Table of Contents
Preface
SECTION 1. FLUORESCENCE
CHAPTER 1
INTRODUCTION
Fluorescence Microscopy
Michael J. Sanderson, Ian Smith, Ian Parker, and Martin D. Bootman
CHAPTER 2
INTRODUCTION
Ca2+-Sensitive Fluorescent Dyes and Intracellular Ca2+ Imaging
Martin D. Bootman, Katja Rietdorf, Tony Collins, Simon Walker, and Michael Sanderson
PROTOCOLS
1 Loading Fluorescent Ca2+ Indicators into Living Cells
Martin D. Bootman, Katja Rietdorf, Tony Collins, Simon Walker, and Michael Sanderson
2 Converting Fluorescence Data into Ca2+ Concentration
Martin D. Bootman, Katja Rietdorf, Tony Collins, Simon Walker, and Michael Sanderson
CHAPTER 3
INTRODUCTION
Properties and Use of Genetically Encoded FRET Sensors for Cytosolic and Organellar Ca2+ Measurements
J. Genevieve Park and Amy E. Palmer
PROTOCOLS
1 Verifying the Function and Localization of Genetically Encoded Ca2+ Sensors and Converting FRET Ratios to Ca2+ Concentrations
J. Genevieve Park and Amy E. Palmer
2 Measuring the In Situ Kd of a Genetically Encoded Ca2+ Sensor
J. Genevieve Park and Amy E. Palmer
CHAPTER 4
INTRODUCTION
Photolysis of Caged Compounds: Studying Ca2+ Signaling and Activation of Ca2+-Dependent Ion Channels
Janos Almassy and David I. Yule
PROTOCOLS
1 Studying the Activation of Epithelial Ion Channels Using Global Whole-Field Photolysis
Janos Almassy and David I. Yule
2 Investigating Ion Channel Distribution Using a Combination of Spatially Limited Photolysis, Ca2+ Imaging, and Patch-Clamp Recording
Janos Almassy and David I. Yule
3 Analyzing Ca2+ Dynamics in Intact Epithelial Cells Using Spatially Limited Flash Photolysis
Janos Almassy and David I. Yule
CHAPTER 5
INTRODUCTION
Electroporation Loading and Flash Photolysis to Investigate Intra- and Intercellular Ca2+ Signaling
Elke Decrock, Marijke De Bock, Nan Wang, Mélissa Bol, Ashish K. Gadicherla, and Luc Leybaert
PROTOCOLS
1 Electroporation Loading of Membrane-Impermeable Molecules to Investigate Intra- and Intercellular Ca2+ Signaling
Elke Decrock, Marijke De Bock, Nan Wang, Mélissa Bol, Ashish K. Gadicherla, and Luc Leybaert
2 Flash Photolysis of Caged IP3 to Trigger Intercellular Ca2+ Waves
Elke Decrock, Marijke De Bock, Nan Wang, Mélissa Bol, Ashish K. Gadicherla, and Luc Leybaert
CHAPTER 6
INTRODUCTION
Investigating Calcium Signaling by Confocal and Multiphoton Microscopy
Lars Kaestner and Peter Lipp
PROTOCOLS
1 Two-Photon Photolysis Combined with a Kilobeam Array Scanner to Probe Calcium Signaling in Cardiomyocytes
Benjamin Sauer, Martin Oberhofer, Peter Lipp, and Lars Kaestner
2 Multibeam Two-Photon Imaging of Fast Ca2+ Signals in the Langendorff Mouse Heart
Karin Hammer, Peter Lipp, and Lars Kaestner
3 Multichannel Imaging of Cellular Signaling: Interplay of Ca2+ and Conventional Protein Kinase C
Peter Lipp, Xin Hui, Gregor Reither, and Lars Kaestner
4 Two-Dimensional Imaging of Fast Intracellular Ca2+ Release
Qinghai Tian, Lars Kaestner, and Peter Lipp
5 Confocal FLIM of Genetically Encoded FRET Sensors for Quantitative Ca2+ Imaging
Benjamin Sauer, Qinghai Tian, Peter Lipp, and Lars Kaestner
CHAPTER 7
INTRODUCTION
Combining Calcium Imaging with Other Optical Techniques
Marco Canepari, Dejan Zecevic, Kaspar E. Vogt, David Ogden, and Michel De Waard
PROTOCOLS
1 Combining Ca2+ and Membrane Potential Imaging in Single Neurons
Marco Canepari, Kaspar E. Vogt, Michel De Waard, and Dejan Zecevic
2 Combining Ca2+ Imaging with L-Glutamate Photorelease
Marco Canepari, Michel De Waard, and David Ogden
CHAPTER 8
INTRODUCTION
High-Throughput Analyses of IP3 Receptor Behavior
Colin W. Taylor, Stephen C. Tovey, and Ana M. Rossi
PROTOCOLS
1 High-Throughput Functional Assays of IP3-Evoked Ca2+ Release
Stephen C. Tovey and Colin W. Taylor
2 High-Throughput Fluorescence Polarization Assay of Ligand Binding to IP3 Receptors
Ana M. Rossi and Colin W. Taylor
SECTION 2. LUMINESCENCE
CHAPTER 9
INTRODUCTION
The Use of Aequorin and Its Variants for Ca2+ Measurements
Veronica Granatiero, Maria Patron, Anna Tosatto, Giulia Merli, and Rosario Rizzuto
PROTOCOLS
1 Using Targeted Variants of Aequorin to Measure Ca2+ Levels in Intracellular Organelles
Veronica Granatiero, Maria Patron, Anna Tosatto, Giulia Merli, and Rosario Rizzuto
CHAPTER 10
INTRODUCTION
Introduction of Aequorin into Zebrafish Embryos for Recording Ca2+ Signaling during the First 48 h of Development
Sarah E. Webb, Ching Man Chan, and Andrew L. Miller
PROTOCOLS
1 Microinjecting Holo-Aequorin into Dechorionated and Intact Zebrafish Embryos
Sarah E. Webb and Andrew L. Miller
2 Reconstitution of Holo-Aequorin with Apoaequorin mRNA and Coelenterazine in Zebrafish Embryos
Ching Man Chan, Andrew L. Miller, and Sarah E. Webb
SECTION 3. RADIOACTIVE TECHNIQUES
CHAPTER 11
INTRODUCTION
Measurement of Intracellular Ca2+ Release in Intact and Permeabilized Cells Using 45Ca2+
Ludwig Missiaen, Tomas Luyten, Geert Bultynck, Jan B. Parys, and Humbert De Smedt
PROTOCOLS
1 Measurement of Intracellular Ca2+ Release in Permeabilized Cells Using 45Ca2+
Tomas Luyten, Geert Bultynck, Jan B. Parys, Humbert De Smedt, and Ludwig Missiaen
2 Measurement of Intracellular Ca2+ Release in Intact Cells Using 45Ca2+
Tomas Luyten, Geert Bultynck, Jan B. Parys, Humbert De Smedt, and Ludwig Missiaen
CHAPTER 12
INTRODUCTION
Measuring Ca2+ Pump Activity in Overexpression Systems and Cardiac Muscle Preparations
Tine Holemans, Ilse Vandecaetsbeek, Frank Wuytack, and Peter Vangheluwe
PROTOCOLS
1 High-Throughput Measurement of the Ca2+-Dependent ATPase Activity in COS Microsomes
Ilse Vandecaetsbeek, Tine Holemans, Frank Wuytack, and Peter Vangheluwe
2 Measuring Ca2+-Dependent Ca2+-Uptake Activity in the Mouse Heart
Tine Holemans, Ilse Vandecaetsbeek, Frank Wuytack, and Peter Vangheluwe
SECTION 4. ELECTROPHYSIOLOGY
CHAPTER 13
INTRODUCTION
Patch-Clamp Recording of Voltage-Sensitive Ca2+ Channels
Mar?á A. Gandini, Alejandro Sandoval, and Ricardo Felix
PROTOCOLS
1 Whole-Cell Patch-Clamp Recordings of Ca2+ Currents from Isolated Neonatal Mouse Dorsal Root Ganglion (DRG) Neurons
Mar?á A. Gandini, Alejandro Sandoval, and Ricardo Felix
2 Whole-Cell Patch-Clamp Recording of Recombinant Voltage-Sensitive Ca2+ Channels Heterologously Expressed in HEK-293 Cells
Mar?á A. Gandini, Alejandro Sandoval, and Ricardo Felix
CHAPTER 14
INTRODUCTION
Patch-Clamp Measurement of ICRAC and ORAI Channel Activity
Dalia Alansary, Tatiana Kilch, Christian Holzmann, Christine Peinelt, Markus Hoth, and Annette Lis
PROTOCOLS
1 Measuring Endogenous ICRAC and ORAI Currents with the Patch-Clamp Technique
Dalia Alansary, Tatiana Kilch, Christian Holzmann, Christine Peinelt, Markus Hoth, and Annette Lis
2 The Minimal Requirements to Use Calcium Imaging to Analyze ICRAC
Dalia Alansary, Tatiana Kilch, Christian Holzmann, Christine Peinelt, Markus Hoth, and Annette Lis
CHAPTER 15
INTRODUCTION
Patch-Clamp Electrophysiology of Intracellular Ca2+ Channels
Don-On Daniel Mak, Horia Vais, King-Ho Cheung, and J. Kevin Foskett
PROTOCOLS
1 Isolating Nuclei from Cultured Cells for Patch-Clamp Electrophysiology of Intracellular Ca2+ Channels
Don-On Daniel Mak, Horia Vais, King-Ho Cheung, and J. Kevin Foskett
2 Nuclear Patch-Clamp Electrophysiology of Ca2+ Channels
Don-On Daniel Mak, Horia Vais, King-Ho Cheung, and J. Kevin Foskett
CHAPTER 16
INTRODUCTION
Bilayer Measurement of Endoplasmic Reticulum Ca2+ Channels
Ilya Bezprozvanny
PROTOCOLS
1 Preparation of Microsomes to Study Ca2+ Channels
Ilya Bezprozvanny
2 Reconstitution of Endoplasmic Reticulum InsP3 Receptors into Black Lipid Membranes
Ilya Bezprozvanny
CHAPTER 17
INTRODUCTION
Measurement of Mitochondrial Ca2+ Transport Mediated by Three Transport Proteins: VDAC1, the Na+/Ca2+ Exchanger, and the Ca2+ Uniporter
Danya Ben-Hail, Raz Palty, and Varda Shoshan-Barmatz
PROTOCOLS
1 Purification of VDAC1 from Rat Liver Mitochondria
Danya Ben-Hail and Varda Shoshan-Barmatz
2 Reconstitution of Purified VDAC1 into a Lipid Bilayer and Recording of Channel Conductance
Danya Ben-Hail and Varda Shoshan-Barmatz
3 Assay of Ca2+ Transport by VDAC1 Reconstituted into Liposomes
Danya Ben-Hail and Varda Shoshan-Barmatz
4 Assays of Mitochondrial Ca2+ Transport and Ca2+ Efflux via the MPTP
Danya Ben-Hail and Varda Shoshan-Barmatz
5 Mitochondrial Na+/Ca2+ Exchange Assays
Raz Palty and Varda Shoshan-Barmatz
CHAPTER 18
INTRODUCTION
Calcium-Sensitive Mini- and Microelectrodes
Roger C. Thomas and Donald M. Bers
PROTOCOLS
1 How to Make Calcium-Sensitive Minielectrodes
Roger C. Thomas and Donald M. Bers
2 How to Make Calcium-Sensitive Microelectrodes
Roger C. Thomas and Donald M. Bers
SECTION 5. SPECIAL TISSUES
CHAPTER 19
INTRODUCTION
The Xenopus Oocyte: A Single-Cell Model for Studying Ca2+ Signaling
Yaping Lin-Moshier and Jonathan S. Marchant
PROTOCOLS
1 Nuclear Microinjection to Assess How Heterologously Expressed Proteins Impact Ca2+ Signals in Xenopus Oocytes
Yaping Lin-Moshier and Jonathan S. Marchant
2 A Rapid Western Blotting Protocol for the Xenopus Oocyte
Yaping Lin-Moshier and Jonathan S. Marchant
CHAPTER 20
PROTOCOLS
Imaging and Manipulating Calcium Transients in Developing Xenopus Spinal Neurons
Nicholas C. Spitzer, Laura N. Borodinsky, and Cory M. Root
CHAPTER 21
INTRODUCTION
A Systematic Approach for Assessing Ca2+ Handling in Cardiac Myocytes
Karin R. Sipido, Niall Macquaide, and Virginie Bito
PROTOCOLS
1 Basic Methods for Monitoring Intracellular Ca2+ in Cardiac Myocytes Using Fluo-3
Virginie Bito, Karin R. Sipido, and Niall Macquaide
2 Characterizing the Trigger for Sarcoplasmic Reticulum Ca2+ Release in Cardiac Myocytes
Virginie Bito, Niall Macquaide, and Karin R. Sipido
3 Measuring Sarcoplasmic Reticulum Ca2+ Content, Fractional Release, and Ca+ Buffering in Cardiac Myocytes
Niall Macquaide, Virginie Bito, and Karin R. Sipido
4 Measuring Ca2+ Sparks in Cardiac Myocytes
Niall Macquaide, Virginie Bito, and Karin R. Sipido
5 Assessing Ca2+-Removal Pathways in Cardiac Myocytes
Virginie Bito, Karin R. Sipido, and Niall Macquaide
CHAPTER 22
INTRODUCTION
Monitoring Ca2+ Signaling in Yeast
Renata Tisi, Enzo Martegani, and Rogelio L. Brandão
PROTOCOLS
1 Monitoring Yeast Intracellular Ca2+ Levels Using an In Vivo Bioluminescence Assay
Renata Tisi, Enzo Martegani, and Rogelio L. Brandão
2 Total Cellular Ca2+ Measurements in Yeast Using Flame Photometry
Renata Tisi, Enzo Martegani, and Rogelio L. Brandão
3 Measurement of Calcium Uptake in Yeast Using 45Ca
Renata Tisi, Enzo Martegani, and Rogelio L. Brandão
CHAPTER 23
INTRODUCTION
Ca2+ Imaging in Plants Using Genetically Encoded Yellow Cameleon Ca2+ Indicators
Smrutisanjita Behera, Melanie Krebs, Giovanna Loro, Karin Schumacher, Alex Costa, and Jörg Kudla
PROTOCOLS
1 Live Cell Imaging of Cytoplasmic Ca2+ Dynamics in Arabidopsis Guard Cells
Smrutisanjita Behera and Jörg Kudla
2 High-Resolution Imaging of Cytoplasmic Ca2+ Dynamics in Arabidopsis Roots
Smrutisanjita Behera and Jörg Kudla
3 Live Cell Imaging of Cytoplasmic and Nuclear Ca2+ Dynamics in Arabidopsis Roots
Melanie Krebs and Karin Schumacher
4 Imaging of Mitochondrial and Nuclear Ca2+ Dynamics in Arabidopsis Roots
Giovanna Loro and Alex Costa
SECTION 6. NAD(P)-DERIVED MESSENGERS
CHAPTER 24
INTRODUCTION
Cyclic ADP-Ribose: Endogenous Content, Enzymology, and Ca2+ Release
Andreas H. Guse, Tanja Kirchberger, and Santina Bruzzone
PROTOCOLS
1 Cycling Assay for Determining Intracellular Cyclic ADP-Ribose Levels Santina Bruzzone and Andreas H. Guse
2 Measuring CD38 (ADP-Ribosyl Cyclase/Cyclic ADP-Ribose Hydrolase) Activity by Reverse-Phase HPLC
Tanja Kirchberger and Andreas H. Guse
3 Measuring Ca2+ Release Evoked by Cyclic ADP-Ribose
Andreas H. Guse
CHAPTER 25
INTRODUCTION
Methods in Nicotinic Acid Adenine Dinucleotide Phosphate Research
Antony Galione, Kai-Ting Chuang, Tim M. Funnell, Lianne C. Davis, Anthony J. Morgan, Margarida Ruas, John Parrington, and Grant C. Churchill
PROTOCOLS
1 Preparation and Use of Sea Urchin Egg Homogenates for Studying NAADP-Mediated Ca2+ Release
Antony Galione, Kai-Ting Chuang, Tim M. Funnell, Lianne C. Davis, Anthony J. Morgan, Margarida Ruas, John Parrington, and Grant C. Churchill
2 Synthesis of NAADP-AM as a Membrane-Permeant NAADP Analog
Antony Galione, Kai-Ting Chuang, Tim M. Funnell, Lianne C. Davis, Anthony J. Morgan, Margarida Ruas, John Parrington, and Grant C. Churchill
3 Measurement of Luminal pH of Acidic Stores as a Readout for NAADP Action
Antony Galione, Kai-Ting Chuang, Tim M. Funnell, Lianne C. Davis, Anthony J. Morgan, Margarida Ruas, John Parrington, and Grant C. Churchill
4 Synthesis of Caged NAADP
Antony Galione, Kai-Ting Chuang, Tim M. Funnell, Lianne C. Davis, Anthony J. Morgan, Margarida Ruas, John Parrington, and Grant C. Churchill
5 Synthesis of [32P]NAADP for Radioreceptor Binding Assay
Antony Galione, Kai-Ting Chuang, Tim M. Funnell, Lianne C. Davis, Anthony J. Morgan, Margarida Ruas, John Parrington, and Grant C. Churchill
SECTION 7. MEASURING AND MODELING CA2+ DYNAMICS
CHAPTER 26
INTRODUCTION
Measuring Ca2+-Binding Kinetics of Proteins
Guido C. Faas and Istvan Mody
PROTOCOLS
1 Measuring the Steady-State Properties of Ca2+ Indicators with a Set of Calibrated [Ca2+] Solutions
Guido C. Faas and Istvan Mody
2 Measuring the Rate Constants of Ca2+ Indicators
Guido C. Faas and Istvan Mody
3 Collecting Data to Determine the Ca2+-Binding Properties of DM-Nitrophen and Proteins
Guido C. Faas and Istvan Mody
CHAPTER 27
INTRODUCTION
Translating Intracellular Calcium Signaling into Models
Rüdiger Thul
PROTOCOLS
1 Exploring Oscillations in a Point Model of the Intracellular Ca2+ Concentration
Rüdiger Thul
2 Time to Blip—Stochastic Simulation of Single Channel Opening
Rüdiger Thul
APPENDIX 1
General Safety and Hazardous Material Information
Index
