Main description:

A variety of powerful techniques for monitoring and analysing events during signal transduction at the single cell level are described in this lab manual. An introductionary section on cell handling includes guidelines for constructing a perfusion chamber. A main section of the book presents protocols on fluorescence techniques such as flow cytometry, microfluorescence, ion imaging and confocal microscopy. The electrophysiological section illustrates multiple applications of the patch-clamp technique in various cell types from both animals and plants. Emphasis is put on calibration and validation of the different techniques to measure changes of membrane potential, and intracellular ion concentration or pH.

Contents:

Part I Handling of Cells in Single Cell Experiments: - 1 Maintaining Cells Under the Microscope, CAN Ince. - 2 Simple Perfusion Chambers for Single Cell Measurements, Kei Inouye. - 3 Temperature Control on the Stage of the Microscope, Arie de Vos, Bert van Duijn, and Anneke Wiltink. - 4 A Modified U-Tube Drug Application System, Misa Dzoljic. - 5 Laser Microsurgery as a Tool in Single Cell Research, Pettie P. Booij and Albertus H. De Boer. Part II Ion Channel and Membrane Potential Measurements Using the Patch-Clamp Technique, II-1 Introduction to the Measurement Technique: - 6 Significance of Ion Channels and Membrane Potential Changes in Cells, Sandor Damjanovich. - 7 Practical Introduction to Patch Clamping by Simulation Experiments with Simple Electrical Circuits, Dirk L. Ypey. - 8 Improved Electrophysiological Measurements by Series Resistance Compensation, A.C.G. van Ginneken and A.O. Verkerk. - 9 Preparation of Patchable Plant Cell Protoplasts and a Procedure for the Improvement of Gigaseal Formation, Sake A. Vogelzang and Margreet Blom-Zandstra. II-2 Single Channel Measurements: - 10 Recording and Analysis of ATP-Sensitive Potassium Channels in Inside-Out Patches of Rabbit Ventricular Myocytes, Marieke W. Veldkamp. - 11 Mechanotransduction and Mechanosensitive Ion Channels in Osteoblasts, Randall L. Duncan. - 12 The Study of (Plant) Ion Channels Reconstituted in Planar Lipid Bilayers, Henk Miedema. - 13 Ion Channel Measurement on the Cell Nucleus, Federica Bertaso, Raffaella Tonini, and Michele Mazzanti. - 14 Monitoring Cytosolic Calcium and Membrane Potential from Cell-Attached Patch-Clamp Currents in Human T Lymphocytes, JOS A.H. VERHEUGEN. II-3 Whole Cell Measurements: - 15 Membrane Potential and Action Potential Measurements in Whole Cell and Perforated Patch Configurations, Zheng Wang, Dirk L. Ypey, and Rutgeris J. van den Berg. - 16 Perforated Patch-Clamp Technique in Heart Cells, E. Etienne Verheijck. - 17 Measurement and Analysis of Different Aspectsof Potassium Currents in Human Lymphocytes, REZSO GSPR JR., ZOLTAN VARGA, GYOERGY PANYI, ZOLTAN KRASZNAI, CARLO PIERI, AND SANDOR DAMJANOVICH. - 18 Ionic Conductances in Chicken Osteoclasts, ZOLTAN KRASZNAI, ADAM F. WEIDEMA, RESZO GSPR, GYOERGY PANYI, AND DIRK L. YPEY. - 19 GABAA Receptor-Mediated Chloride Currents in Acutely Dissociated Hippocampal Neurons, MISA DZOLJIC AND WYTSE J. WADMAN. - 20 Measurement of Whole Cell Potassium Currents in Protoplasts from Tobacco Cell Suspensions, BERT VAN DUIJN. - 21 Inward Rectifying Potassium Conductance in Barley Aleurone Protoplasts, MARCEL T. FLIKWEERT AND MEI WANG. Part II Fluorescence to Measure Intracellular Ions, II-1 Introduction to the Measurement Technique: - 22 An Introduction to the Use of Fluorescent Probes in Ratiometric Fluorescence Microscopy, PIET VIS AND JOLANDA LEMMERS. - 23 How To Use Video Cameras To Acquire Images, JOCHEM HERRMANN. - 24 Strategies for Studying Intracellular pH Regulation, JAN H. RAVESLOOT. Part III Fluorescence to Measure Intracellular Ions, III-2 Flow Cytometer Measurements: - 25 An Introduction to the Working Principles of the Flow Cytometer, SANDOR DAMJANOVICH, CARLO PIERI, LASZLO BENE, ATTILA JENEI, AND REZSOE GSPR, JR.. - 26 Flow Cytometric Membrane Potential Measurements, SANDOR DAMJANOVICH, CARLO PIERI, LASZLO BENE, ATTILA JENEI, AND REZSOE GSPR, JR.. - 27 Flow Cytometric Measurement of Cytosolic Calcium in Lymphocytes, ERIKA BAUS, JACQUES URBAIN, OBERDAN LEO AND FABIENNE ANDRIS. III-3 Microfluorescence Measurements: - 28 Cytosolic pH and Cell Movement Measurement in Dictyostelium, Kei Inouye. - 29 Analysis of Agonist-Induced Cell Recruitment in Terms of Intracellular Calcium Mobilization in a Population of Enzymatically Dispersed Pancreatic Acinar Cells, ROLF L.L. SMEETS, REMKO R. BOSCH, AND PETER H.G.M. WILLEMS. II-4 Confocal Microscopy: - 30 Quantitative Confocal Fluorescence Measurements in Living Tissue, MARK FRICKER, RACHEL ERRINGTON, JULIAN WOOD, MONIKA TLALKA, MIKE MAY