Ferroptosis: Methods and Protocols (Methods in Molecular Biology, 2712)

✍ Scribed by Guido Kroemer (editor), Daolin Tang (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 258
• Edition: 1st ed. 2023
• Category: Library

✦ Synopsis

This volume provides a comprehensive collection of experimental protocols for investigating ferroptosis in different systems, including cultured cells, animal models, and human tissues. The techniques covered in this book look at various aspects of ferroptosis ranging from the detection of lipid peroxidation to the measurement of glutathione peroxidase activity and the evaluation of mitochondrial morphology. Chapters also discuss basic molecular biology methods such as quantitative PCR and immunoblotting, and advanced imaging techniques such as transmission electron microscopy and confocal fluorescence microscopy. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and authoritative, Ferroptosis: Methods and Protocols is a valuable resource for researchers who are interested in studying ferroptosis in different contexts including basic research, drug discovery, and clinical applications.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Fluorogenic Probes for Intracellular Iron Detection
1 Introduction
2 Materials
3 Methods
3.1 Iron Probe for Measuring Fe2+
3.2 Iron Probe for Measuring LIP
3.2.1 Calcein-AM Assay
3.2.2 FIP-1 Assay
4 Notes
References
Chapter 2: Stratifying Ferroptosis Sensitivity in Cells and Tissues with PALP
1 Introduction
2 Materials
2.1 Cell Culture and Tissue Sectioning
2.2 Preparation of Fluorescent Lipid Peroxidation Reporter
2.3 Measurement of Lipid Peroxidation
2.3.1 Required Software
2.3.2 Required Equipment
3 Methods
3.1 Collecting and Snap-Freezing the Tissue Samples
3.2 Cryosectioning Tissues (see Note 1)
3.3 Pre-staining Sample with BODIPY-C11 (see Notes 4-6)
3.4 Inducing Lipid Peroxidation and Stratifying Ferroptosis Sensitivity (see Notes 6 and 7)
3.5 Data Processing: Quantify PALP Signals on Regions of Interest (see Notes 11 and 12)
4 Notes
References
Chapter 3: ChIP and ChIRP Assays in Ferroptosis
1 ChIP Assay in Ferroptosis
1.1 Introduction
1.2 Materials
1.2.1 Materials and Equipments
1.2.2 Reagents and Buffers
1.3 Methods
1.4 Notes
2 ChIRP Assay in Ferroptosis
2.1 Introduction
2.2 Materials
2.2.1 Materials and Equipments
2.2.2 Reagents and Buffers
2.3 Methods
2.4 Notes
References
Chapter 4: PAR-CLIP Assay in Ferroptosis
1 Introduction
2 Materials
3 Methods and Protocols
3.1 Cells Preparation
3.2 UV-Crosslinking
3.2.1 UV-Crosslinking for Adherent Cells
3.2.2 UV-Crosslinking for Cells Grown in Suspension
3.3 Cell Lysis and RNase T1 Digestion
3.4 Immunoprecipitation (IP) and Recovery of Crosslinked Target RNA Fragments
3.4.1 Preparation of Magnetic Beads
3.4.2 IP, Second RNase T1 Digestion, and Dephosphorylation
3.4.3 Radiolabeling of RNA Segments Crosslinked to Immunoprecipitated Proteins
3.4.4 SDS-PAGE and Electroelution of Crosslinked RNA-Protein Complexes from Gel Slices or Electrophoresis, Transfer, and Recov...
Electroelution of Crosslinked RNA-Protein Complexes from Gel Slices
Electrophoresis, Transfer, and Recovery of RNA from Nitrocellulose Membrane
3.4.5 Proteinase K Digestion
Selection of Electroelution in the Previous Step
Selection of Nitrocellulose Membrane in the Previous Step
3.5 cDNA Library Preparation and Deep Sequencing
3.5.1 3′ Adapter Ligation
3.5.2 5′ Adapter Ligation
3.5.3 Reverse Transcription
3.5.4 PCR Amplification
3.6 Bioinformatic Analysis
4 Notes
References
Chapter 5: Organoids Models of Pancreatic Duct Adenocarcinoma
1 Introduction
2 Materials
2.1 Required Material or Equipment (see Note 1)
2.2 Buffers and Reagents to Prepare Before Starting Isolation (see Note 2)
3 Methods (see Note 4)
3.1 Conditioned Medium Preparation (see Note 5)
3.1.1 Generating Wnt3a Conditioned Medium
3.1.2 Generating R-Spondin Conditioned Medium
3.2 Generation of Human Organoids from PDAC Tumor Tissue and Normal Pancreatic Tissue
3.3 Generation of Mouse Organoids from Normal Pancreatic Tissues of Mice
3.4 Isolation of Human or Mouse Organoids from Matrigel by Generation of Pellets
3.5 Preserving Organoids at Low Temperatures
3.6 Passaging Organoids
3.7 Extraction of RNA and Protein from Organoids
3.8 Fixation and Embedding of Organoids
4 Notes
References
Chapter 6: Probing Lipid Peroxidation in Ferroptosis: Emphasizing the Utilization of C11-BODIPY-Based Protocols
1 Introduction
2 Materials
2.1 Reagents and Consumables
2.2 Equipments and Softwares
3 Methods
3.1 Detection of Lipid Peroxidation Using Flow Cytometry
3.2 Detection of Lipid Peroxidation Using Fluorescence Microscopy
4 Notes
References
Chapter 7: Membrane Integrity Assay in Ferroptosis
1 Introduction
2 Materials
3 Methods
3.1 TEM Analysis
3.2 Flow Cytometry Analysis
3.3 Assessing Oxidoreductase-Mediated Membrane Damages
3.3.1 Preparation of Phospholipid Films
3.3.2 Generation of Liposomes
3.3.3 Initiation of Reaction and Recording
4 Notes
References
Chapter 8: LC-MS-Based Redox Phosphoipidomics Analysis in Ferroptosis
1 Introduction
2 Materials
2.1 Lipid Extraction
2.2 Phosphate Content Determination
2.3 Liquid Chromatography-Mass Spectrometry Analysis
3 Methods
3.1 Lipid Extraction Using Folch Method
3.2 Phosphate Content Determination
3.3 Liquid Chromatography-Mass Spectrometry Analysis
3.3.1 Preparation of Standard and Sample Solutions
3.3.2 Perform LC-MS Analysis)
3.4 Data Analysis
3.4.1 Feature Extraction
3.4.2 Identification of Oxidized Phospholipid
3.4.3 Semi-Quantitative Analysis of Oxidized Phospholipid (See Note 7)
4 Notes
References
Chapter 9: Monitoring Lysosome Function in Ferroptosis
1 Introduction
2 Materials
3 Methods
3.1 Monitoring Lysosomal Integrity
3.1.1 Nuclear and Cytoplasmic Protein Extraction
3.1.2 Western Blotting
3.2 Monitoring Lysosomal Membrane Permeabilization
3.2.1 Fluorescent Dextran
3.2.2 Galectin Puncta Assay
3.2.3 Acridine Orange Staining
3.3 Monitoring Lysosomal Activity
3.4 Monitoring Lysosomal pH
3.5 Monitoring Lysosomal Reactive Oxygen Species
4 Notes
References
Chapter 10: Monitoring Mitochondria Function in Ferroptosis
1 Introduction
2 Materials
3 Methods
3.1 Monitoring Mitochondrial Proteins
3.1.1 Protein Extraction
3.1.2 Western Blotting
3.2 Monitoring Energy Production
3.2.1 ATP Assay
3.2.2 OCR Assay
3.2.3 ECAR Assay
3.3 Monitoring Mitochondrial Membrane Potential
3.3.1 TMRM Assay
3.3.2 JC-1 Assay
3.4 Monitoring Cell Viability
3.5 Monitoring Mitochondrial ROS
3.6 Monitoring Mitophagy
4 Notes
References
Chapter 11: Generation of Organoids and Analysis of Ferroptosis in Organoids
1 Introduction
2 Materials
2.1 Generation of Liver Organoids from iPSCs
2.2 Generation of Human Cancer Organoids
2.3 Monitoring Ferroptosis of Organoids
3 Methods
3.1 Generation of Liver Organoids from iPSCs
3.1.1 iPSCs Generation
3.1.2 Hepatocytes Generation
3.1.3 Liver Organoids Generation
3.2 Generation of Human Cancer Organoids
3.2.1 Generation of Hepatocellular Carcinoma-Derived Organoids
3.3 Monitoring Ferroptosis of Organoids
3.3.1 SYTOX Green Assay
3.3.2 Cell-Titer Glo Assay
3.3.3 MDA Assay
3.3.4 Iron Assay
3.3.5 GSH Assay
4 Notes
References
Chapter 12: Analysis of Protein Degradation in Ferroptosis
1 Introduction
2 Materials
3 Methods
3.1 Monitoring UPS Protein Degradation Using Western Blotting
3.2 Monitoring UPS Protein Degradation Using Co-immunoprecipitation
3.3 Monitoring UPS Protein Degradation Using In Vitro Ubiquitination Assay
3.4 Monitoring UPS Protein Degradation Using Proteasome Assay
3.4.1 Proteasome 26S Assay
3.4.2 Proteasome 20S Assay
4 Notes
References
Chapter 13: Lipidomics Analysis in Ferroptosis
1 Introduction
2 Materials
2.1 For LC-MS/MS
2.2 For GC-MS
3 Methods
3.1 LC-MS/MS
3.2 GC-MS
4 Notes
References
Chapter 14: In-Cell Western Assay in Ferroptosis
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 15: Flow Cytometric Analysis of Regulated Cell Death
1 Introduction
2 Materials
3 Methods
3.1 Flow Cytometry Assay in Apoptosis
3.1.1 Annexin V-PI Co-dyeing Assay
3.1.2 Fluoregenic Caspase Assay
3.1.3 FLICA Assay
3.1.4 TUNEL Assay
3.2 Flow Cytometry Assay in Autophagy
3.2.1 AO Assay
3.2.2 LC3-II Assay
3.2.3 LysoTracker Assay
3.3 Flow Cytometry Assay in Ferroptosis
3.3.1 DCFH-DA Assay
3.3.2 C11-BODIPY 581/591 Assay
3.3.3 Liperfluo Assay
3.3.4 PGSK Assay
3.3.5 FerroOrange Assay
3.4 Flow Cytometry Assay in Pyroptosis
3.5 Flow Cytometry Assay in Immunogenic Cell Death
4 Notes
References
Chapter 16: Thermal Shift Assay in Ferroptosis
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 17: Detection of Ferroptosis in Patient-Derived Tumor Models
1 Introduction
2 Materials
3 Methods
3.1 Transmission Electron Microscopy Assay
3.1.1 Fixation (see Note 1)
3.1.2 Dehydration
3.1.3 Embedding
3.1.4 Sectioning (see Note 2)
3.1.5 Staining (see Note 3)
3.2 Immunohistochemistry Assay
3.2.1 Tissue Fixation
3.2.2 Tissue Embedding in Paraffin Wax (see Note 4)
3.2.3 Paraffin Wax Sectioning (see Note 5)
3.2.4 Deparaffinization/Rehydration
3.2.5 Antigen Retrieval (see Note 6)
3.2.6 Blocking (see Note 7)
3.2.7 Immunohistochemical Staining with Universal SAP Kit (ZSGB-Bio, SAP-9100)
3.2.8 Nuclear Counterstaining
3.2.9 Mounting Sections and Sample Visualization
3.3 Iron Assay
3.3.1 Sample Preparation (see Note 8)
3.3.2 Standard Preparation
3.3.3 Assay Procedure
3.4 Western Blotting
3.4.1 Protein Extraction
3.4.2 Polyacrylamide Gel Electrophoresis (see Note 9)
3.4.3 Western Blotting (see Note 10)
3.4.4 Antibody Staining and Enhanced Chemiluminescence (see Note 11)
4 Notes
References
Chapter 18: Assessment of Ferroptosis in Hematopoietic Stem and Progenitor Cells
1 Introdution
2 Materials
3 Flow Cytometry to Detect HSC Ferroptosis In Vivo
3.1 Overview
3.2 The Measurement of the Number and Viability of HSPCs
3.3 The Measurement of the Cell Cycle of HSPCs
3.4 ROS Measurements
4 Detection of HSPC Ferroptosis In Vitro
4.1 Overview
4.2 Sorting and Culturing HSPCs In Vitro
4.3 Lipid ROS Measurement
4.4 Cell Viability Measurement
5 Notes
References
Chapter 19: Detection of Ferroptosis by Immunohistochemistry and Immunofluorescence
1 Introduction
2 Materials
2.1 Tissue and Antibody Preparation
2.2 Immunohistochemistry Buffer (see Note 3)
2.3 Immunofluorescence Buffer
2.4 Equipment
3 Methods
3.1 Monitoring Ferroptosis Using IHC
3.2 Monitoring Ferroptosis Using IF
4 Notes
References
Chapter 20: Analysis of MicroRNAs in Ferroptosis
1 Introduction
2 Materials
3 Methods
3.1 Discovering and Screening for miRNAs Involved in Ferroptosis with RT-PCR or MicroRNA Sequencing
3.1.1 Isolation of Total RNA
3.1.2 Reverse Transcription (RT) Reaction
3.1.3 Real-time PCR
3.2 Validating the Functional Significance of miRNAs Involved in Ferroptosis
3.3 Evaluating miRNA Targets by Luciferase Reporter Assay
3.3.1 Co-transfection of Plasmids
3.3.2 Dual-Luciferase Reporter Assay
4 Notes
References
Chapter 21: Detection of Ferroptosis in Models of Brain Diseases
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
2.3 Consumables
3 Methods
3.1 Gene and Protein Expression Assay
3.1.1 Real-Time PCR
3.1.2 Western Blotting
3.2 Lipid Peroxidation
3.2.1 C11-BODIPY
3.2.2 MDA Assay
3.2.3 4-HNE Assay
3.2.4 Immunofluorescence Staining for Detection of MDA and/or 4-HNE
3.3 GPX Activity Assay
3.4 Quantification of GSH Content and GSH/GSSG Ratio
3.5 Iron Concentration
3.5.1 Perl´s Staining
3.5.2 Iron Elements Determination with Inductive Coupled Plasma-Mass Spectrometry (ICP-MS)
3.5.3 Iron Colorimetric Assay (Fe2+, Fe3+, Total Iron)
3.6 Transmission Electron Microscopy (TEM)
4 Notes
References
Index