Phagocytosis and Phagosomes: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Bacterial Binding, Phagocytosis, and Killing Capacity: Measurements Using Colony Forming Units
1 Introduction
2 Materials
2.1 Bacteria and Microbiology Components
2.2 Macrophage Components
2.3 General Assay Components
3 Methods
3.1 Bacterial Binding Assay
3.2 Gentamicin Protection Assay for Bacterial Phagocytosis
3.3 Particle Binding Assay
3.4 Bacterial Killing Assay: Macrophage
4 Notes
References
Chapter 2: Analysis of Human and Mouse Neutrophil Phagocytosis by Flow Cytometry
1 Introduction
2 Materials
3 Methods
3.1 Phagocytosis by Human Blood Neutrophils
3.1.1 Preparation of pHrodo Particles
3.1.2 Acquisition of Blood Samples
3.1.3 Blood Neutrophil Stimulation
3.1.4 Fluorescent Labeling
3.1.5 Sample Acquisition
3.2 Mouse Peritoneal Recruitment and Phagocytosis
3.2.1 Preparation of pHrodo Particles and Intraperitoneal Injection
3.2.2 Peritoneal Lavage
3.2.3 Sample Fixation
3.2.4 Labeling
4 Notes
References
Chapter 3: Quantifying Phagocytosis by Immunofluorescence and Microscopy
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Opsonizing Particles with IgG
2.2.1 Polystyrene Beads
2.2.2 Fluorescent Polystyrene Beads
2.2.3 Escherichia coli
2.2.4 Fluorescent Escherichia coli
2.2.5 Sheep Red Blood Cells
2.3 Inside-Outside Staining of Phagocytosed Opsonized Particles
2.4 Inside-Outside Staining of Phagocytosed Non-opsonized Particles
2.5 Microscopy and Quantification Phagocytosis
3 Methods
3.1 Cell Culture of RAW 264.7 Macrophage
3.2 Opsonization of Particles with IgG
3.2.1 Opsonization of Polystyrene Beads with IgG
3.2.2 Opsonization of E. coli with IgG
3.2.3 Opsonization of Sheep RBCs with IgG
3.3 Phagocytosis and Inside-Out Staining of Non-opsonized E. coli
3.4 Phagocytosis of Opsonized Fluorescent Particles
3.5 Phagocytosis and Inside-Out Staining of Opsonized Phagocytosed Particles
3.6 Quantification of Phagocytosis
4 Notes
References
Chapter 4: Methods for Quantitative Efferocytosis Assays
1 Introduction
2 Materials
2.1 Primary Human Macrophage Preparation
2.2 J774.2 Cell Culture and Transfection
2.3 Synthetic Efferocytic Targets
2.4 Preparation of Apoptotic Jurkat T Cells
2.5 Preparation of Apoptotic Neutrophils
2.6 Efferocytosis Assays
2.7 Immunostaining
2.8 Microscopy
3 Methods
3.1 Cell Culture and Cell Transfection
3.1.1 Primary Human Macrophage Preparation
3.1.2 Culture and Transfection of the J774.2 Macrophage Cell Line
3.2 Generation of Synthetic and Natural Efferocytic Targets
3.2.1 Synthetic Lipid-Coated Apoptotic Cell Targets
3.2.2 Synthetic Protein-Coated Apoptotic Cell Targets
3.2.3 Preparation of Apoptotic Jurkat T Cells
3.2.4 Preparation of Apoptotic Neutrophils
3.3 Efferocytosis Assays with Synthetic and Natural Targets
3.3.1 Synthetic Target-Based Efferocytosis Assay
3.3.2 Efferocytosis Assay Using Apoptotic Cells
3.3.3 Immunostaining
3.3.4 Fixed Sample Imaging and Quantification of Efferocytosis
3.3.5 Live-Cell Imaging
4 Notes
References
Chapter 5: Analysis of Efferocytic Receptor Dynamics and Synapse Formation in a Frustrated Efferocytosis Model
Abbreviations
1 Introduction
2 Materials
2.1 Cell Culture and THP-1 Differentiation
2.2 Preparation of Supported Lipid Bilayers
2.3 Immunostaining
2.4 Frustrated Efferocytosis and Image Acquisition
2.5 Image Analysis for Single Particle Tracking
3 Methods
3.1 Cell Culture and THP-1 Differentiation
3.2 Functionalization of Coverslips with Supported Lipid Bilayers
3.3 Immunostaining
3.4 Frustrated Efferocytosis and Image Acquisition
3.5 Single Particle Tracking Analysis
3.6 Morphological Analysis
4 Notes
References
Chapter 6: Super-Resolution Spinning-Disk Confocal Microscopy Using Optical Photon Reassignment (SoRa) to Visualize the Actin ...
1 Introduction
2 Materials
2.1 RAW 264.7 Murine Macrophage Cell Preparation
2.2 Transient Transfection of RAW Cells
2.3 Electroporation of RAW Cells
2.4 Opsonization of Polystyrene Beads
2.5 Phagocytosis Live Imaging and Microscopy
2.6 Immunofluorescence and Phalloidin Staining
2.7 Image Analysis
3 Methods
3.1 RAW 264.7 Murine Macrophage Cell Preparation
3.2 Transient Transfection of RAW Cells (See Note 14)
3.3 Electroporation of RAW Cells (See Note 14)
3.4 Opsonization of Polystyrene Beads
3.5 Phagocytosis Live Imaging and Microscopy
3.6 Immunofluorescence of Podosomes in Resting Macrophages
3.7 Image Analysis
4 Notes
References
Chapter 7: Filamentous Bacteria as Targets to Study Phagocytosis
1 Introduction
2 Materials
3 Methods
3.1 Obtaining Filamentous Bacterial Targets
3.1.1 Generating Filamentous Bacterial Targets of Escherichia coli or Salmonella typhimurium Using Antibiotics
3.1.2 Generating Filamentous Bacterial Targets of a Thermosensitive ftsZ Escherichia coli Mutant Strain
3.1.3 Engineering E. coli PAT84 to Express Fluorescent Proteins
3.1.4 Preparing Fixed Filaments of E. coli PAT84
3.1.5 Generating Filamentous Bacterial Targets of L. pneumophila in BCYE Agar
3.1.6 Generating Filamentous L. pneumophila in BYE Media
3.1.7 Preparing Fixed Filaments of L. pneumophila
3.2 Phagocytosis Assay Using Filamentous Bacterial Targets
3.3 Measuring Phagocytic Internalization Using Filamentous Bacteria
3.4 Remodeling and Maturation of Phagocytic Cups and Phagosomes
3.4.1 Phagocytic Cup Remodeling
3.4.2 Phagocytic Cup Maturation
3.4.3 Assessing the Luminal Environment of Phagocytic Cups and Phagosomes During Maturation
4 Notes
References
Chapter 8: The Derivation and Use of HoxB8-Driven Conditionally Immortalized Macrophages
1 Introduction
2 Materials
2.1 ER-HoxB8 DNA Isolation
2.2 ER-HoxB8 Lentivirus Production
2.3 Bone Marrow Isolation
2.4 ER-HoxB8 Lentiviral Transduction
2.5 Macrophage Differentiation
3 Methods
3.1 ER-HoxB8 and Lentiviral Packaging Constructs DNA Isolation
3.2 Generating ER-HoxB8 Lentivirus
3.2.1 Plating HEK293T Cells
3.2.2 Transfection
3.2.3 Virus Harvesting
3.2.4 Ultracentrifugation
3.3 Isolating Bone Marrow
3.4 Transduction with ER-HoxB8 Lentivirus
3.5 Macrophage Differentiation
4 Notes
References
Chapter 9: Quantitative Immunofluorescence to Study Phagosome Maturation and Resolution
1 Introduction
2 Materials
3 Methods
3.1 Cell Culture
3.2 Particle Preparation
3.2.1 mCherry-Escherichia coli (E. coli) Production and Fluorescent Protein Expression
3.2.2 Preparing IgG-Coated Particles
3.3 Phagocytosis, Phagosome Maturation, and Phagosome Resolution
3.3.1 Phagocytosis of IgG-Coated Beads
3.3.2 Phagocytosis of mCherry-E. coli and Fixation
3.4 Antibody Staining of Whole Phagocytes
3.5 Microscopy and Analysis of Whole Phagocytes
3.5.1 Maturation of Intact Phagosomes
3.5.2 Imaging and Quantification of Fragmented Phagosomes
3.6 Immunofluorescence and Flow Cytometry Analysis of Isolated Phagosomes
3.6.1 Cell Culture for Phagosome Isolation
3.6.2 Phagosome Isolation
3.6.3 LAMP1 and Clathrin Staining of Isolated Phagosomes
3.6.4 Microscopy and Analysis of Isolated Phagosomes
3.6.5 Flow Cytometry and Analysis of Isolated Phagosomes
4 Notes
References
Chapter 10: Approaches to Measuring Reductive and Oxidative Events in Phagosomes
1 Introduction
2 Materials
2.1 Preparation of Redox Reporter Beads
2.2 Cell Preparation and Handling
2.3 Performing Redox Measurements on Live Cells
3 Methods
3.1 Preparation of Redox Reporter Beads
3.1.1 Preparation of Dextran-Linked Reductase-Reporter Beads
3.1.2 Preparation of ROS-Reactive OxyBURST Beads
3.2 Cell Preparation and Handling
3.2.1 BMMØs and BMDCs
3.2.2 GB8-MØs
3.2.3 J774s
3.3 Performing Redox Measurements in Live Cells
3.3.1 Assessment of Phagosome-Specific Disulfide Reduction
3.3.2 Assessment of Phagosome-Specific ROS Production
3.3.3 Measurement of Extracellular H2O2
3.3.4 Measurement of Reactive Oxygen Species Production
4 Notes
References
Chapter 11: Measuring Phagosomal pH by Fluorescence Microscopy
1 Introduction
1.1 Instrumentation
1.2 Probe Selection
1.3 Calibration
2 Materials
2.1 Cell Lines
2.2 Reagents
2.3 Microscopy System Requirements
3 Methods
3.1 Labeling of Phagocytic Targets with pH Sensors
3.2 IgG Opsonization of Zymosan
3.3 Phagocytosis Assay
3.4 In Situ Calibration
4 Notes
References
Chapter 12: Multiplexed Phagosomal Assays for the Detection and Quantification of Bidirectional Exchange Between the Phagolyso...
1 Introduction
2 Materials
2.1 Cells, Reagents, and Buffers
2.2 Imaging Instrument and Analysis Software
3 Methods
3.1 Preparation of Experimental Reporter Particles
3.2 Preparation of Cellulase Reporter Beads
3.3 Preparation of DQ Green BSA and pHrodo Reporter Beads
3.4 Cell Preparation and Handling
3.5 Imaging System Setup
3.6 Acquisition of Images
3.7 Analysis
3.8 Using Spotfire DecisionSite to Visualize Data in Graphical Format
4 Notes
References
Chapter 13: Quantitative Spatio-temporal Analysis of Phagosome Maturation in Live Cells
1 Introduction
2 Materials
2.1 Cells, Buffers, and Solutions
2.1.1 Cells
2.1.2 Culture Media
2.1.3 Reagents for the Preparation of Phagocytic Targets
2.1.4 Labelling of Intracellular Organelles
2.1.5 Preparation of BMM
2.1.6 Preparation of HMDM
2.1.7 Preparation of Human and EB
2.2 Equipment
2.2.1 Preparation of BMM
2.2.2 Preparation of HMDM
2.2.3 Preparation of iPSDM
2.2.4 Live Cell Dishes
2.2.5 Microscope and Environmental Chamber
3 Methods
3.1 Preparation of Macrophages
3.1.1 Preparation of BMM
3.1.2 Preparation of HMDM (As Alternative to BMM)
3.1.3 Preparation of iPSDM (As Alternative to BMM or HMDM)
3.1.4 Preparing iPSDM for Experiments
3.2 Preparation of Phagocytic Targets
3.2.1 Polystyrene and Silica Beads
3.2.2 Preparation of Mycobacteria for Infection
3.3 Labelling of Intracellular Organelles
3.3.1 LysoTracker Delivery into Phagosomes
3.3.2 Cell Transfection
3.4 Live Cell Imaging
3.5 Image Analysis
3.5.1 Analysis of the Association of Various Markers to IgG-Coated Beads
3.5.2 Analysis of the Association of Various Markers to Mycobacteria
4 Notes
References
Chapter 14: Measurement of Salmonella enterica Internalization and Vacuole Lysis in Epithelial Cells
1 Introduction
2 Materials
3 Methods
3.1 Gentamicin Protection Assay
3.2 Chloroquine Resistance Assay
4 Notes
References
Chapter 15: Microscopy-Based Tracking and Quantification Methods to Study Phagosome Resolution
1 Introduction
2 Materials
3 Methods
3.1 Opsonization of Fixed Bacteria
3.2 Phagocytosis Assay
3.3 Live Cell Imaging
3.4 Fixation and Immunostaining
3.5 Determining the Total Volume of Phagosomes and Phagosome-Derived Vesicles (PDVs) per Cell
3.6 Determining the Co-occurrence of Various Membrane Markers with PDVs
4 Notes
References
Chapter 16: Isolation of Polystyrene Bead-Induced Phagosomes for Western Blotting
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Induction of Phagocytosis and Cell Lysis
2.3 Phagosome Purification
2.4 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)/Components
2.5 Immunoblotting
2.6 Immuno-detection
3 Methods
3.1 Induction of Phagocytosis
3.2 Cell Lysis
3.3 Phagosome Purification
3.4 Sample Preparation and SDS-PAGE Electrophoresis
3.5 Protein Transfer
3.6 Immunoblotting
3.7 Signal Detection
4 Notes
References
Chapter 17: Biochemically Reconstituted Fusion of Phagosomes with Endosomes and Lysosomes
1 Introduction
2 Materials
3 Methods
3.1 Preparation of BSA-Rhodamine
3.2 Coating of 1 μm Latex Beads with BSA
3.3 Preparation of Cytosol from J774E Macrophages
3.4 Preparation of Mowiol
3.5 Fluorescent and Paramagnetic Labeling of Lysosomes
3.6 Fluorescent and Paramagnetic Labeling of Late Endosomes
3.7 Fluorescent and Paramagnetic Labeling of Early Endosomes
3.8 Purification of Fluorescently and Paramagnetically Labeled Early Endosomes, Late Endosomes, or Lysosomes
3.9 Purification of Latex Bead Phagosomes
3.10 Cell-Free Fusion Between Phagosomes and Endosomes/Lysosomes
4 Notes
References
Chapter 18: An In Vitro System to Analyze Generation and Degradation of Phagosomal Phosphatidylinositol Phosphates
1 Introduction
2 Materials
2.1 Purification of Glutathione-S-Transferase (GST) or GST-fused PIP-Binding Proteins from E. coli BL21(DE3)
2.2 Detection of PIPs on Purified LBPs by Immunofluorescence Microscopy
3 Methods
3.1 Purification of Glutathione-S-Transferase (GST) or GST-fused PIP-Binding Proteins from E. coli BL21(DE3), Genotype E. coli...
3.2 Coating Latex Beads with BSA
3.3 Preparation of Cytosol from J774E Macrophages
3.4 Preparation of Mowiol
3.5 Purification of Latex Bead Phagolysosomes from J774E Macrophages
3.6 Incubation of Phagosomes with Lipid-Binding Probes
3.7 Fluorescence Microscopy Analysis and Quantification of the Amounts of Lipid-Binding Probes on Phagosomes
4 Notes
References
Chapter 19: Dissecting Phagosomal Pattern Recognition Receptor-Dependent Signaling and Antigen MHC-II Presentation from Phagos...
1 Introduction
2 Materials
2.1 Equipment
2.2 Mice
2.3 Reagents and Supplies
2.3.1 Cell Culture
2.3.2 Assays
Bead Preparation and Phagocytosis
PRR Signaling
Eα Ag Presentation
3 Methods
3.1 Cell Isolation or Differentiation
3.2 Signaling
3.2.1 Preparation of LPS-Coated Beads
3.2.2 DC Harvesting
3.2.3 DC Stimulation
3.3 Eα Antigen Presentation Assay
3.3.1 Eα-YFP-Coated Bead Preparation
3.3.2 Eα-YFP-Coated Bead Phagocytosis, Antigen Loading, and Presentation
3.3.3 Immunofluorescence
3.3.4 Flow Cytometry
4 Notes
References
Chapter 20: Examining the Kinetics of Phagocytosis-Coupled Inflammasome Activation in Murine Bone Marrow-Derived Dendritic Cel...
1 Introduction
2 Materials
2.1 Mice
2.2 DNA Constructs
2.3 Cell Culture
2.4 NLRC4/NLRP3 Inflammasome Stimuli
2.5 NLRP3 Inflammasome Stimuli
2.6 Other Reagents and Materials
2.7 Instruments and Software
3 Methods
3.1 Preparation of Reagents
3.1.1 Preparation of Toxin-Coated Beads
3.2 BM Isolation and BMDC Differentiation
3.3 Seeding and Splitting BM Cells
3.4 Transfecting Plat-E Cells and Seeding BM Cells for Retroviral Transduction
3.5 Salmonella Typhimurium Culture
3.6 Harvesting and Plating BMDCs
3.7 Cell Priming and Stimulation in 96-Well Plate
3.8 Caspase-11 Detection by TCA Precipitation
3.9 Cell Priming and Stimulation in Glass-Bottom p35 Dishes for ASC Speck Visualization by Fluorescence Microscopy or Live Cel...
3.10 ASC Speck Counting
3.11 Count Nuclei
4 Notes
References
Chapter 21: Analysis of LC3-Associated Phagocytosis and Antigen Presentation in Macrophages and B Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.1.1 Isolation of Human Peripheral Blood Mononuclear Cells
2.1.2 Isolation and EBV Transformation of Human B Cells
EBV Production and Titration
2.1.3 Isolation of Human Macrophages
2.1.4 Isolation of Human Whole Blood CD4+ T Cells
2.2 CRISPR/Cas9 Knock-out of ATG4B in LCLs
2.3 Lentiviral Constructs of ATG4Bwt or ATG4BC78S Overexpression
2.4 Western Blot
2.4.1 Reagents
2.4.2 Buffers
2.5 Immunofluorescence and Confocal Microscopy
2.6 Role of LC3-Associated Phagocytosis During MHC Class II Antigen Presentation
2.7 Reagents
2.7.1 Cell Culture
2.7.2 Antibodies
2.7.3 Other Probes
3 Methods
3.1 Cell Culture
3.1.1 EBV Production and Titration
3.1.2 Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)
3.1.3 Isolation and EBV Transformation of Human B Cells
3.1.4 Isolation of Monocyte-Derived Macrophages
3.1.5 Isolation of Autologous CD4+ T Cells for Antigen Presentation
3.2 CRISPR/Cas9 Knock-out of ATG4B in LCLs
3.3 Lentivirus Transduction of Human Macrophages for ATG4 Overexpression
3.4 Trigger LC3-Associated Phagocytosis in Human Macrophages Using Candida albicans Extract
3.4.1 Candida albicans Extract Coated Beads
3.4.2 LAP Triggered with Candida albicans Extract Coated Beads
3.5 Western Blot to Validate ATG4 Knockout or Overexpression in Human Antigen Presenting Cells
3.6 An Immunofluorescence Protocol to Investigate the Role of ATG4 During LAP in Human Macrophages and Its Role During Autopha...
3.6.1 Immunofluorescence Protocol for Adherent Cells
3.6.2 Immunofluorescence Protocol for Cells in Suspensions
3.6.3 Image Acquisition and Analysis
3.7 Assess the Involvement of ATG4 in LAPosome Stabilization to Allow Macrophages to Sustain MHC Class II Antigen Presentation
4 Notes
References
Chapter 22: Visualizing Phagocytic Cargo In Vivo from Engulfment to Resolution in Caenorhabditis elegans
1 Introduction
2 Materials
2.1 Worm Strains and Maintenance
2.2 RNA Interference (RNAi) Reagents
2.3 Microscopy
3 Methods
3.1 Preparing Buffers and Media
3.1.1 Luria Broth (LB)
3.1.2 Nematode Growth Media (NGM) Plates
3.1.3 NGM Lite Plates
3.1.4 M9 Buffer
3.1.5 Egg Salts
3.1.6 Agarose Pads
3.2 Mounting Embryos for Time-Lapse Imaging
3.2.1 Mounting Embryos for Time-Lapse Imaging (Agarose Pad)
3.2.2 Mounting Embryos for Time-Lapse Imaging (Well)
3.3 Time-Lapse Imaging
3.4 Normalization of Time-Lapse Series
3.5 Cargo Signaling for Engulfment
3.6 Engulfment
3.7 Phagosome Maturation
3.8 Phagosome-Lysosome Fusion
3.9 Phagosome and Phagolysosome Acidification
3.10 Cargo Membrane Breakdown
3.10.1 Direct Membrane Breakdown Assay
3.10.2 Indirect Chromosome Assay for Membrane Breakdown
3.11 Phagolysosome Shrinkage
3.12 Phagolysosome Tubulation, Vesiculation, and Resolution
4 Notes
References
Chapter 23: Assessing the Phagosome Proteome by Quantitative Mass Spectrometry
1 Introduction
2 Materials
2.1 Equipment
2.2 Cell Lysis, Reduction, Alkylation, and Digestion
2.3 Sample Clean-up, TMT Labelling, and MS
2.4 Data Analysis
3 Methods
3.1 Cell Lysis, Reduction, Alkylation, and Digestion for Quantitative Proteomic Analysis
3.2 Quantitative Proteomics of Phagosomes Using TMTpro 16plex
3.3 Offline HPLC Fractionation for TMTpro 16plex Labelled Samples
3.4 Mass Spectrometry Analysis for TMT Samples
3.5 Mass Spectrometry Analysis for Label-Free Samples
3.6 Data Processing and Analysis
3.7 Quality Control
4 Notes
References
Chapter 24: Using Ion Substitution and Fluid Indicators to Monitor Macropinosome Dynamics in Live Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Primary Macrophage Derivation
2.3 Buffers
2.4 Chemicals
2.5 Confocal Microscope
3 Methods
3.1 Deriving BMDM
3.1.1 Preparation of M-CSF
3.1.2 Isolation of BMDMs from Murine Long Bones
3.2 Seeding BMDM onto Coverslips
3.3 Inducing Macropinocytosis
3.4 Imaging and Recording
3.5 Data Analysis
4 Notes
References
Index