List of Contributors xiii

1 Basic Light Microscopy 1
Minnie O’Farrell

Introduction 1

Key components of the compound microscope 2

Techniques of microscopy 6

Protocols

1.1 Setting up the microscope for bright field microscopy 7

1.2 Setting K¨ohler illumination 8

1.3 Focusing procedure 9

1.4 Setting up the microscope for phase contrast microscopy 11

1.5 Setting up the microscope for epifluorescence 14

1.6 Poly-L-lysine coating 18

References 19

2 Basic Electron Microscopy 21
J. Robin Harris

Introduction 21

EM methods available 22

Protocols

2.1 Preparation of carbon-formvar, continuous carbon and holey carbon support films 25

2.2 The ‘droplet’ negative staining procedure (using continuous carbon, formvar–carbon and holey carbon support films) 27

2.3 Immunonegative staining 29

2.4 The negative staining-carbon film technique: cell and organelle cleavage 31

2.5 Preparation of unstained and negatively stained vitrified specimens 33

2.6 Metal shadowing of biological specimens 35

2.7 A routine schedule for tissue processing and resin embedding 37

2.8 Agarose encapsulation for cell and organelle suspensions 39

2.9 Routine staining of thin sections for electron microscopy 40

2.10 Post-embedding indirect immunolabelling of thin sections 42

2.11 Imaging the nuclear matrix and cytoskeleton by embedment-free electron microscopy 44
Jeffrey A. Nickerson and Jean Underwood

References 50

3 Cell Culture 51
Anne Wilson and John Graham

Cells: isolation and analysis 51
Anne Wilson

Mechanical disaggregation of tissue 52

Protocols

3.1 Tissue disaggregation by mechanical mincing or chopping 54

3.2 Tissue disaggregation by warm trypsinization 56

3.3 Cold trypsinization 58

3.4 Disaggregation using collagenase or dispase 60
Anne Wilson

3.5 Recovery of cells from effusions 63
Anne Wilson

3.6 Removal of red blood cells by snap lysis 64

3.7 Removal of red blood cells and dead cells using isopycnic centrifugation 65
Anne Wilson

3.8 Quantitation of cell counts and viability 67
Anne Wilson

3.9 Recovery of cells from monolayer cultures 71
Anne Wilson

3.10 Freezing cells 74

3.11 Thawing cells 76
John Graham

3.12 Purification of human PBMCs on a density barrier 80

3.13 Purification of human PBMCs using a mixer technique 82

3.14 Purification of human PBMCs using a barrier flotation technique 83

References 84

4 Isolation and Functional Analysis of Organelles 87
John Graham

Introduction 88

Homogenization 88

Differential centrifugation 90

Density gradient centrifugation 91

Nuclei and nuclear components 92

Mitochondria 93

Lysosomes 94

Peroxisomes 94

Rough and smooth endoplasmic reticulum (ER) 95

Golgi membranes 96

Plasma membrane 96

Chloroplasts 97

Protocols

4.1 Isolation of nuclei from mammalian liver in an iodixanol gradient (with notes on cultured cells) 98

4.2 Isolation of metaphase chromosomes 100

4.3 Isolation of the nuclear envelope 102

4.4 Nuclear pore complex isolation 104
J. Robin Harris

4.5 Preparation of nuclear matrix 106

4.6 Preparation of nucleoli 107

4.7 Isolation of a heavy mitochondrial fraction from rat liver by differential centrifugation 108

4.8 Preparation of a light mitochondrial fraction from tissues and cultured cells 110

4.9 Purification of yeast mitochondria in a discontinuous Nycodenz^® gradient 112

4.10 Purification of mitochondria from mammalian liver or cultured cells in a median-loaded discontinuous Nycodenz^® gradient 114

4.11 Succinate–INT reductase assay 116

4.12 Isolation of lysosomes in a discontinuous Nycodenz^® gradient 117

4.13 β-Galactosidase (spectrophotometric assay) 119

4.14 β-Galactosidase (fluorometric assay) 120

4.15 Isolation of mammalian peroxisomes in an iodixanol gradient 121

4.16 Catalase assay 123

4.17 Analysis of major organelles in a preformed iodixanol gradient 124

4.18 Separation of smooth and rough ER in preformed sucrose gradients 127

4.19 Separation of smooth and rough ER in a self-generated iodixanol gradient 129

4.20 NADPH-cytochrome c reductase assay 131

4.21 Glucose-6-phosphatase assay 132

4.22 RNA analysis 133

4.23 Isolation of Golgi membranes from liver 134

4.24 Assay of UDP-galactose galactosyl transferase 136

4.25 Purification of human erythrocyte ‘ghosts’ 137

4.26 Isolation of plasma membrane sheets from rat liver 139

4.27 Assay for 5’-nucleotidase 141

4.28 Assay for alkaline phosphodiesterase 143

4.29 Assay for ouabain-sensitive Na⁺/K⁺-ATPase 144

4.30 Isolation of chloroplasts from green leaves or pea seedlings 145

4.31 Measurement of chloroplast chlorophyll 147

4.32 Assessment of chloroplast integrity 148

5 Fractionation of Subcellular Membranes in Studies on Membrane Trafficking and Cell Signalling 153
John Graham

Introduction 154

Methods available 154

Plasma membrane domains 155

Analysis of membrane compartments in the endoplasmic reticulum–Golgi–plasma membrane pathway 156

Separation of membrane vesicles from cytosolic proteins 157

Endocytosis 158

Protocols

5.1 Separation of basolateral and bile canalicular plasma membrane domains from mammalian liver in sucrose gradients 160

5.2 Isolation of rat liver sinusoidal domain using antibody-bound beads 162

5.3 Fractionation of apical and basolateral domains from Caco-2 cells in a sucrose gradient 163

5.4 Fractionation of apical and basolateral domains from MDCK cells in an iodixanol gradient 165

5.5 Isolation of lipid rafts 167

5.6 Isolation of caveolae 170

5.7 Analysis of Golgi and ER subfractions from cultured cells using discontinuous sucrose–D₂O density gradients 172

5.8 Analysis of Golgi, ER, ERGIC and other membrane compartments from cultured cells using continuous iodixanol density gradients 174

5.9 Analysis of Golgi, ER, TGN and other membrane compartments in sedimentation velocity iodixanol density gradients (continuous or discontinuous) 177

5.10 SDS–PAGE of membrane proteins 180

5.11 Semi-dry blotting 182

5.12 Detection of blotted proteins by enhanced chemiluminescence (ECL) 183

5.13 Separation of membranes and cytosolic fractions from (a) mammalian cells and (b) bacteria 185

5.14 Analysis of early and recycling endosomes in preformed iodixanol gradients; endocytosis of transferrin in transfected MDCK cells 188

5.15 Analysis of clathrin-coated vesicle processing in self-generated iodixanol gradients; endocytosis of asialoglycoprotein by rat liver 191

5.16 Polysucrose–Nycodenz^® gradients for the analysis of dense endosome–lysosome events in mammalian liver 194

References 196

6 In Vitro Techniques 201
Edited by J. Robin Harris

Introduction 203

Protocols

Nuclear components

6.1 Nucleosome assembly coupled to DNA repair synthesis using a human cell free system 204
Geneviève Almouzni and Doris Kirschner

6.2 Single labelling of nascent DNA with halogenated thymidine analogues 210
Daniela Dimitrova

6.3 Double labelling of DNA with different halogenated thymidine analogues 214

6.4 Simultaneous immunostaining of proteins and halogen-dU-substituted DNA 217

6.5 Uncovering the nuclear matrix in cultured cells 220
Jeffrey A. Nickerson, Jean Underwood and Stefan Wagner

6.6 Nuclear matrix–lamin interactions: in vitro blot overlay assay 228
Barbara Korbei and Roland Foisner

6.7 Nuclear matrix–lamin interactions: in vitro nuclear reassembly assay 230

6.8 Preparation of Xenopus laevis egg extracts and immunodepletion 234
Tobias C. Walther

6.9 Nuclear assembly in vitro and immunofluorescence 237
Martin Hetzer

6.10 Nucleocytoplasmic transport measurements using isolated Xenopus oocyte nuclei 240
Reiner Peters

6.11 Transport measurements in microarrays of nuclear envelope patches by optical single transporter recording 244
Reiner Peters

Cells and membrane systems

6.12 Cell permeabilization with Streptolysin O 248
Ivan Walev

6.13 Nanocapsules: a new vehicle for intracellular delivery of drugs 250
Anton I. P. M. de Kroon, Rutger W. H. M. Staffhorst, Ben de Kruijff and Koert N. J.Burger

6.14 A rapid screen for determination of the protective role of antioxidant proteins in yeast 255
Luis Eduardo Soares Netto

6.15 In vitro assessment of neuronal apoptosis 259
Eric Bertrand

6.16 The mitochondrial permeability transition: PT and Δѱm loss determined in cells or isolated mitochondria with confocal laser imaging 265
Judie B. Alimonti and Arnold H. Greenberg

6.17 The mitochondrial permeability transition: measuring PT and Δѱm loss in isolated mitochondria with Rh123 in a fluorometer 268
Judie B. Alimonti and Arnold H. Greenberg

6.18 The mitochondrial permeability transition: measuring PT and Δѱm loss in cells and isolated mitochondria on the FACS 270
Judie B. Alimonti and Arnold H. Greenberg

6.19 Measuring cytochrome c release in isolated mitochondria by Western blot analysis 271
Judie B. Alimonti and Arnold H. Greenberg

6.20 Protein import into isolated mitochondria 272
Judie B. Alimonti and Arnold H. Greenberg

6.21 Formation of ternary SNARE complexes in vitro 274
Jinnan Xiao, Anuradha Pradhan and Yuechueng Liu

6.22 In vitro reconstitution of liver endoplasmic reticulum 277
Jacques Paiement and Robin Young

6.23 Asymmetric incorporation of glycolipids into membranes and detection of lipid flip-flop movement 280
Félix M. Goñi, Ana-Victoria Villar, F.-Xabier Contreras and Alicia Alonso

6.24 Purification of clathrin-coated vesicles from rat brains 286
Brian J. Peter and Ian G. Mills

6.25 Reconstitution of endocytic intermediates on a lipid monolayer 288
Brian J. Peter and Matthew K. Higgins

6.26 Golgi membrane tubule formation 293
William J. Brown, K. Chambers and A. Doody

6.27 Tight junction assembly 296
C. Yan Cheng and Dolores D. Mruk

6.28 Reconstitution of the major light-harvesting chlorophyll a/b complex into liposomes 300
Chunhong Yang, Helmut Kirchhoff, Winfried Haase, Stephanie Boggasch and Harald Paulsen

6.29 Reconstitution of photosystem 2 into liposomes 305
Julie Benesova, Sven-T. Liffers and Matthias Rögner

6.30 Golgi–vimentin interaction in vitro and in vivo 307
Ya-sheng Gao and Elizabeth Sztul

Cytoskeletal and fibrillar systems

6.31 Microtubule peroxisome interaction 313
Meinolf Thiemann and H. Dariush Fahimi

6.32 Detection of cytomatrix proteins by immunogold embedment-free electron microscopy 317
Robert Gniadecki and Barbara Gajkowska

6.33 Tubulin assembly induced by taxol and other microtubule assembly promoters 326
Susan L. Bane

6.34 Vimentin production, purification, assembly and study by EPR 331
John F. Hess, John C. Voss and Paul G. FitzGerald

6.35 Neurofilament assembly 337
Shin-ichi Hisanaga and Takahiro Sasaki

6.36 α-Synuclein fibril formation induced by tubulin 342
Kenji Uéda and Shin-ichi Hisanaga

6.37 Amyloid-β fibril formation in vitro 345
J. Robin Harris

6.38 Soluble Aβ_1–42 peptide induces tau hyperphosphorylation in vitro 348
Terrence Town and Jun Tan

6.39 Anti-sense peptides 353
Nathaniel G. N. Milton

6.40 Interactions between amyloid-β and enzymes 359
Nathaniel G. N. Milton

6.41 Amyloid-β phosphorylation 364
Nathaniel G. N. Milton

6.42 Smitin–myosin II coassembly arrays in vitro 369
Richard Chi and Thomas C. S. Keller III

6.43 Assembly/disassembly of myosin filaments in the presence of EF-hand calcium-binding protein S100A4 in vitro 372
Marina Kriajevska, Igor Bronstein and Eugene Lukanidin

6.44 Collagen fibril assembly in vitro 375
David F. Holmes and Karl E. Kadler

7 Selected Reference Data for Cell and Molecular Biology 379
David Rickwood

Chemical safety information 379

Centrifugation data 386

Radioisotope data 388

Index 391