دانلود کتاب Chemotaxis: Methods and Protocols (Methods in Molecular Biology, 571)

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FM1_O.pdf
1_O.pdf
Chapter 1
Microfluidic Techniques for the Analysis of Bacterial Chemotaxis
1. Introduction
1.1. Brief Review of Bacterial Motility and Chemotaxis
1.2. Established Methods for Assaying Bacterial Chemotaxis
1.2.1. Swim and Swarm Plates
1.2.2. Capillary Assays
1.2.3. Monitoring Movement of Bacteria in Stable Gradients
1.2.4. Tracking Individual Swimming Bacteria
1.2.5. Tethered Cell Assay
1.2.6. Microfluidic Assays
2. Materials
2.1. Microfluidic Device
2.2. Microscopy and Imaging
2.3. Bacteria, Plasmids, Culture Media, and Buffers
3. Methods
3.1. Microfluidic Device Design
3.1.1. Generation of Device Molds
3.1.2. Device Fabrication
3.2. Growth of Highly Motile E. coli
3.3. mPlug Assay
3.3.1. Results with the mPlug Device
3.3.2. Data Analysis: mPlug
3.4. Microfluidic Chemotaxis (mFlow)
References
2_O.pdf
2
Prokaryotic Phototaxis
1.2.3. Twitching Motility in Synechocystis
Anchor 14
2.1. Growth of Halobacterium salinarum
3.2. Phototaxis of Rhodobacter sphaeroides
3.2.2. Surface Tethering of Rhodobacter sphaeroides
Anchor 49
References
3_O.pdf
Chapter 3
Photoorientation in Photosynthetic Flagellates
1. Introduction
2. Photoorientation
2.1. Photokinesis
2.2. Photophobic Responses or Photoshock Response
2.3. Phototaxis
3. Phototaxis in Chlamydomonas
4. Phototaxis in Euglena
References
4_O.pdf
Chapter 4
Dictyostelium Slug Phototaxis
References
5_O.pdf
Chapter 5
Electrotaxis and Wound Healing: Experimental Methods to Study Electric Fields as a Directional Signal for Cell Migration
2.4. Timelapse Video Microscopy
3.2.3. Application of a Direct Current Electric Field
3.4. Quantitative Analysis of Cell Migration
3.4.1. Semiautomatic Analysis
References
6_O.pdf
Chapter 6
Chemotropism During Yeast Mating
References
7_O.pdf
Chapter 7
Group Migration and Signal Relay in Dictyostelium
1. Introduction
2. Materials
2.1. Development of Dictyostelium Cells
2.2. Group Migration Assays
2.3. Adenylyl Cyclase Activation Assay
3. Methods
3.1. Development of Dictyostelium cells
3.1.1. Cell Development on Non-Nutrient Agar Dishes
3.1.2. Cell Development in Non-Nutrient Buffer with cAMP Pulses
3.1.3. Assessing Development by Western Analysis
3.2. Group Migration Assays
3.2.1. Self-Streaming Assay
3.2.2. Streaming to a Micropipette
3.2.3. Streaming Under Agar
3.3. Adenylyl Cyclase Activity Assay
3.3.1. Preparation of Dowex and Alumina Chromatography Columns
3.3.2. Cell Preparation
3.3.3. cAMP-Mediated Adenylyl Cyclase Activation
3.3.4. Mn2+ and GTPgS-Mediated Adenylyl Cyclase Activation
3.3.5. Chromatography
3.3.6. Calculations
4. Notes
References
8_O.pdf
Chapter 8
Quantitative Analysis of Distal Tip Cell Migration in C. elegans
1. Introduction
2. Materials
2.1. Obtaining a Construct for Feeding RNAi
2.2. Feeding RNAi
2.3. Preparing C. elegans Embryos
2.4. DIC Microscopy
3. Methods
3.1. Obtaining a Construct for Feeding RNAi
3.2. Feeding RNAi
3.3. Preparing C. elegans Embryos
3.4. DIC Microscopy
4. Notes
References
9_O.pdf
Chapter 9
Inflammation and Wound Healing in Drosophila
1. Introduction
2. Materials
2.1. Embryo Collection
2.2. Embryo Mounting and Culture
2.2.1. Method A (see Fig. 2a)
2.2.2. Method B (see Fig. 2b)
2.3. Epithelial Wound Assay
2.4. Hemocyte Migration Assays
2.4.1. Hemocyte Developmental Dispersal
2.4.2. Hemocyte Wound Chemotaxis
2.4.3. Hemocyte Bead Chemotaxis
2.5. Postmicroscopy Editing
3. Methods
3.1. Embryo Collection
3.2. Embryo Mounting and Culture
3.2.1. Method A
3.2.2. Method B
3.3. Epithelial Wound Assay
3.4. Hemocyte Migration Assays
3.4.1. Hemocyte Developmental Dispersal
3.4.2. Hemocyte Wound Chemotaxis
3.4.3. Hemocyte Bead Chemotaxis
3.5. Postmicroscopy Editing
4. Notes
References
10_O.pdf
Chapter 10
Neutrophil Motility In Vivo Using Zebrafish
1. Introduction
2. Materials
2.1. Zebrafish Maintenance and Mating
2.2. Endogenous MPO Activity Assay
2.3. Live Microscopy of Zebrafish Embryos
3. Methods
3.1. Zebrafish Maintenance and Mating
3.2. Endogenous MPO Activity Assay Following Wounding
3.3. Live Microscopy of Zebrafish Embryos After Wounding
4. Notes
References
11_O.pdf
Chapter 11
Chemotaxis in Neutrophil-Like HL-60 Cells
1. Introduction
2. Materials
2.1. HL-60 Cell Culture and Differentiation
2.2. Amaxa Nucleofection of HL-60 cells
2.3. Plating Cells for Microscopy
2.4. Micropipette Assay
2.5. EZ-TAXIScan Assay
2.6. Staining the Actin Cytoskeleton
3. Methods
3.1. Maintenance of HL-60 Cell Culture Line
3.2. Transient Transfection of DNA into HL-60 Cells
3.3. Preparing for live Cell Microscopy
3.4. Micropipette-Stimulated Cell Migration
References
12_O.pdf
Chapter 12
Chemokine Receptor Dimerization and Chemotaxis
References
13_O.pdf
Chapter 13
Intravital Two-Photon Imaging of Adoptively Transferred B Lymphocytes in Inguinal Lymph Nodes
3.2. Labeling B Lymphocytes andIn Vivo Transfer of Them
3.3. Anesthesia
3.4. Operation for Exposing the Inguinal Lymph Node (iLN)
3.5. Imaging using Multiphoton Microscopy (See Fig.1)
3.6. Analysis
4. Notes
References
14_O.pdf
Chapter 14
Breast Cancer Cell Movement: Imaging Invadopodia by TIRF and IRM Microscopy
References
15_O.pdf
Chapter 15
In Vivo Assay for Tumor Cell Invasion
1. Introduction
2. Materials
2.1. Instruments for Needle Cell Collection Setup
2.2. Animal Models Used for Cell Collection
2.3. Reagents for Cell Collection
2.4. Reagents for Cell Counting
2.5. Reagents for Cell Typing by Immuno-fluorescence
3. Methods
3.1. Preparing Solutions for Needle Collection
3.2. Preparing the Needle Collection Setup
3.3. Performing the Assay
3.4. Identification of the Invasive Population
4. Notes
References
16_O.pdf
Chapter 16
Quantitative Studies of Neuronal Chemotaxis in 3D
1. Introduction
2. Materials
2.1. Tissue Preparation
2.2. Collagen Preparation
2.3. Tissue Embedding in Collagen Gel
2.4. Gradient Printing
3. Methods
3.1. Tissue Preparation
3.2. Collagen Gel Preparation
3.3. Tissue Embedding in the Collagen Gel
3.4. Gradient Printing
4. Notes
References
17_O.pdf
Chapter 17
Assays for Chemotaxis and Chemoattractant-Stimulated TorC2 Activation and PKB Substrate Phosphorylation in Dictyostelium
2. Materials
2.1. Cell Culture Media, Buffer, and Solutions
2.2. Micropipette Assay
2.3. Two-Drop Assay
2.4. Western Blotting
2.5. Primary Antibodies for Western Blotting
2.6. Indirect Immunofluorescence
2.7. Immunopurification of PKB Substrates
2.8. PIP3 Detection Biochemically
2.9. Genes and Dictyostelium Data Base (DDB) Numbers
3. Methods
3.1. Preparation of Chemotactically Competent Cells
3.1.1. Starvation of Dictyo-stelium discoideum Cells
3.1.2. “Basalation” of Cells
3.2. Under Buffer Assay
3.3. Chemotaxis Assays
3.4. Micropipette Assay
3.4.1. Two-Drop Assay
3.5. Detection of PIP3 Production
3.5.1. PIP3 Detection by a Biosensor, PH-GFP (Fluorescent Microscope)
3.5.2. PIP3 Detection by a Biosensor, PH-GFP (Biochemically)
3.6. Detection of PKB and TorC2 Activity
3.6.1. cAMP Stimulation and Sample Preparation
3.6.2. Western Blotting
3.6.3. Indirect Immuno-fluorescence
3.7. Purification of Substrates of PKB
4. Notes
References
18_O.pdf
Chapter 18
Biochemical Responses to Chemoattractants in Dictyostelium: Ligand–Receptor Interactions and Downstream Kinase Activation
1. Introduction
2. Materials
2.1. Solutions
2.2. Supplies
3. Methods
3.1. Development of Aggregation-Competent Dictyostelium in Shaking Culture (See Note 1)
3.2. Binding of Surface Cell Receptors for cAMP in Phosphate Buffer (See Note 2)
3.3. Binding Affinity of Surface Cell Receptors for cAMP in Phosphate Buffer (See Note 2)
3.4. Relative Binding Affinities for cAMP Analogs in Phosphate Buffer (See Note 2)
3.5. Binding of Surface Cell Receptors for cAMP in the Presence of Saturated (NH4 )2 SO4 (See Note 2)
3.6. Binding Affinity of Surface Cell Receptors for cAMP in the Presence of Saturated (NH4 )2SO4 (See Note 2)
3.7. Relative Binding Affinities for cAMP Analogs in the Presence of Saturated (NH4 )2SO4 (See Note 2)
3.8. GTP g S Inhibition of cAMP Receptor Binding
3.9. cAMP Stimulation of Developed Cells
3.10. cAMP-Stimulated Synthesis of PI(3,4,5)P3 [Phosphatidylinositol (3,4,5)-Trisphosphate]
3.11. cAMP Stimulation of GSK3 Activity
3.12. cAMP Stimulation of ERK2 Phosphorylation
3.13. Folic Acid Stimulation of ERK2 Phosphorylation
4. Notes
References
19_O.pdf
Chapter 19
Quantifying In Vivo Phosphoinositide Turnover in Chemotactically Competent Dictyostelium Cells
2.1. Equipment
2.2. Cell Culturing and Preparation
2.3. Labelling and Isolation of Phospholipids
2.4. Separation of Phospholipids via Thin-Layer Chromatography (TLC)
2.5. Staining Total Lipids
3.1. Cell Preparation and Labelling of Phospholipids
3.2. Separation of Phospholipids via TLC
3.3. Staining Total Lipids
References
20_O.pdf
Chapter 20
In Vivo Measurements of Cytosolic Calcium in Dictyostelium discoideum
2.1. Aequorin Method
2.1.1. Cell Culture and Loading
2.1.2. Calcium Measurements
2.2. Fura-2-Dextran Loading Method
2.3. 45Ca 2+Method
2.3.1. Cell Culture and Loading
3.1. Aequorin Method
3.2. Fura-2-Dextran Loading Method
3.2.1. Scrape Loading of Cells with Fura-2 Dextran
3.2.2. Loading with Fura-2-Dextran by Electroporation
3.2.3. Stimulation with Chemoattractant
3.2.4. Microscopy, Image Acquisition and Analysis
3.2.5. Fluorescence Ratio: [Ca 2+] Calibration
3.3 45Ca2+Method
3.3.1. Cell Culture
3.3.2. 45Ca 2+Uptake due to Chemoattractant Stimulation
3.3.3. Counting
3.3.4. Non-specific Calcium Uptake
References
21_O.pdf
Chapter 21
Chemokine Receptor Signaling and HIV Infection
References
22_O.pdf
Chapter 22
Spatiotemporal Stimulation of Single Cells Using Flow Photolysis
References
23_O.pdf
Chapter 23
Spatiotemporal Regulation of Ras-GTPases During Chemotaxis
2.1. Buffers and Materials for GST-RBD Preparation
2.2. Cell Culture and GST-Pull Down Assay
2.3. Time-Lapse and Simultaneous Imaging
3.1. Biochemical Analysis of Ras Activation
3.1.1. Preparation of GST-RBD from E. coli
3.1.2. GST-Pull Down Assay for Chemoattractant-Induced Ras Activation
3.2. Analyzing Ras and Rap1 Activation by Time-Lapse Imaging
3.2.1. Ras and Rap1 Activation in Response to Global Stimulation
3.2.2. Ras and Rap1 Activation During Chemotaxis
3.2.3. Ras and Rap1 Activation During Random Movement
3.3. Simultaneous Analysis of Ras Activation and Other Signaling Events
References
24_O.pdf
Chapter 24
FRAP Analysis of Chemosensory Components of Dictyostelium
1.1. Overview of FRAP
1.2. Applications in Dictyostelium
1.2.1. Diffusion of Molecules and the Role of the Cytoskeleton
1.2.2. Membrane FRAP
1.2.3. Selective FRAP
3.1. Preparation of Cells
3.2. Instruments Used
3.3. Determining Cell Imaging Parameters for Confocal FRAP
3.4. Selecting the Region of Interest
3.5. Determining Proper FRAP Experimental Conditions
3.6. Performing the Actual Experiment
3.7. Analyzing the Data
3.8. Detailed Methods for Membrane FRAP of PTEN-YFP
3.9. The Future of FRAP
References
25_O.pdf
Chapter 25
Monitoring Dynamic GPCR Signaling Events Using Fluorescence Microscopy, FRET Imaging, and Single-Molecule Imaging
2. Materials
2.1. D. discoideum Cell Culture
2.2. D. discoideum Development
2.3. HEK 293 Cell Culture
2.4. Dyes for Chemoattractant Imaging and Chemoattractant Delivery
2.5. Confocal Fluorescent Microscope
2.6. TIR Fluorescent Microscope
3.1. Dictyostelium Growth and Development
3.2. Measurements of Applied Chemoattractant Stimulations
3.3. FRET Time-Lapse Imaging in Live Cells
3.4. Imaging the Dynamics of GPCR and G-Protein Subunits at the Single-Molecule Level by TIRF Microscopy
3.4.1. Preparation of the Cover Glass
3.4.2. Preparation of Labtek Eight-Well Chambers
3.4.3. Preparation of Cells
3.4.4. Adjustment of the Laser Beam Angle to Achieve TIR
References
26_O.pdf
Chapter 26
Imaging Actin Cytoskeleton Dynamics in Dictyostelium Chemotaxis
1. Introduction
1.1. Overview of Rapid Actin Responses in Chemotaxis
1.2. Cell Strains Expressing Fluorescent Proteins
1.2.1. Fluorescent Actin Labels
GFP-Actin
Fluorescent LimE∆ Constructs
1.2.2. Proteins Associated with the Leading Edge in Chemotaxing Cells
Myosin-IB at the Cutting Edge of Lamellipodia
Subunits of the Arp2/3 Complex
Coronin, an Indicator of Actin Depolymerization
1.2.3. Labeling Filopodia
1.3. Practical Considerations for Imaging Actin Dynamics
1.3.1. Expression Levels
1.3.2. Background Fluorescence
1.3.3. Introducing a Reference for the Cell Border
Labeling the Plasma Membrane
Marking the Cell-to-Substrate Interspace
1.4. Imaging Techniques
1.4.1. Fast Recording of Actin Dynamics by TIRF or Spinning-Disc Confocal Microscopy
TIRF Microscopy
Spinning-Disc Microscopy
1.4.2. Choice of Simulta-neous or Consecutive Image Acquisition in Dual-Wavelength Imaging
Consecutive Image Acquisition
Simultaneous Image Acquisition
1.5. Recording Actin Dynamics in Response to Chemoattractant
2. Materials
2.1. Cell Culture
2.2. Fluorescence Microscopy
3. Methods
3.1. Cell Growth and Development to Aggregation Competence
3.1.1. Simplified Procedure
3.1.2. Standardized Procedure
3.2. Actin Dynamics in Changing Gradients of Attractant
3.2.1. Stimulation Through a Micropipette (see Note 9)
3.2.2. Image Analysis of the Gain and Loss of Actin Structures
3.3. Response to Temporal Programs of Attractant Concentration
3.3.1. Stimulation of Cells in a Flow Chamber
3.3.2. Evaluating the Recruitment of Actin and Associated Proteins to the Cell Cortex
4. Notes
References
27_O.pdf
Chapter 27
Analysis of Actin Assembly by In Vitro TIRF Microscopy
1. Introduction
1.1. The Principle of TIRF Microscopy
1.3. TIRF Microscopy Reveals Novel Insights into the Function of VASP
2.1. Preparation and Modification of Actin and Myosin from Rabbit Skeletal Muscle
2.1.1. Preparation of Actin from Rabbit Skeletal Muscle
2.1.2. Preparation of G-actin from Acetone Powder
2.1.3. Labeling of Actin with Alexa-Fluor-Maleimide Dyes
2.1.4. Preparation of NEM-Heavy-Mero-Myosin II (NEM-HMM)
2.2. Purification of GST Fusion Proteins
2.3. TIRF Microscopy
2.3.1. Microscopic Setup
2.3.2. Preparation of Flow Cells
2.3.3. Actin Polymerization Assays
3.1. Preparation and Labeling of Actin and Myosin from Rabbit Skeletal Muscle
3.1.1. Preparation of Acetone Powder from Rabbit Skeletal Muscle
3.1.2. Preparation of G-actin from Acetone Powder
3.1.3. Labeling of Actin with Alexa-Fluor Maleimide Dyes
3.1.4. Preparation of NEM-Heavy-Mero-Myosin II (NEM-HMM)
3.2. Purification of GST-DdVASP
3.3. TIRF Microscopy
3.3.1. Preparation of Flow Cells
3.3.2. Actin Polymerization Assays
References
28_O.pdf
Chapter 28
Single-Molecule Imaging Techniques to Visualize Chemotactic Signaling Events on the Membrane of Living Dictyostelium Cells
3.1. Experimental Setup of TIRFM
3.2. Cell Preparation
3.2.1. Culture and Development
3.2.2. Transformation of Cells
3.2.3. Labeling with HaloTag Ligand
3.3. Visualizing Single Molecules Under TIRFM
3.4. Single-Molecule Tracking
3.5. Lifetime Analysis
3.6. Short-Range Diffusion Coefficients Analysis
3.7. Diffusion Analysis Based on Distribution Function of Displacements
References
29_O.pdf
Chapter 29
Imaging B-Cell Receptor Signaling by Single-Molecule Techniques
2.1. Preparation of Planar Lipid Bilayers
2.1.1. Preparation of Small Unilamellar Vesicles
2.1.2. Preparation of Planar Lipid Bilayers
2.2. Preparation of Histidine-Tagged Ligands
2.3. Labeling Receptors by Fluorescent Fab Antibody Fragments
2.4. Single-Molecule Image Acquisition
3.1. Preparation of Planar Lipid Bilayers
3.1.1. Preparation of Small Unilamellar Vesicles
3.1.2. Preparation of Planar Lipid Bilayers
3.2. Preparation of Histidine-Tagged Ligands and Their Attachment to Planar Lipid Bilayers
3.3. Fluorescent Labeling of Receptors
3.4. Single-Molecule Detection and Analysis
References
30_O.pdf
Chapter 30
Light Microscopy to Image and Quantify Cell Movement
3.1. 2D-DIAS Analysis of Basic Motile Behavior
3.1.1. Sample Preparation for Analysis of Basic Motile Behavior
3.1.2. Image Capture
3.1.3. Outlining with DIAS
3.1.4. DIAS Path Files
3.1.5. Shape Analysis
3.1.6. Windowing to Compute Relative Flow
3.1.7. Computing Parameters
3.2. 3D-DIAS for the 3D Reconstruction of Live Cells During Basic Motile Behavior
3.2.1. Sample Preparation
3.2.2. Optical Sectioning, Outlining, and Reconstructing Live Cells Using 3D-DIAS
3.2.3. Motility and Dynamic Morphology Parameters Computed by 3D-DIAS
3.3. 3D-DIAS for the Reconstruction of Filopodia
3.3.1. Sample Preparation for the 3D Reconstruction of Filopodia
3.3.2. Optical Sectioning, Outlining, and Reconstructing Filopodia in 3D
3.4. 3D Reconstruction of Live Cells from Confocal Images for Localization of Tagged Molecules During Basic Motile Behavior
3.4.1. Sample Preparation
3.4.2. Image Acquisition
3.4.3. Outlining Images Using Trace Slots
3.5. 3D-DIAS for Analysis of Surface Microsphere Movements
3.5.1. Preparation of Sample
3.5.2. Optical Sectioning, Image Acquisition, Outlining, 3D Reconstruction with 3D DIAS, and Analysis of Surface Microsphere Mo
References
31_O.pdf
Chapter 31
Mathematics of Experimentally Generated Chemoattractant Gradients
2.1. Experimental Setup for Zigmond Chamber
2.2. Measurement/Analysis for Zigmond Chamber
2.3. Diffusion Equations for Zigmond Chamber
3.1. Experimental Setup for Pipette Assay
3.2. Measurement/Analysis for Pipette Assay with and Without Flow
3.3. Diffusion Equations for Pipette Gradients Without Flow
3.4. Diffusion Equations for Pipette Gradients with Flow
References
32_O.pdf
Chapter 32
Modeling Spatial and Temporal Dynamics of Chemotactic Networks
1.1. Modeling Temporal Dynamics
1.1.1. Binding Reaction
1.1.2. Enzymatic Reaction Using Michaelis–Menten Dynamics
1.2. Modeling Spatial Dynamics
1.3. LEGI Model of Gradient Sensing
2.1. Compartmental Models
2.2. Spatial Models
3.1. Specifying the Components
3.2. Specifying the Reactions
3.3. Specifying the Geometry
3.4. Linking the Physiological and Geometrical Models
3.5. Specifying Initial Conditions
3.6. Running a Spatial Simulation
3.7. Running a Temporal Simulation
References
33_O.pdf
Chapter 33
Computational Modeling of Signaling Networks for Eukaryotic Chemosensing
3.1. How to Define Properties of Molecule Types, Multimolecular Complexes, Cell Types, Extracellular Space, and Simulations
3.2. Fundamentals of a Detailed Model of Eukaryotic Chemosensing and Its Implementation
3.3. Simulating Exposure of D. discoideum to Gradients of cAMP
3.4. Analyzing the Behavior of the Simulated Signaling Pathway
References
BM1_O.pdf