This extensively updated and expanded second edition contains new chapters on the elctrophysiological measurement of Ca2+ channel activity, enhanced coverage of confocal microscopy, and practical tutorials on two of the most common industrial high-throughput machines, the Fluormetric Imaging Plate Reader (FLIPR), and the FlexStation. All experimental chapters provide readily reproducible techniques for measuring the many dacets of Ca2+ signaling. The protocols follow the successful Methods in Molecular Biology(TM) series format, each offering step-by-step laboratory instructions, an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.

Part I. General

Fluorescent Measurement of [Ca2+]c: Basic Practical Considerations
Alec W. M. Simpson

Measurement of [Ca2+]i in Whole Cell Suspensions Using Fura-2
Robert A. Hirst, Charlotte Harrison, Kazuyoshi Hirota, and David G. Lambert

Measurement of [Ca2+]i in Cell Suspensions Using Indo-1
Adriaan Nelemans

Part II. Specialist Measurement Systems

Confocal Microscopy: Theory and Applications for Cellular Signaling
Stephen C. Tovey, Paul J. Brighton, and Gary B. Willars

Single-Cell and Subcellular Measurement of Intracellular Ca2+ Concentration
Anthony J. Morgan and Andrew P. Thomas

Ratiometric Ca2+ Measurements Using the FlexStation® Scanning Fluorometer
Ian C. B. Marshall, Izzy Boyfield, and Shaun McNulty

Measuring Ca2+ Changes in Multiwell Format Using the Fluorometric Imaging Plate Reader
Ian C. B. Marshall, Davina E. Owen, and Shaun McNulty

Part III. Measurement of Ca2+ Channel Activity

Measurement of Ca2+ Entry Using 45Ca2+
Mercedes Villarroya, Manuela G. López, María F. Cano-Abad, and Antonio G. García

Measurement of [3H]PN200-110 and [125I]w-Conotoxin MVIIA Binding
Kazuyoshi Hirota and David G. Lambert

Whole-Cell Patch Clamp Recording of Voltage-Sensitive Ca2+ Channel Currents: Heterologous Expression Systems and Dissociated Brain Neurons
Atticus H. Hainsworth, Andrew D. Randall, and Alessandro Stefani

Part IV. Measurement of Ins(1,4,5)P3 and Ca2+ Release From Intracellular Stores

Measurement of Phospholipase C by Monitoring Inositol Phosphates Using [3H]Inositol-Labeling Protocols in Permeabilized Cells
Alison Skippen, Philip Swigart, and Shamshad Cockcroft

Measurement of Inositol(1,4,5)Triphosphate Using a Stereospecific Radioreceptor Mass Assay
Darren Smart

Measurement of Calcium Fluxes in Permeabilized Cells Using a45Ca2+ Uptake and Release Assay
Robert A. Wilcox

Microinjection of myo-Inositol(1,4,5)Trisphosphate and Other Calcium-Mobilizing Agents Into Intact Adherent Cells
Robert A. Wilcox, Ian D. Forsythe, and Terence J. McCann

Measurement of Free [Ca2+] Changes in Agonist-Sensitive Internal Stores Using Compartmentalized Fluorescent Indicators
Aldebaran M. Hofer

Part V. Specialist Measurement Techniques

Measurement of [Ca2+]i in Smooth Muscle Strips Using Front-Surface Fluorimetry
Hideo Kanaide

Measurement of Calcium and Movement in Heart Cells
Leong L. Ng and Paulene A. Quinn

Simultaneous Analysis of Intracellular pH and Ca2+ From Cell Populations
Raul Martinez-Zaguilan, Linda S. Tompkins, Robert J. Gillies, and Ronald M. Lynch

Measurement of Cytosolic-Free Ca2+ in Plant Tissue
Martin R. McAinsh and Carl K.-Y. Ng

Part VI. Ca2+-Sensitive Targets

Assay and Purification of Calmodulin-Dependent Protein Kinase
Rajendra K. Sharma

Assay and Purification of Calmodulin-Dependent Cyclic Nucleotide Phosphodiesterase and Isozyme Separation
Rajendra K. Sharma

Measurement of Ca2+-ATPase Activity (in PMCA and SERCA1)
Danuta Kosk-Kosicka

Index