Cell Biology Protocols 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.

Features:

• Features helpful notes and safety information for practical application
• Favours easy use at the bench with space for notes and important safety information
• Contains essential analytical information that will prove invaluable to those working on all aspects of cell biology

Contents

Basic Light Microscopy

• Setting up the microscope for bright field microscopy.
• Setting Köhler illumination.
• Focusing procedure.
• Setting up the microscope for phase contrast microscopy.
• Setting up the microscope for epifluorescence.
• Poly-L-lysine coating

Basic Electron Microscopy

• Preparation of carbon-formvar, continuous carbon and holey carbon support films.
• The ‘droplet’ negative staining procedure (using continuous carbon, formvar-carbon and holey carbon support films).
• Immunonegative staining.
• The negative staining-carbon film technique: cell and organelle cleavage.
• Preparation of unstained and negatively stained vitrified specimens.
• Metal shadowing of biological specimens.
• A routine schedule for tissue processing and resin embedding.
• Agarose encapsulation for cell and organelle suspensions.
• Routine staining of thin sections for electron microscopy.
• Post-embedding indirect immunolabelling of thin sections.
• Imaging the nuclear matrix and cytoskeleton by embedment-free electron microscopy

Cell Culture

• Tissue disaggregation by mechanical mincing or chopping.
• Tissue disaggregation by warm trypsinization.
• Cold trypsinization.
• Disaggregation using collagenase or dispase.
• Recovery of cells from effusions.
• Removal of red blood cells by snap lysis.
• Removal of red blood cells (rbc) and dead cells using isopycnic centrifugation.
• Quantification of cell counts and viability.
• Recovery of cells from a monolayer.
• Freezing cells.
• Thawing cells.
• Purification of human PBMCs on a density barrier.
• Purification of human PBMCs using a mixer technique.
• Purification of human PBMCs using a barrier flotation technique

Isolation and Functional Analysis of Organelles

• Isolation of nuclei from mammalian liver in an iodixanol gradient (with notes on cultured cells).
• Isolation of metaphase chromosomes.
• Isolation of the nuclear envelope J. Robin Harris.
• Nuclear pore complex isolation J. Robin Harris.
• Preparation of nuclear matrix.
• Preparation of nucleoli.
• Isolation of a heavy mitochondrial fraction from rat liver by differential centrifugation.
• Preparation of a light mitochondrial fraction from tissues and cultured cells.
• Purification of yeast mitochondria in a discontinuous Nycodenz® gradient.
• Purification of mitochondria from mammalian liver or cultured cells in a median-loaded discontinuous Nycodenz® gradient.
• Succinate--INT reductase assay.
• Isolation of lysosomes in a discontinuous Nycodenz® gradient.
• ß-Galactosidase (spectrophotometric assay).
• ß-Galactosidase (fluorometric assay).
• Isolation of mammalian peroxisomes in an iodixanol gradient.
• Catalase assay.
• Analysis of major organelles in a preformed iodixanol gradient.
• Separation of smooth and rough ER in preformed sucrose gradients.
• Separation of smooth and rough ER in a self-generated iodixanol gradient.
• NADPH-cytochrome c reductase assay.
• Glucose-6-phosphatase assay.
• RNA analysis.
• Isolation of Golgi membranes from liver.
• Assay of UDP-galactose galactosyl transferase.
• Purification of human erythrocyte ‘ghosts’.
• Isolation of plasma membrane sheets from rat liver.
• Assay for 5'-nucleotidase.
• Assay for alkaline phosphodiesterase.
• Assay for ouabain-sensitive Na+/K+-ATPase.
• Isolation of chloroplasts from green leaves or pea seedlings
• Measurement of chloroplast chlorophyll.
• Assessment of chloroplast integrity

Fractionation of Subcellular Membranes in Studies on Membrane Trafficking and Cell Signalling

• Separation of basolateral and bile canalicular plasma membrane domains from mammalian liver in sucrose gradients.
• Isolation of rat liver sinusoidal domain using antibody-bound beads.
• Fractionation of apical and basolateral domains from Caco-2 cells in a sucrose gradient.
• Fractionation of apical and basolateral domains from MDCK cells in an iodixanol gradient.
• Isolation of lipid rafts.
• Isolation of caveolae.
• Analysis of Golgi and ER subfractions from cultured cells using discontinuous sucrose--D2O density gradients.
• Analysis of Golgi, ER, ERGIC and other membrane compartments from cultured cells using continuous iodixanol density gradients.
• Analysis of Golgi, ER, TGN and other membrane compartments in sedimentation velocity iodixanol density gradients (continuous or discontinuous).
• SDS-PAGE of membrane proteins.
• Semi-dry blotting.
• Detection of blotted proteins by enhanced chemiluminescence (ECL).
• Separation of membranes and cytosolic fractions from (a) mammalian cells and (b) bacteria.
• Analysis of early and recycling endosomes in preformed iodixanol gradients; endocytosis of transferrin in transfected MDCK cells.
• Analysis of clathrin-coated vesicle processing in self-generated iodixanol gradients; endocytosis of asialoglycoprotein by rat liver.
• Polysucrose--Nycodenz® gradients for the analysis of dense endosome--lysosome events in mammalian liver

In Vitro Techniques in Cell Biology

• Nucleosome assembly coupled to DNA repair synthesis using a human cell free system
• Single labelling of nascent DNA with halogenated thymidine analogues
• Double labelling of DNA with different halogenated thymidine analogues
• Simultaneous immunostaining of proteins and halogen-dU-substituted DNA
• Uncovering the nuclear matrix in cultured cells
• Nuclear matrix--lamin interactions: in vitrooverlay assay
• Nuclear matrix--lamin interactions: in vitro nuclear reassembly assay
• Preparation of Xenopus laevis egg extracts and immunodepletion
• Nuclear assembly in vitroand immunofluorescence
• Nucleocytoplasmic transport measurements using isolated Xenopusoocyte nuclei
• Transport measurements in microarrays of nuclear envelope patches by optical single transporter recording
• Cell permeabilization with Streptolysin
• Nanocapsules: a new vehicle for intracellular delivery of drugs
• A rapid screen for determination of the protective role of antioxidant proteins in yeast
• In vitro assessment of neuronal apoptosis
• The mitochondrial permeability transition: PT and [Gk cap delta] [Gk cap psi]m loss determined in cells or isolated mitochondria with confocal laser imaging
• The mitochondrial permeability transition: measuring PT and [Gk cap delta][Gk cap psi]m loss in isolated mitochondria with Rh123 in a fluorometer
• The mitochondrial permeability transition: measuring PT and [Gk cap delta][Gk cap psi]m loss in cells and isolated mitochondria on the FACS
• Measuring cytochrome c release in isolated mitochondria by Western blot analysis
• Protein import into isolated mitochondria
• Formation of ternary SNARE complexes in vitro
• Isolation of rough and smooth membrane domains of the endoplasmic reticulum from rat liver
• In vitro reconstitution of liver endoplasmic reticulum
• Asymmetric incorporation of glycolipids into membranes and detection of lipid flip-flop movement
• Purification of clathrin-coated vesicles from rat brains
• Reconstitution of endocytic intermediates on a lipid monolayer
• Golgi membrane tubule formation
• Tight junction assembly
• Reconstitution of the major light-harvesting chlorophyll a/b complex into liposomes
• Reconstitution of Photosystem 2 into liposomes
• Golgi--vimentin interaction in vitro and in vivo
• Microtubule peroxisome interaction
• Detection of cytomatrix proteins by immunogold embedment-free electron microscopy
• Tubulin assembly induced by taxol and other microtubule assembly promoters
• Vimentin production, purification, assembly and study
• Neurofilament assembly
• [Gk lowercase alpha]-Synuclein fibril formation induced
• Amyloid-ß fibril formation in vitro
• Soluble Aß1-42 peptide induces tau hyperphosphorylation in vitro
• Anti-sense peptides
• Interactions between amyloid-ß and enzymes
• Amyloid-ß phosphorylation
• Smitin--myosin II coassembly arrays in vitro
• Assembly/disassembly of myosin filaments in the presence of EF-hand calcium-binding protein S100A4 in vitro
• Collagen fibril assembly in vitro

Selected Reference Data for Cell and Molecular Biology

• Chemical safety information
• Centrifugation data
• Radioisotope data

Index

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