparation of HKP1 Cells for Injection
		3.3 Xenograft Tumor Model: Subcutaneous Inoculation of Cells
		3.4 Xenograft Tumor Model: Tumor Excision and Embedding
		3.5 Sample Preparation for MALDI Imaging: Cryo-sectioning
		3.6 Sample Preparation for MALDI Imaging: Matrix Deposition
		3.7 MALDI Imaging: MALDI-TOF Measurement
		3.8 MALDI Imaging: Hematoxylin and Eosin Staining
		3.9 MALDI Imaging: MALDI Imaging Data Processing
	4 Notes
	References
Chapter 23: Assessment of Metabolic Pathways and Parameters in Extracellular Matrix-Detached Cells
	1 Introduction
	2 Materials
		2.1 U-13C Glucose and/or U-13C Glutamine Tracing
		2.2 Glucose Uptake Assay
		2.3 Measuring Fatty Acid Oxidation
		2.4 Measurement of ATP Levels
		2.5 Assays to Measure ROS in ECM Detachment
	3 Methods
		3.1 U-13C Glucose and/or U-13C Glutamine Tracing in ECM Detachment
		3.2 Glucose Uptake Assay
		3.3 Measuring Fatty Acid Oxidation
		3.4 Measurement of ATP Levels
		3.5 Assay to Measure ROS Using DCFDA
		3.6 Assay to Measure ROS Using CellROX (via Fluorescence Microscopy)
		3.7 Assay for Measuring ROS Using MitoSOX (via Fluorescence Microscopy)
	4 Notes
	References
Chapter 24: Metabolic Networks: Weighted Gene Correlation Network Analysis
	1 Introduction
	2 Reagents
	3 Methods
	4 Notes
	References
Index