List of protocols                                  xv
Abbreviations                                      xxi
1 Cell culture                                     1  (34)
  Samantha Touhey and Mary Heenan
    1 Introduction                                 1  (1)
    2 Safety                                       1  (1)
    3 Good laboratory technique                    2  (1)
    4 Types of cell cultures                       3  (4)
      Primary cell cultures                        3  (3)
      Finite life-span cell cultures               6  (1)
      Continuous cell lines                        6  (1)
    5 Media requirements                           7  (1)
    6 Serum-free medium                            7  (3)
    7 Subculturing cells                           10 (4)
      Subculturing adherent cells                  10 (1)
      Subculture of suspension cell systems        11 (1)
      Subculturing cells in serum-free medium      12 (2)
    8 Cryopreservation of cells                    14 (3)
      Cryopreservation of adherent cells           14 (2)
      Cryopreservation of cells in serum-free      16 (1)
      medium
    9 Monitoring of sterility of cell culture      17 (1)
    solutions
    10 Mycoplasma analysis of cell lines           17 (5)
      Collection of samples for mycoplasma         18 (1)
      testing
      Indirect staining procedure for              18 (2)
      mycoplasma analysis
      Direct microbial culture for mycoplasma      20 (2)
      analysis
    11 Cloning animal cells                        22 (3)
      Isolation of clonal populations using        22 (2)
      cloning rings
      Cloning procedure with the limiting          24 (1)
      dilution assay
      Semi-solid media cloning                     24 (1)
    12 The application of cell culture to in       25 (7)
    vitro toxicity testing
      Miniaturized in vitro methods for            26 (2)
      toxicity testing
      MTT assay                                    28 (1)
      Acid phosphatase assay                       29 (1)
      Neutral red assay                            29 (1)
      Protein staining assays                      30 (1)
      Growth stimulation                           31 (1)
    Additional reading                             32 (1)
    References                                     32 (3)
2 Basic light microscopy                           35 (28)
  Timo Zimmermann, Rainer Pepperkok, David
  Stephens, Andreas Girod, and Jens Rietdorf
    1 Introduction                                 35 (1)
    2 The sample                                   35 (9)
      Volume and length measurements and           36 (1)
      principal problems of sample preparation
      Sample preparation                           37 (7)
    3 The light microscope                         44 (10)
      Objectives 45 Magnification                  46 (1)
      Illumination                                 47 (1)
      Contrast methods for transmission imaging    47 (3)
      Fluorescence microscopy                      50 (4)
    4 3D microscopy                                54 (2)
      3D sectioning microscopes                    54 (1)
      Confocal microscopes                         54 (2)
    5 In vivo microscopy                           56 (1)
    6 Image acquisition                            56 (2)
      CCD cameras                                  57 (1)
      Intensified CCD cameras                      58 (1)
      Colour cameras                               58 (1)
    7 Data analysis, image processing, and data    58 (1)
    presentation
    8 Online resources                             59 (1)
    References                                     59 (2)
    Appendix Preparation of stock solutions        61 (2)
3 Electron microscopy in cell biology              63 (50)
  John Lucocq
    1 Introduction                                 63 (1)
    2 Overview of sectioning methods for           64 (2)
    transmission EM
      Methods using aldehyde fixation              65 (1)
      Methods using rapid/high pressure freezing   65 (1)
    3 Aldehyde fixation and preliminary            66 (3)
    specimen preparation
      Perfusion fixation of tissue                 69 (1)
      Embedding protocols                          69 (1)
    4 Conventional epoxy resin embedding           69 (6)
      Contrasting epoxy resin sections             74 (1)
    5 Thawed ultrathin cryosections                75 (4)
      Principle                                    75 (1)
      Preparing the blocks of pellet, tissue,      75 (1)
      or gelatin embedded material
      Gelatin embedding                            76 (2)
      Mounting the specimen block on the           78 (1)
      specimen holder
      Sectioning                                   78 (1)
      Picking up the sections                      78 (1)
    6 Progressive lowering of temperature          79 (5)
      Sectioning hydrophilic resins                82 (1)
      Contrasting of Lowicryl sections             82 (2)
    7 Freeze substitution of aldehyde fixed        84 (1)
    specimens
    8 Cryofixed specimens                          85 (2)
      High pressure freezing                       85 (1)
      Rapid freezing                               86 (1)
    9 Freeze substitution                          87 (1)
      Contrasting                                  88 (1)
    10 Frozen hydrated sections                    88 (1)
    11 Immunogold labelling                        89 (10)
      Preparation of reagents                      89 (2)
      Immunolabelling                              91 (3)
      Double labelling                             94 (2)
      Adsorption of particulate/membrane           96 (2)
      organelle fractions to grids
      Pre-embedding labelling                      98 (1)
    12 Quantitation of gold labelling on           99 (6)
    ultrathin sections
      Labelling proportions                        100(1)
      Density of gold labelling over an            101(3)
      organelle (profile)
      Controls for the specificity of labelling    104(1)
    13 Methods for visualizing molecules and       105(2)
    macromolecular assemblies
      Negative staining                            105(2)
    14 Cryofracture techniques: freeze fracture    107(2)
    and freeze etching
      Cryofracture labelling                       108(1)
    15 Scanning electron microscopy                109(3)
    Acknowledgements                               112(1)
    References                                     112(1)
4 Subcellular fractionation                        113(20)
  Alois Hodel and J. Michael Edwardson
    1 Introduction                                 113(1)
    2 Basic principles                             113(1)
    3 Choice of starting material                  114(1)
    4 Homogenization of tissue and cultured        114(4)
    cells
    5 Fractionation by centrifugation              118(5)
      Differential pelleting                       119(1)
      Velocity gradient centrifugation             119(1)
      Isopycnic gradient centrifugation            120(3)
    6 Free-flow electrophoresis                    123(1)
    7 Immunological methods of organelle           124(2)
    purification
      Selection of antigen                         124(1)
      Selection of solid support                   125(1)
      Selection of coupling mode                   125(1)
    8 Monitoring purity                            126(1)
    9 Monitoring organelle structural integrity    127(3)
    and activity
    Acknowledgement                                130(1)
    References                                     130(3)
5 Plant cell biology                               133(30)
  Aldo Ceriotti, Nadine Paris, Stefan Hillmer,
  Lorenzo Frigerio, Jean-Marc Neuhaus,
  Alessandro Vitale, and David G. Robinson
    1 Introduction                                 133(1)
    2 Expression and analysis of heterologous      134(9)
    proteins in plant cells
      Transient or permanent expression?           134(1)
      Tobacco mesophyll protoplasts                135(1)
      Choice of expression vector for transient    136(1)
      expression
      Preparation of DNA and amount used in        137(2)
      transfection
      Labelling of protoplasts                     139(2)
      Protoplast subcellular fractionation         141(2)
    3 Immunolabelling, GFP, and confocal           143(8)
    microscopy
      Sample preparation for fluorescence          143(3)
      immunostaining
      Fluorescence immunolabelling                 146(2)
      Confocal microscopy                          148(1)
      GFP marker                                   149(2)
    4 Immunogold electron microscopy               151(10)
      General remarks                              151(1)
      Assessing 疣tigenic stability                151(2)
      Chemical fixation for immunocytochemical     153(1)
      studies
      Dehydration                                  154(2)
      Polymerization                               156(1)
      Rapid freezing and freeze substitution       156(1)
      Immunogold labelling of acrylic sections     157(1)
      Preparation and immunogold labelling of      158(3)
      cryosections
    References                                     161(2)
6 Protein purification                             163(34)
  Susan E. Slade and Howard Dalton
    1 Introduction                                 163(1)
    2 General principles of protein purification   163(1)
    3 Determination of protein concentrations      164(5)
      Spectroscopic methods-UV absorption          165(1)
      Colorimetric dye-binding assays              165(4)
    4 Fractionation of proteins using              169(8)
    adsorptive techniques
      Fractionation by charge-ion exchange         169(3)
      chromatography
      Fractionation by hydrophobicity              172(2)
      Hydroxyapatite chromatography                174(1)
      Immobilized metal affinity chromatography    175(2)
      (IMAO)
    5 Affinity purification                        177(5)
      Immobilized natural ligands                  177(1)
      Group-specific adsorbents                    178(4)
    6 Fractionation by size-gel filtration         182(2)
    chromatography
    7 Chromatofocusing                             184(1)
    8 Purification of specific types of proteins   185(4)
      Purification of membrane proteins            185(2)
      Recombinant proteins                         187(2)
    9 Example purification                         189(6)
      Preparation of cell-free extract and ion     189(2)
      exchange chromatography to fraction sMMO
      proteins
      Gel filtration chromatography of             191(1)
      hydroxylase protein
      High resolution ion exchange purification    192(1)
      of the hydroxylase
      Separation of the hydroxylase subunits by    192(3)
      reverse phase chromatography
    References                                     195(1)
    Recommended reading                            195(2)
7 Gel electrophoresis of proteins                  197(72)
  David E. Garfin
    1 Introduction                                 197(1)
    2 Gel electrophoresis                          198(29)
      Gels                                         199(4)
      Buffers                                      203(11)
      Pore-size gradient gels                      214(1)
      Apparatus                                    215(3)
      Resolution and separation                    218(1)
      Detergents                                   219(1)
      Choice of system                             220(1)
      Sample preparation                           221(2)
      Molecular mass estimation                    223(2)
      Precast gels                                 225(2)
    3 Isoelectric focusing                         227(7)
      Establishing pH gradients                    228(1)
      Gels for isoelectric focusing                229(3)
      Power conditions and resolution in           232(1)
      isoelectric focusing
      Protein solubilization for isoelectric       232(2)
      focusing
    4 Two-dimensional gel electrophoresis          234(3)
    5 Detection of proteins in gels                237(11)
      Dye staining                                 239(2)
      Silver staining                              241(2)
      Copper and zinc staining                     243(2)
      Fluorescent stains                           245(1)
      Detection of proteins in isoelectric         246(2)
      focusing gels
    6 Image acquisition and analysis               248(1)
    7 Blotting                                     249(7)
      Immunoblotting                               249(7)
    8 Preparative gel electrophoresis              256(9)
      Extraction from gel slices                   256(3)
      The whole gel eluter                         259(3)
      Continuous elution electrophoresis           262(3)
    Acknowledgements                               265(1)
    References                                     265(4)
8 Biophysical methods in structural cell biology   269(24)
  Mavis Agband je-McKenna, Arthur S. Edison,
  and Robert McKenna
    1 Introduction                                 269(1)
    2 Nuclear magnetic resonance spectroscopy      270(6)
      Sample preparation                           272(1)
      Instrumentation hardware                     273(1)
      Data acquisition and structure               273(3)
      determination
    3 X-ray crystallography                        276(9)
      Sample preparation for crystallization       277(2)
      experiments
      Crystal growth                               279(3)
      Cryo-cooling of crystals for data            282(1)
      collection
      Instrumentation hardware                     283(1)
      Phasing of X-ray diffraction data            283(2)
      Structure determination                      285(1)
    4 Electron microscopy                          285(4)
      Sample preparation                           286(2)
      Instrumentation hardware                     288(1)
      Recording images, analysis, and              288(1)
      reconstruction
    5 The protein database                         289(1)
    Suggested reading                              289(4)
9 Nucleic acids                                    293(24)
  T.A. Brown
    1 Introduction                                 293(1)
    2 Purification of nucleic acids                293(1)
    3 The polymerase chain reaction                294(5)
      PCR in outline                               295(1)
      Planning and carrying out a PCR              295(4)
    4 Agarose gel electrophoresis                  299(4)
      DNA gels                                     299(3)
      RNA gels                                     302(1)
    5 Southern hybridization                       303(6)
      The Southern blot                            304(2)
      Hybridization analysis of a Southern blot    306(3)
    6 DNA sequencing                               309(3)
      Sequencing PCR products                      309(2)
      Thermal cycle sequencing of cloned DNA       311(1)
    7 Other DNA and RNA manipulations              312(3)
    Acknowledgements                               315(1)
    References                                     315(2)
10 Cell lipids: from isolation to functional       317(32)
dynamics
  Robert Jan Veldman, Eve-Isabelle P馗heur,
  Sven C.D. van IJzendoorn, Jan Willem Kok, and
  Dick Hoekstra
    1 Introduction                                 317(1)
    2 Analysis of cellular sphingolipids           318(12)
      Extraction and hydrolysis                    318(2)
      Sphingolipid mass measurements               320(6)
      Sphingolipid analysis by metabolic           326(4)
      radiolabelling
    3 Sphingolipid trafficking in living cells     330(8)
      Incorporation of C6-NBD-labelled             332(3)
      sphingolipids into the plasma membrane of
      eukaryotic cells
      Processing of membrane-inserted              335(1)
      sphingolipid by endocytosis
      Determination of recycling of                335(1)
      internalized sphingolipids
      Biosynthetic transport of C6-NBD-labelled    336(2)
      sphingolipids to the plasma membrane
    4 Lipid vesicles and their use in studies      338(7)
    of membrane fusion
      Analysis of phospholipid purity              339(1)
      Preparation of liposomes, anchor lipid,      339(3)
      and peptide-coupled vesicles
      Fusion assay based upon lipid mixing         342(3)
    References                                     345(4)
11 Extracellular matrix protocols for the study    349(16)
of complex phenotypes
  Calvin D. Roskelley and Shoukat Dedhar
    1 Introduction                                 349(1)
    2 Mammary gland development in vivo            350(1)
      Embryonic development                        350(1)
      Postnatal development                        350(1)
      Lobulo-aveolar development                   350(1)
      Involution                                   351(1)
    3 Mammary epithelial cell morphogenesis,       351(1)
    differentiation, and apoptosis in vitro
    4 Experimental models and approaches           351(10)
      Functional cell lines                        351(1)
      Monolayer culture                            352(1)
      Three-dimensional spheroidogenesis and       353(2)
      differentiation
      Extracellular matrix overlays                355(2)
      Cell shape                                   357(1)
      Differentiative rotegrin signalling          358(2)
      Suspension-mediated apoptosis                360(1)
    References                                     361(4)
12 The cytoskeleton                                365(26)
  Theresia B. Stradal, Antonio S. Sechi, J�rgen
  Wehland, and Klemens Rottner
    1 Introduction                                 365(3)
      Intermediate filaments                       365(1)
      The actin cytoskeleton                       366(1)
      The microtubule system                       366(2)
    2 Visualization of the cytoskeleton            368(11)
      Plating of cells on different substrates     369(2)
      Localization of cytoskeletal components      371(3)
      by indirect immunofluorescence microscopy
      Cytoskeletal dynamics in living cells        374(5)
      Cytoskeletal drugs                           379(1)
    3 Basic biochemical analysis of actin and      379(7)
    actin binding proteins
      Purification of actin from smooth muscle     379(3)
      Fluorescent labelling of purified proteins   382(1)
      Co-sedimentation of actin binding and        383(2)
      bundling proteins with F-actin
      Actin polymerization assay                   385(1)
    4 Buffers and stock solutions                  386(1)
    Acknowledgements                               387(1)
    References                                     387(4)
A1 List of suppliers                               391(6)
Index                                              397