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.

I Handling of Cells in Single Cell Experiments

1 Maintaining Cells Under the Microscope
2 Simple Perfusion Chambers for Single Cell Measurements
3 Temperature Control on the Stage of the Microscope
4 A Modified U-Tube Drug Application System
5 Laser Microsurgery as a Tool in Single Cell Research
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
7 Practical Introduction to Patch Clamping by Simulation Experiments with Simple Electrical Circuits
8 Improved Electrophysiological Measurements by Series Resistance Compensation
9 Preparation of Patchable Plant Cell Protoplasts and a Procedure for the Improvement of Gigaseal Formation
II-2 Single Channel Measurements
10 Recording and Analysis of ATP-Sensitive Potassium Channels in Inside-Out Patches of Rabbit Ventricular Myocytes
11 Mechanotransduction and Mechanosensitive Ion Channels in Osteoblasts
12 The Study of (Plant) Ion Channels Reconstituted in Planar Lipid Bilayers
13 Ion Channel Measurement on the Cell Nucleus
14 Monitoring Cytosolic Calcium and Membrane Potential from Cell-Attached Patch-Clamp Currents in Human T Lymphocytes
II-3 Whole Cell Measurements
15 Membrane Potential and Action Potential Measurements in Whole Cell and Perforated Patch Configurations
16 Perforated Patch-Clamp Technique in Heart Cells
17 Measurement and Analysis of Different Aspects of Potassium Currents in Human Lymphocytes
18 Ionic Conductances in Chicken Osteoclasts
19 GABAA Receptor-Mediated Chloride Currents in Acutely Dissociated Hippocampal Neurons
20 Measurement of Whole Cell Potassium Currents in Protoplasts from Tobacco CellSuspensions
21 Inward Rectifying Potassium Conductance in Barley Aleurone Protoplasts
III Fluorescence to Measure Intracellular Ions
III-1 Introduction to the Measurement Technique
22 An Introduction to the Use of Fluorescent Probes in Ratiometric Fluorescence Microscopy
23 How To Use Video Cameras To Acquire Images
24 Strategies for Studying Intracellular pH Regulation
III-2 Flow Cytometer Measurements
25 An Introduction to the Working Principles of the Flow Cytometer
26 Flow Cytometric Membrane Potential Measurements
27 Flow Cytometric Measurement of Cytosolic Calcium in Lymphocytes
III-3 Microfluorescence Measurements
28 Cytosolic pH and Cell Movement Measurement in Dictyostelium
29 Analysis of Agonist-Induced Cell Recruitment in Terms of Intracellular Calcium Mobilization in a Population of Enzymatically Dispersed Pancreatic Acinar Cells
III-4 Confocal Microscopy
30 Quantitative Confocal Fluorescence Measurements in Living Tissue
31 Cytosolic Calcium Measurements with Confocal Microscopy.