Cell Biology Protocols

As a modern composite scientific discipline, Cell Biology has expanded and moved forward rapidly in recent years. Cell Biologists now require a wide range of techniques, including those of analytical biochemistry and microscopy in all its diverse forms. These are often used alongside the techniques of molecular biology and molecular genetics. This book contains numerous useful protocols, covering light and electron microscopy, cell culture, cell separation, subcellular fractionation, organelle and membrane isolation, and the use of in vitro reassembly systems in Cell Biology. Many of these protocols feature helpful notes and safety information for practical application. The format favours easy use at the bench with space for notes and important safety information. An appendix contains essential analytical information that will prove invaluable to those working on all aspects of cell biology. This book will be of interest to students and more experienced cell biologists, as well as molecular biologists and those working in genomics and proteomics who are looking for cellular techniques to validate their findings within intact cells.

Table of Contents:
Preface xi 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 Aβ1–42 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

About the Author :
J. Robin Harris is the editor of Cell Biology Protocols, published by Wiley. John M. Graham is the editor of Cell Biology Protocols, published by Wiley. David Rickwood is the editor of Cell Biology Protocols, published by Wiley.

Review :
"A valuable possession for every cell biologist’s library." (BSCB Newsletter, September 2006)