Chromaffin Cells: Methods and Protocols

Preface
Contents
Contributors
Part I: Culturing and Morphology
Chapter 1: Rat Pheochromocytoma PC12 Cells in Culture
1 Introduction
2 Materials
2.1 Maintenance of Cell Culture
2.1.1 Coating Dishes for Cell Culture
2.1.2 Pass Cells
2.1.3 Freeze and Thaw Cell Stocks
2.2 Transfection
2.3 Seeding Single Cells
2.3.1 Coating Coverslips or 3.5-cm Culture Dishes (See Note 6)
2.3.2 Disperse and Replate Cells
2.4 Loading of Norepinephrine
2.5 Cell Permeabilization (Optional)
2.6 Single-Vesicle Amperometry
2.7 Single-Cell Biochemical Assays: The Proximity Ligation Assay with Immunostaining
3 Methods
3.1 Maintenance of Cell Culture
3.1.1 Coating Dishes for Cell Culture
3.1.2 Pass Cells
3.1.3 Freeze and Thaw Cell Stocks
3.2 Transfection
3.3 Seeding Single Cells
3.3.1 Coating Coverslips or 3.5-cm Culture Dishes
3.3.2 Disperse and Replate Cells
3.4 Loading of Norepinephrine
3.5 Cell Permeabilization (Optional)
3.6 Single-Vesicle Amperometry
3.7 Single-Cell Biochemical Assays: The Proximity Ligation Assay with Immunostaining
4 Notes
References
Chapter 2: Adrenal Grafts in the Central Nervous System: Chromaffin and Chromaffin Progenitor Cell Transplantation
1 Introduction
2 Materials
2.1 Chromaffin and Chromaffin Progenitor Cell Isolation
2.2 Chromosphere Formation and Expansion
2.3 Cell Transplantation
2.4 Perfusion and Brain Tissue Processing
3 Methods
3.1 Preculture Procedures: Isolation of Chromaffin and Chromaffin Progenitor Cells from Adult Cattle Adrenal Glands (See Fig. ...
3.1.1 Collection and Cleaning of Adrenal Glands
3.1.2 Enzymatic and Mechanical Dissociation of Adrenal Medulla Cells
3.1.3 Differential Plating for Separation of Chromaffin and Chromaffin Progenitor Cells from Other Adrenal Medulla Cell Types ...
3.2 Chromaffin and Chromaffin Progenitor Cells Culture (See Fig. 1c)
3.2.1 Chromaffin Cell Culture
3.2.2 Chromaffin Progenitor Cell Culture (Chromospheres Formation and Expansion)
3.3 Preparation of the Cells for Transplantation into the CNS
3.3.1 Chromaffin Cells Preparation
3.3.2 Chromaffin Progenitor Cells Preparation
3.3.3 Transplantation into the Central Nervous System of Adult Wistar Rats
3.4 Analysis of the Transplanted Cells in the Brain Tissue
4 Notes
References
Chapter 3: Immunocytochemistry of Acutely Isolated Adrenal Medullary Chromaffin Cells
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 4: Transmission Electron Microscopy: A Method for Studying the Adrenal Chromaffin Cells
1 Introduction
2 Materials
2.1 Removal of the Adrenal Glands and Fixation
2.2 Dehydration and Embedding
2.3 Slicing
2.4 Staining
3 Methods
3.1 Removal of the Adrenal Glands and Fixation
3.2 Fixation
3.3 Dehydration and Embedding
3.4 Slicing
3.5 Staining
4 Notes
References
Chapter 5: Immunogold for Protein Location in Chromaffin Cells
1 Introduction
2 Materials
2.1 Buffers
2.2 Tissue and Fixation
2.3 Sectioning of the Adrenal Gland
2.4 Membrane Permeabilization
2.5 Cryofixation
2.6 Freeze-Substitution
2.7 Pre-Embedding Immunohistochemistry
2.8 Post-Embedding Immunohistochemistry
2.9 Processing for Electron Microscopy in Pre-Embedding Immunohistochemistry
2.10 Ultrathin Sectioning
2.11 Staining of Ultrathin Sections
3 Methods
3.1 Tissue and Fixation
3.2 Sectioning of the Adrenal Gland
3.3 Membrane Permeabilization
3.3.1 Freeze-Thaw
3.3.2 Detergent Treatment: Triton X-100
3.4 Cryofixation
3.5 Freeze-Substitution
3.6 Pre-Embedding Immunogold
3.7 Post-Embedding Immunogold
3.8 Processing Sections for Electron Microscopy
3.9 Ultrathin Sectioning of Pre-Embedding Reactions
3.10 Staining of Ultrathin Sections
4 Notes
References
Chapter 6: Visualization of Exo- and Endocytosis Membrane Dynamics with Super-Resolution STED Microscopy
1 Introduction
2 Materials
2.1 Primary Culture of Bovine Adrenal Chromaffin Cells
2.2 Bovine Chromaffin Cells Transfection
2.3 Stimulated Emission Depletion (STED) Microscopy
3 Methods
3.1 Primary Culture of Bovine Adrenal Chromaffin Cells
3.2 Bovine Chromaffin Cells Transfection
3.3 Stimulated Emission Depletion (STED) Microscopy
3.4 STED Image Deconvolution
4 Notes
References
Part II: Stimulus-Secretion Coupling
Chapter 7: Online Detection of Catecholamine Release from the Perfused Rat Adrenal Gland
1 Introduction
2 Materials
2.1 Rats
2.2 Surgical Material
2.3 Other Materials
2.4 Krebs-Bicarbonate Solution
3 Methods
3.1 General Setup
3.2 Surgical Procedure
3.3 Perfusion
3.4 Splanchnic Nerve Stimulation
3.4.1 Electrical Stimulation Parameters
3.5 Online Recording of Secretion
3.6 Perfusion Chamber Design
4 Notes
References
Chapter 8: Real Time Recording of Perifused Chromaffin Cells
1 Introduction
2 Materials
2.1 General SetUp
2.2 Krebs HEPES (KH) Solution
2.3 KH-35 mM K+ (35K-KH)
2.4 KH-59 mM K+ (59K-KH)
2.5 KH-70 mM K+ (70K-KH)
2.6 KH-ACh
3 Methods
3.1 Online Amperometric Recordings of Catecholamine Release from Cell Populations
4 Notes
References
Chapter 9: Recording of Chromaffin Cell Electrical Activity In Situ in Acute Adrenal Slices
1 Introduction
2 Materials
2.1 Dissection of the Adrenal Glands
2.2 Adrenal Slice Preparation
2.3 Slice Storage After Cutting
2.4 Electrophysiological Recordings (Patch-Clamp)
3 Methods
3.1 Acute Slice Preparation
3.2 Electrophysiological Recordings (Patch-Clamp)
4 Notes
References
Chapter 10: Calcium Imaging and Amperometric Recording in Cultured Chromaffin Cells and Adrenal Slices from Normotensive, Wist...
1 Introduction
2 Materials
2.1 Animals
2.2 Materials for Noninvasive Blood Pressure and Heart Rate Measurements
2.2.1 Materials for Blood Pressure and Heart Rate Measurements with Panlab (Harvard Apparatus) or Similar
2.2.2 Materials for Blood Pressure and Heart Rate Measurements with CODA (Kent Scientific Corporation) or Similar
2.3 Materials for the Preparation of Primary Cultures of Chromaffin Cells
2.4 Materials for the Preparation of Adrenal Gland Slices
2.5 Materials for Amperometry
2.5.1 Materials for the manufacturing of Carbon Fiber Microelectrodes
2.5.2 Materials for Amperometry in Cultured Cells and Adrenal Slices
2.5.3 Materials for Intracellular Calcium Imaging
3 Methods
3.1 Blood Pressure and Heart Rate Measurement Methods Using Panlab
3.2 Blood Pressure and Heart Rate Measurement Methods Using CODA
3.3 Preparation of Primary Cultures of Chromaffin Cells
3.4 Preparation of Adrenal Gland Slices
3.5 Manufacture of Plastic-Encased Carbon Fiber Microelectrodes
3.6 Manufacture of Glass-Encased Carbon Fiber Microelectrodes
3.7 Single-Cell Amperometry Recordings
3.7.1 Amperometric Recordings
3.7.2 Amperometric Recordings in Primary Cultures of Chromaffin Cells
3.7.3 Amperometric Recordings in Chromaffin Cells of Adrenal Gland Slices
3.8 Amperometric Spike Analysis
3.9 Single-Cell Intracellular Calcium Imaging
3.9.1 Intracellular Calcium Imaging in Primary Cultured Chromaffin Cells
3.9.2 Intracellular Calcium Imaging in Chromaffin Cells of Adrenal Gland Slices
3.9.3 Intracellular Calcium Imaging
3.9.4 Recording Spontaneous and Evoked Intracellular Calcium Signals
3.10 Data Analysis and Statistics
4 Notes
References
Chapter 11: Measurements of Calcium in Chromaffin Cell Organelles Using Targeted Aequorins
1 Introduction
2 Materials
2.1 Instrument/Luminometer
2.2 Solutions
2.3 Transduction and Expression System
2.4 Adenoviral Particles
3 Methods
3.1 Endoplasmic Reticulum Calcium Measurements
3.1.1 Recording ER Ca2+ Dynamics in Intact Cells
3.1.2 Recording ER Ca2+ Dynamics in Permeabilized Cells
3.2 Mitochondrial Calcium Measurements
3.2.1 Recording Mitochondrial Ca2+ Dynamics in Intact Cells
3.2.2 Recording Mitochondrial Ca2+ Dynamics in Permeabilized Cells
3.3 Secretory Vesicles Calcium Measurements
3.3.1 Recording Secretory Vesicle Ca2+ Dynamics in Intact Cells
3.3.2 Recording Secretory Vesicle Ca2+ Dynamics in Permeabilized Cells
3.4 Transforming Luminescence in [Ca2+]
4 Notes
References
Chapter 12: Quantification of Secretory Granule Exocytosis by TIRF Imaging and Capacitance Measurements
1 Introduction
2 Materials
2.1 Cells
2.2 Labeling of Secretory Granules
2.3 Solutions
2.4 TIRF Microscope
2.5 Patch-Clamp Setup
2.6 Pressure Application
3 Methods
3.1 Coverslip Preparation
3.2 Cell Preparation
3.3 TIRF Imaging of Single Granule Exocytosis
3.4 Patch-Clamp Measurements of Exocytosis and Ca2+ Currents
4 Notes
References
Chapter 13: Membrane Capacitance Measurements of Stimulus-Evoked Exocytosis in Adrenal Chromaffin Cells
1 Introduction
2 Materials
2.1 Solutions and Small Parts
2.2 Equipment
3 Methods
3.1 Fabrication of Patch Pipettes
3.2 Whole-Cell Patch Clamp Recording and Cm Measurements of Exocytosis
3.3 Data Analysis
4 Notes
References
Chapter 14: Quantal Release Analysis of Electrochemically Active Molecules Using Single-Cell Amperometry
1 Introduction
2 Material
3 Methods
3.1 Chromaffin Cells Plating
3.2 Amperometric Detection of Exocytosis
3.3 Amperometry-Spike Analysis
3.4 Statistical Analysis
4 Notes
References
Chapter 15: Methodologies for Detecting Quantal Exocytosis in Adrenal Chromaffin Cells Through Diamond-Based MEAs
1 Introduction
2 Materials
2.1 Diamond Material
2.2 Solutions for Bovine Chromaffin Cells
3 Methods
3.1 μG-D-MEA Microfabrication
3.2 Characteristics of the Electronic Chain
3.3 Bovine Chromaffin Cells Culturing on μ-D-MEAs
3.3.1 Isolation and Culture of Bovine Chromaffin Cells.
3.4 How to Carry Out the Measurement
3.5 Cleaning Procedure of MEA Devices
4 Notes
References
Chapter 16: Vesicle Collision Protocols for the Study of Quantum Size and Exocytotic Fraction Released
1 Introduction
2 Materials
2.1 Microelectrode Fabrication
2.1.1 Carbon Disk Electrodes
2.1.2 Carbon Nanotip Electrodes (CNEs)
2.1.3 Carbon Nanopipette Electrodes
2.1.4 ITO Microelectrode Arrays
2.2 Cell Culture and Solutions
2.2.1 PC12 Cells
2.2.2 Chromaffin Cells and Vesicle
2.2.3 β Cells
2.2.4 Homogenizing Buffer
2.2.5 Isotonic Solution
2.3 Instruments
2.3.1 Amperometric Experiments (See Note 4)
2.3.2 Positioning System
2.3.3 Microscopes
2.3.4 Temperature Control
2.3.5 Vibration and Electromagnetic Radiation Isolation
3 Methods
3.1 Vesicle Impact Electrochemistry Cytometry (VIEC)
3.1.1 Carbon Disk Electrodes
3.1.2 VIEC at Carbon Nanopipettes (CNPs)
3.2 Intracellular Impact Electrochemistry Cytometry (IVIEC)
3.2.1 Carbon Nanotip Electrodes and Carbon Nanopipettes
3.3 Combined Techniques
3.3.1 Two Electrodes to Simultaneously Carry out SCA and IVIEC
3.3.2 VIEC with Combined Confocal Fluorescence Microscopy (See Note 7)
Vesicle Labeling
Electrochemical Measurements
3.3.3 Confocal Measurements
4 Notes
References
Chapter 17: Patch Amperometry and Intracellular Patch Electrochemistry
1 Introduction
2 Materials
2.1 Solutions and Small Parts
2.2 Equipment
2.3 Equipment for PA Only
3 Method
3.1 Rig Setup for PA
3.2 Rig Setup for IPE
3.3 Pipette Preparation and Assembly
3.4 Carbon Fiber Electrode (CFE) Fabrication
3.5 Electrode and Holder Assembly
3.6 Patching and Recording Exocytotic Events with PA
3.7 Measuring Cytosolic Catecholamines with IPE
4 Notes
References
Chapter 18: Artificial Cells for Dissecting Exocytosis
1 Introduction
2 Materials
2.1 Preparation of Small Unilamellar Vesicles (SUVs) from Soybean Lipid Extract
2.2 Preparation of Neurotransmitter-Filled Large Unilamellar Vesicles (LUV)
2.3 Preparation of Carbon Fiber Microelectrodes for Amperometry Detection
2.4 Preparation of Glass Microinjection Pipette and Electroinjection of Pipette Solution
3 Methods
3.1 Amperometry Measurement, an Artificial Cell of the Later Stages of Exocytosis (See Note 11)
3.2 Monitoring Exocytosis Vesicle Fusion and Release from an Artificial Cell
3.3 Data Analysis of the Amperometry Recordings
4 Notes
References
Part III: Chromaffin Granules and Membrane Traffic
Chapter 19: Isolation and Purification of Chromaffin Granules from Adrenal Glands and Cultured Neuroendocrine Cells
1 Introduction
2 Materials
2.1 Tissue Samples
2.2 Reagents and Solutions
2.3 Glass and Plastic Supplies
2.4 Equipment and Instruments
3 Methods
3.1 Adrenal Medulla Dissection
3.2 Adrenal Medulla Homogenization
3.3 Chromaffin Granule Isolation and Purification
3.4 Chromaffin Granule Membrane Enrichment
3.5 Obtention of Granules from Other Sources
3.5.1 Primary Culture of Chromaffin Cells from Bovine Adrenal Glands
3.5.2 Mouse Chromaffin Granule Isolation
4 Notes
References
Chapter 20: Confocal Microscopy Studies of F-Actin Cytoskeleton Distribution and Dynamics Using Fluorescent LifeAct Constructs...
1 Introduction
2 Materials
2.1 Adrenal Glands
2.2 Chromaffin Cells Reagents, Solutions, and Fungibles
2.3 Basal and Depolarizing Solutions
2.4 Fluorescent Protein Expression and Fluorochrome Dyes
2.5 DNA Electroporation Kit
2.6 Perfusion System
2.7 Confocal Microscopy
2.8 Data Analysis
3 Methods
3.1 Isolation, Culture, and Transfection of Bovine Chromaffin Cells
3.2 Fluorochrome Mitochondrial Labeling
3.3 Dynamic Confocal Experiments-1: Expansions and Retractions
3.4 Data Analysis of Expansions and Retractions
3.5 Dynamic Confocal Experiments-2: F-Actin Tail Comets
3.6 Data Analysis of F-Actin Tail Comets
4 Notes
References
Chapter 21: Unveiling the Nanoscale Dynamics of the Exocytic Machinery in Chromaffin Cells with Single-Molecule Imaging
1 Introduction
2 Materials
2.1 Chromaffin Cell Culture
2.2 Transfection (Electroporation)
2.3 Imaging Buffer
2.4 Microscope Setup for Imaging
2.5 Software for Data Analysis
3 Methods
3.1 Chromaffin Cell Preparation
3.2 Transfection of Chromaffin Cells
3.3 Single-Molecule Imaging Preparation
3.4 Single-Molecule Data Acquisition (sptPALM)
3.5 Data Processing
3.6 In-Depth Data Analysis
4 Notes
References
Part IV: Miscellanea
Chapter 22: Determination of Catecholamines in a Small Volume (25 μL) of Plasma from Conscious Mouse Tail Vein
1 Introduction
2 Materials
2.1 Standard Curve
2.2 Tris Buffer
2.3 Phosphate-Citrate Buffer
2.4 Mobile Phase
2.5 Adsorption and Desorption of Catecholamines
2.6 Instrument
2.7 Software
3 Methods
3.1 Mouse Tail Vein Plasma Collection
3.2 Preparation of Plasma Samples
3.3 Electrochemical Detection of Catecholamines
3.4 Standard Curve
3.5 Detection of Catecholamines in Different Plasma Volumes
3.6 Comparable Plasma Catecholamine Levels When Blood Was Collected from the Tail Vein in Conscious Mice and from the Heart in...
4 Notes
References
Chapter 23: Quantification of Chromogranin A and Its Fragments in Biological Fluids
1 Introduction
2 Materials
2.1 Reagents
2.2 Cells and Plasmids
2.3 Chromatographic Resins and Columns
3 Methods
3.1 Preparation of CgA1-439 Expression Plasmid
3.2 Production of Recombinant Human CgA1-439
3.2.1 Preparation of Pre-inoculum
3.2.2 Cell Culture and Protein Expression
3.2.3 Disruption of Bacteria by Sonication and Preparation of the ``Heat Stable´´ Fraction
3.2.4 Ion Exchange Chromatography-1
3.2.5 Dialysis-1
3.2.6 Affinity Chromatography-1
3.2.7 Ion Exchange Chromatography-2
3.2.8 Dialysis-2
3.2.9 Affinity Chromatography-2 (Endotoxin Removal)
3.3 Preparation of CgA1-373 Expression Plasmid
3.4 Production of CgA1-373
3.4.1 Fermentation and Preparation of the Heat Stable Fraction
3.4.2 Immobilized Metal Chelate Affinity chromatography (IMAC)
3.4.3 Dialysis-1
3.4.4 Immunoaffinity Chromatography
3.4.5 Dialysis-2
3.4.6 Affinity Chromatography (Endotoxin Removal)
3.5 Biochemical Characterization of CgA1-439 and CgA1-373
3.6 Preparation of Antibodies
3.6.1 Preparation and Purification of Anti-CgA368-373 Antibodies
3.6.2 Preparation and Purification of Anti-CgA434-439 Antibodies
3.6.3 Preparation and Purification of Anti-CgA1-439 Antibodies
3.7 Characterization of Antibody Specificity
3.8 ELISAs
3.8.1 Total CgA-ELISA
3.8.2 CgA439-ELISA
3.8.3 CgA373-ELISA
4 Notes
References
Chapter 24: Cytotoxicity Models in Chromaffin Cells to Evaluate Neuroprotective Compounds
1 Introduction
1.1 Calcium Overload Models
1.2 Oxidative Stress Models
2 Materials
3 Methods
3.1 Preparation and Plating of BCC
3.2 Evaluation of Neuroprotectants in Different Cytotoxicity Models
3.2.1 Veratridine Model (Fig. 1)
3.2.2 Thapsigargin Model (Fig. 2)
3.2.3 Rotenone Plus Oligomycin A Model (Fig. 3)
3.2.4 6-OHDA Model (Fig. 4)
3.3 Evaluation of Cell Viability: Lactate Dehydrogenase (LDH) Assay (See Note 8)
4 Notes
References
Index