Cell Biology Protocols

, by Harris, J. Robin; Graham, John M.; Rickwood, David

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ISBN: 9780470847589 | 0470847581
Cover: Hardcover
Copyright: 3/6/2006

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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.

Contents.

List of Contributors.

Preface.

1 Basic Light Microscopy Minnie O’Farrell.

Introduction.

Key components of the compound microscope.

Techniques of microscopy.

Protocols.

1.1 Setting up the microscope for bright field microscopy.

1.2 Setting Köhler illumination.

1.3 Focusing procedure.

1.4 Setting up the microscope for phase contrast microscopy.

1.5 Setting up the microscope for epifluorescence.

Preparation and staining of specimens.

Protocol.

1.6 Poly-L-lysine coating.

References.

2 Basic Electron Microscopy J. Robin Harris.

Introduction.

EM methods available.

Protocols.

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

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

2.3 Immunonegative staining.

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

2.5 Preparation of unstained and negatively stained vitrified specimens.

2.6 Metal shadowing of biological specimens.

2.7 A routine schedule for tissue processing and resin embedding.

2.8 Agarose encapsulation for cell and organelle suspensions.

2.9 Routine staining of thin sections for electron microscopy.

2.10 Post-embedding indirect immunolabelling of thin sections.

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

References.

3 Cell Culture Anne Lewis and John Graham.

Cells: isolation and analysis -- Anne Lewis.

Mechanical disaggregation of tissue.

Protocols.

3.1 Tissue disaggregation by mechanical mincing or chopping.

3.2 Tissue disaggregation by warm trypsinization.

3.3 Cold trypsinization.

3.4 Disaggregation using collagenase or dispase.

Isolation of cells from body fluids -- Anne Lewis.

Protocol.

3.5 Recovery of cells from effusions.

Removal of red blood cells -- Anne Lewis.

Protocols.

3.6 Removal of red blood cells by snap lysis.

3.7 Removal of red blood cells (rbc) and dead cells using isopycnic centrifugation.

Cell counting and cell viability -- Anne Lewis.

Protocol.

3.8 Quantification of cell counts and viability.

Use of cell cultures -- Anne Lewis.

Protocol.

3.9 Recovery of cells from a monolayer.

Cryopreservation -- Anne Lewis.

Protocols.

3.10 Freezing cells.

3.11 Thawing cells.

Isolation of human peripheral blood mononuclear cells -- John Graham.

Protocols.

3.12 Purification of human PBMCs on a density barrier.

3.13 Purification of human PBMCs using a mixer technique.

3.14 Purification of human PBMCs using a barrier flotation technique.

References.

4 Isolation and Functional Analysis of Organelles John Graham.

Introduction.

Homogenization.

Differential centrifugation.

Density gradient centrifugation.

Nuclei and nuclear components.

Mitochondria.

Lysosomes.

Peroxisomes.

Rough and smooth endoplasmic reticulum (ER).

Golgi membranes.

Plasma membrane.

Chloroplasts.

Protocols.

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

4.2 Isolation of metaphase chromosomes.

4.3 Isolation of the nuclear envelope J. Robin Harris.

4.4 Nuclear pore complex isolation J. Robin Harris.

4.5 Preparation of nuclear matrix.

4.6 Preparation of nucleoli.

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

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

4.9 Purification of yeast mitochondria in a discontinuous Nycodenz(r) gradient.

4.10 Purification of mitochondria from mammalian liver or cultured cells in a median-loaded discontinuous Nycodenz(r) gradient.

4.11 Succinate--INT reductase assay.

4.12 Isolation of lysosomes in a discontinuous Nycodenz(r) gradient.

4.13 ß-Galactosidase (spectrophotometric assay).

4.14 ß-Galactosidase (fluorometric assay).

4.15 Isolation of mammalian peroxisomes in an iodixanol gradient.

4.16 Catalase assay.

4.17 Analysis of major organelles in a preformed iodixanol gradient.

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

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

4.20 NADPH-cytochrome c reductase assay.

4.21 Glucose-6-phosphatase assay.

4.22 RNA analysis.

4.23 Isolation of Golgi membranes from liver.

4.24 Assay of UDP-galactose galactosyl transferase.

4.25 Purification of human erythrocyte ‘ghosts’.

4.26 Isolation of plasma membrane sheets from rat liver.

4.27 Assay for 5'-nucleotidase.

4.28 Assay for alkaline phosphodiesterase.

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

4.30 Isolation of chloroplasts from green leaves or pea seedlings

4.31 Measurement of chloroplast chlorophyll.

4.32 Assessment of chloroplast integrity.

References.

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

Introduction.

Methods available.

Plasma membrane domains.

Analysis of membrane compartments in the endoplasmic reticulum-Golgi-plasma membrane pathway.

Separation of membrane vesicles from cytosolic proteins.

Endocytosis.

Protocols.

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

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

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

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

5.5 Isolation of lipid rafts.

5.6 Isolation of caveolae.

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

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

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

5.10 SDS-PAGE of membrane proteins.

5.11 Semi-dry blotting.

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

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

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

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

5.16 Polysucrose--Nycodenz(r) gradients for the analysis of dense endosome--lysosome events in mammalian liver.

References.

6 In Vitro Techniques in Cell Biology Edited by J Robin Harris.

Introduction.

Nuclear components.

Protocols.

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

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

6.3 Double labelling of DNA with different halogenated thymidine analogues Daniela S. Dimitrova.

6.4 Simultaneous immunostaining of proteins and halogen-dU-substituted DNA Daniela S. Dimitrova.

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

Nuclear matrix--lamin interactions.

Protocols.

6.6 Nuclear matrix--lamin interactions: in vitrooverlay assay Barbara Korbei and Roland Foisner.

6.7 Nuclear matrix--lamin interactions: in vitro nuclear reassembly assay Barbara Korbei and Roland Foisner.

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

6.9 Nuclear assembly in vitroand immunofluorescence Martin Hetzer.

6.10 Nucleocytoplasmic transport measurements using isolated Xenopusoocyte nuclei Reiner Peters.

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

Cells and membrane systems.

Protocols.

6.12 Cell permeabilization with Streptolysin O Ivan Walev.

6.13 Nanocapsules: a new vehicle for intracellular delivery of drugs 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 Luis Eduardo Soares Netto.

6.15 In vitro assessment of neuronal apoptosis Eric Bertrand.

6.16 The mitochondrial permeability transition: PT and [Gk cap delta] [Gk cap psi]m loss determined in cells or isolated mitochondria with confocal laser imaging Judie B. Almonti and Arnold H. Greenberg.

6.17 The mitochondrial permeability transition: measuring PT and [Gk cap delta][Gk cap psi]m loss in isolated mitochondria with Rh123 in a fluorometer Judie B. Almonti and Arnold H. Greenberg.

6.18 The mitochondrial permeability transition: measuring PT and [Gk cap delta][Gk cap psi]m loss in cells and isolated mitochondria on the FACS Judie B. Almonti and Arnold H. Greenberg.

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

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

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

6.22 Isolation of rough and smooth membrane domains of the endoplasmic reticulum from rat liver Jacques Paiement and Robin Young.

6.23 In vitro reconstitution of liver endoplasmic reticulum Jacques Paiement and Robin Young.

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

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

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

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

6.28 Tight junction assembly C. Yan Cheng and Dolores D. Mruk.

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

6.30 Reconstitution of Photosystem 2 into liposomes Julie Benesova, Sven-T. Liffers and Matthias Rögner.

Cytoskeletal and fibrillar systems.

Protocols.

6.31 Golgi--vimentin interaction in vitro and in vivo Ya-sheng Gao and Elizabeth Sztul.

6.32 Microtubule peroxisome interaction Meinolf Thiemann and H. Dariush Fahimi.

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

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

6.35 Vimentin production, purification, assembly and study by EPR John F. Hess, John C. Voss and Paul G. Fitzgerald.

6.36 Neurofilament assembly Shin-ichi Hisanaga and T. Sasaki.

6.37 [Gk lowercase alpha]-Synuclein fibril formation induced by tubulin Kenji Uéda and Shin-ichi Hisanaga.

6.38 Amyloid-ß fibril formation in vitro J. Robin Harris.

6.39 Soluble Aß1-42 peptide induces tau hyperphosphorylation in vitro Terrence Town and Jun Tan.

6.40 Anti-sense peptides Nathaniel G.N. Milton.

6.41 Interactions between amyloid-ß and enzymes Nathaniel G.N. Milton.

6.42 Amyloid-ß phosphorylation Nathaniel G.N. Milton.

6.43 Smitin--myosin II coassembly arrays in vitro Richard Chi and Thomas C.S. Keller III.

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

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

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

Chemical safety information.

Centrifugation data.

Radioisotope data.

Index.

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Cell Biology Protocols

Summary

Author Biography

Table of Contents

Supplemental Materials