Cell Biology Protocols

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.

• Preface xiList of Contributors xiii1 Basic Light Microscopy 1Minnie O’FarrellIntroduction 1Key components of the compound microscope 2Techniques of microscopy 6Protocols1.1 Setting up the microscope for bright field microscopy 71.2 Setting K¨ohler illumination 81.3 Focusing procedure 91.4 Setting up the microscope for phase contrast microscopy 111.5 Setting up the microscope for epifluorescence 141.6 Poly-L-lysine coating 18References 192 Basic Electron Microscopy 21J. Robin HarrisIntroduction 21EM methods available 22Protocols2.1 Preparation of carbon-formvar, continuous carbon and holey carbon support films 252.2 The ‘droplet’ negative staining procedure (using continuous carbon, formvar–carbon and holey carbon support films) 272.3 Immunonegative staining 292.4 The negative staining-carbon film technique: cell and organelle cleavage 312.5 Preparation of unstained and negatively stained vitrified specimens 332.6 Metal shadowing of biological specimens 352.7 A routine schedule for tissue processing and resin embedding 372.8 Agarose encapsulation for cell and organelle suspensions 392.9 Routine staining of thin sections for electron microscopy 402.10 Post-embedding indirect immunolabelling of thin sections 422.11 Imaging the nuclear matrix and cytoskeleton by embedment-free electron microscopy 44Jeffrey A. Nickerson and Jean UnderwoodReferences 503 Cell Culture 51Anne Wilson and John GrahamCells: isolation and analysis 51Anne WilsonMechanical disaggregation of tissue 52Protocols3.1 Tissue disaggregation by mechanical mincing or chopping 543.2 Tissue disaggregation by warm trypsinization 563.3 Cold trypsinization 583.4 Disaggregation using collagenase or dispase 60Anne Wilson3.5 Recovery of cells from effusions 63Anne Wilson3.6 Removal of red blood cells by snap lysis 643.7 Removal of red blood cells and dead cells using isopycnic centrifugation 65Anne Wilson3.8 Quantitation of cell counts and viability 67Anne Wilson3.9 Recovery of cells from monolayer cultures 71Anne Wilson3.10 Freezing cells 743.11 Thawing cells 76John Graham3.12 Purification of human PBMCs on a density barrier 803.13 Purification of human PBMCs using a mixer technique 823.14 Purification of human PBMCs using a barrier flotation technique 83References 844 Isolation and Functional Analysis of Organelles 87John GrahamIntroduction 88Homogenization 88Differential centrifugation 90Density gradient centrifugation 91Nuclei and nuclear components 92Mitochondria 93Lysosomes 94Peroxisomes 94Rough and smooth endoplasmic reticulum (ER) 95Golgi membranes 96Plasma membrane 96Chloroplasts 97Protocols4.1 Isolation of nuclei from mammalian liver in an iodixanol gradient (with notes on cultured cells) 984.2 Isolation of metaphase chromosomes 1004.3 Isolation of the nuclear envelope 1024.4 Nuclear pore complex isolation 104J. Robin Harris4.5 Preparation of nuclear matrix 1064.6 Preparation of nucleoli 1074.7 Isolation of a heavy mitochondrial fraction from rat liver by differential centrifugation 1084.8 Preparation of a light mitochondrial fraction from tissues and cultured cells 1104.9 Purification of yeast mitochondria in a discontinuous Nycodenz® gradient 1124.10 Purification of mitochondria from mammalian liver or cultured cells in a median-loaded discontinuous Nycodenz® gradient 1144.11 Succinate–INT reductase assay 1164.12 Isolation of lysosomes in a discontinuous Nycodenz® gradient 1174.13 β-Galactosidase (spectrophotometric assay) 1194.14 β-Galactosidase (fluorometric assay) 1204.15 Isolation of mammalian peroxisomes in an iodixanol gradient 1214.16 Catalase assay 1234.17 Analysis of major organelles in a preformed iodixanol gradient 1244.18 Separation of smooth and rough ER in preformed sucrose gradients 1274.19 Separation of smooth and rough ER in a self-generated iodixanol gradient 1294.20 NADPH-cytochrome c reductase assay 1314.21 Glucose-6-phosphatase assay 1324.22 RNA analysis 1334.23 Isolation of Golgi membranes from liver 1344.24 Assay of UDP-galactose galactosyl transferase 1364.25 Purification of human erythrocyte ‘ghosts’ 1374.26 Isolation of plasma membrane sheets from rat liver 1394.27 Assay for 5’-nucleotidase 1414.28 Assay for alkaline phosphodiesterase 1434.29 Assay for ouabain-sensitive Na+/K+-ATPase 1444.30 Isolation of chloroplasts from green leaves or pea seedlings 1454.31 Measurement of chloroplast chlorophyll 1474.32 Assessment of chloroplast integrity 1485 Fractionation of Subcellular Membranes in Studies on Membrane Trafficking and Cell Signalling 153John GrahamIntroduction 154Methods available 154Plasma membrane domains 155Analysis of membrane compartments in the endoplasmic reticulum–Golgi–plasma membrane pathway 156Separation of membrane vesicles from cytosolic proteins 157Endocytosis 158Protocols5.1 Separation of basolateral and bile canalicular plasma membrane domains from mammalian liver in sucrose gradients 1605.2 Isolation of rat liver sinusoidal domain using antibody-bound beads 1625.3 Fractionation of apical and basolateral domains from Caco-2 cells in a sucrose gradient 1635.4 Fractionation of apical and basolateral domains from MDCK cells in an iodixanol gradient 1655.5 Isolation of lipid rafts 1675.6 Isolation of caveolae 1705.7 Analysis of Golgi and ER subfractions from cultured cells using discontinuous sucrose–D2O density gradients 1725.8 Analysis of Golgi, ER, ERGIC and other membrane compartments from cultured cells using continuous iodixanol density gradients 1745.9 Analysis of Golgi, ER, TGN and other membrane compartments in sedimentation velocity iodixanol density gradients (continuous or discontinuous) 1775.10 SDS–PAGE of membrane proteins 1805.11 Semi-dry blotting 1825.12 Detection of blotted proteins by enhanced chemiluminescence (ECL) 1835.13 Separation of membranes and cytosolic fractions from (a) mammalian cells and (b) bacteria 1855.14 Analysis of early and recycling endosomes in preformed iodixanol gradients; endocytosis of transferrin in transfected MDCK cells 1885.15 Analysis of clathrin-coated vesicle processing in self-generated iodixanol gradients; endocytosis of asialoglycoprotein by rat liver 1915.16 Polysucrose–Nycodenz® gradients for the analysis of dense endosome–lysosome events in mammalian liver 194References 1966 In Vitro Techniques 201Edited by J. Robin HarrisIntroduction 203ProtocolsNuclear components6.1 Nucleosome assembly coupled to DNA repair synthesis using a human cell free system 204Geneviève Almouzni and Doris Kirschner6.2 Single labelling of nascent DNA with halogenated thymidine analogues 210Daniela Dimitrova6.3 Double labelling of DNA with different halogenated thymidine analogues 2146.4 Simultaneous immunostaining of proteins and halogen-dU-substituted DNA 2176.5 Uncovering the nuclear matrix in cultured cells 220Jeffrey A. Nickerson, Jean Underwood and Stefan Wagner6.6 Nuclear matrix–lamin interactions: in vitro blot overlay assay 228Barbara Korbei and Roland Foisner6.7 Nuclear matrix–lamin interactions: in vitro nuclear reassembly assay 2306.8 Preparation of Xenopus laevis egg extracts and immunodepletion 234Tobias C. Walther6.9 Nuclear assembly in vitro and immunofluorescence 237Martin Hetzer6.10 Nucleocytoplasmic transport measurements using isolated Xenopus oocyte nuclei 240Reiner Peters6.11 Transport measurements in microarrays of nuclear envelope patches by optical single transporter recording 244Reiner PetersCells and membrane systems6.12 Cell permeabilization with Streptolysin O 248Ivan Walev6.13 Nanocapsules: a new vehicle for intracellular delivery of drugs 250Anton I. P. M. de Kroon, Rutger W. H. M. Staffhorst, Ben de Kruijff and Koert N. J.Burger6.14 A rapid screen for determination of the protective role of antioxidant proteins in yeast 255Luis Eduardo Soares Netto6.15 In vitro assessment of neuronal apoptosis 259Eric Bertrand6.16 The mitochondrial permeability transition: PT and Δѱm loss determined in cells or isolated mitochondria with confocal laser imaging 265Judie B. Alimonti and Arnold H. Greenberg6.17 The mitochondrial permeability transition: measuring PT and Δѱm loss in isolated mitochondria with Rh123 in a fluorometer 268Judie B. Alimonti and Arnold H. Greenberg6.18 The mitochondrial permeability transition: measuring PT and Δѱm loss in cells and isolated mitochondria on the FACS 270Judie B. Alimonti and Arnold H. Greenberg6.19 Measuring cytochrome c release in isolated mitochondria by Western blot analysis 271Judie B. Alimonti and Arnold H. Greenberg6.20 Protein import into isolated mitochondria 272Judie B. Alimonti and Arnold H. Greenberg6.21 Formation of ternary SNARE complexes in vitro 274Jinnan Xiao, Anuradha Pradhan and Yuechueng Liu6.22 In vitro reconstitution of liver endoplasmic reticulum 277Jacques Paiement and Robin Young6.23 Asymmetric incorporation of glycolipids into membranes and detection of lipid flip-flop movement 280Félix M. Goñi, Ana-Victoria Villar, F.-Xabier Contreras and Alicia Alonso6.24 Purification of clathrin-coated vesicles from rat brains 286Brian J. Peter and Ian G. Mills6.25 Reconstitution of endocytic intermediates on a lipid monolayer 288Brian J. Peter and Matthew K. Higgins6.26 Golgi membrane tubule formation 293William J. Brown, K. Chambers and A. Doody6.27 Tight junction assembly 296C. Yan Cheng and Dolores D. Mruk6.28 Reconstitution of the major light-harvesting chlorophyll a/b complex into liposomes 300Chunhong Yang, Helmut Kirchhoff, Winfried Haase, Stephanie Boggasch and Harald Paulsen6.29 Reconstitution of photosystem 2 into liposomes 305Julie Benesova, Sven-T. Liffers and Matthias Rögner6.30 Golgi–vimentin interaction in vitro and in vivo 307Ya-sheng Gao and Elizabeth SztulCytoskeletal and fibrillar systems6.31 Microtubule peroxisome interaction 313Meinolf Thiemann and H. Dariush Fahimi6.32 Detection of cytomatrix proteins by immunogold embedment-free electron microscopy 317Robert Gniadecki and Barbara Gajkowska6.33 Tubulin assembly induced by taxol and other microtubule assembly promoters 326Susan L. Bane6.34 Vimentin production, purification, assembly and study by EPR 331John F. Hess, John C. Voss and Paul G. FitzGerald6.35 Neurofilament assembly 337Shin-ichi Hisanaga and Takahiro Sasaki6.36 α-Synuclein fibril formation induced by tubulin 342Kenji Uéda and Shin-ichi Hisanaga6.37 Amyloid-β fibril formation in vitro 345J. Robin Harris6.38 Soluble Aβ1–42 peptide induces tau hyperphosphorylation in vitro 348Terrence Town and Jun Tan6.39 Anti-sense peptides 353Nathaniel G. N. Milton6.40 Interactions between amyloid-β and enzymes 359Nathaniel G. N. Milton6.41 Amyloid-β phosphorylation 364Nathaniel G. N. Milton6.42 Smitin–myosin II coassembly arrays in vitro 369Richard Chi and Thomas C. S. Keller III6.43 Assembly/disassembly of myosin filaments in the presence of EF-hand calcium-binding protein S100A4 in vitro 372Marina Kriajevska, Igor Bronstein and Eugene Lukanidin6.44 Collagen fibril assembly in vitro 375David F. Holmes and Karl E. Kadler7 Selected Reference Data for Cell and Molecular Biology 379David RickwoodChemical safety information 379Centrifugation data 386Radioisotope data 388Index 391

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