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Adaptation of High Five™ Cells to HyQ© SFX Insect Medium - http://www.hyclone.com/pdf/procedure_insect_highfive.pdf

Category: Cell Culture Protocols: Insect Cell Culture Protocols

This protocol is designed to adapt High Five™ (BTI-Tn-4) cells to serum-free suspension culture in HyQ SFX-Insect in 5 passages. The entire adaptation process takes approximately two weeks and the resulting adapted cells have doubling times of 16 and 20 hours and minimal clumping in suspension culture. - [Read Adaptation of High Five™ Cells to HyQ© SFX Insect Medium]

Assay of Cytokines in Tissue Culture Supernatants - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000223.pdf?pid=PP00000223

Category: Signalling Signal Transduction Protocols

Assay of cytokines in tissue culture supernatants describes a liquid suspension array for quantification of cytokines in tissue culture supernatants or serum. With this assay, it is possible to profile the level of multiple cytokines in a single well. The principle of this cytokine assay is similar to a capture sandwich immunoassay. Includes: Preparation for the Assay, Cytokine Assay, Reagents and Materials. - [Read Assay of Cytokines in Tissue Culture Supernatants]

Assay of Cytokines in Tissue Culture Supernatants Protocol - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000223.pdf?pid=PP00000223

Category: Immunology: Immunoassays

The Bio-Plex cytokine assay employs a liquid suspension array for quantification of cytokines in tissue culture supernatants or serum. Using this 96-well microtiter plateformatted assay, it is possible to profile the level of multiple cytokines in a single well. - [Read Assay of Cytokines in Tissue Culture Supernatants Protocol]

Attaching Suspension Cells to Slides Using a Cytocentrifuge Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4291

Category: Cell Culture Protocols: Cell Staining Protocols

A simple method for handling suspension cells is to attach them to a solid substrate before fixation, and one way to do this is to use a cytocentrifuge. - [Read Attaching Suspension Cells to Slides Using a Cytocentrifuge Protocol]

Cell Fractionation of a Mixed Population of Cells - http://www.axis-shield.com/densityhome/optiprep/C14.pdf

Category: Cell Biology and Cell Culture

Cell fractionation techniques are presented for the design of gradient systems for separating one or more cell types from lavages of body cavities or from mechanically or enzymically-dissociated tissues. Includes: Preparation of cell suspension for gradient loading; Fractionation by buoyant density; Fractionation on the basis of cell size. - [Read Cell Fractionation of a Mixed Population of Cells]

Chromatin Immunoprecipitation (ChIP) of Protein Complexes Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4560

Category: Immunology: Immunoprecipitation Protocols: Chromatin Immunoprecipitation Protocols

Protocol describes the use of chromatin immunoprecipitation technology (ChIP) to analyze interactions of proteins or protein complexes with DNA in vivo. In this approach, the material is fixed with formaldehyde to preserve DNA-protein and protein-protein associations, the cells are lysed, and the chromatin is cut and solubilized. The chromatin suspension is immunoprecipitated with an antibody against the protein(s) of interest, and the coimmunoprecipitated DNA fragments are analyzed. - [Read Chromatin Immunoprecipitation (ChIP) of Protein Complexes Protocol]

Differentiation of Embryonic Stem (ES) Cells Using the Hanging Drop Method - http://www.cshprotocols.org/cgi/content/extract/2006/18/pdb.prot4485

Category: Stem Cell Protocols: Stem Cell Culture Protocols

In vitro differentiation of ES cells occurs when the cells are allowed to aggregate in suspension culture in the absence of mouse embryonic fibroblast (MEF) feeders and leukemia inhibitory factor (LIF). Hanging drops provide a uniform aggregate size, which is then expanded by continued growth in suspension culture. The embryoid bodies are then plated and allowed to differentiate further in culture. - [Read Differentiation of Embryonic Stem (ES) Cells Using the Hanging Drop Method]

Electron Microscopy Protocols - http://www.emlab.ubc.ca/p_em.htm

Category: Molecular Imaging: Electron Microscopy Protocols

Electron Microscopy Protocols ... Chemical Fixation Protocol for Suspension-Cultured Plant Cells for TEM; Standard Glutaraldehyde Fixation for TEM of Animal ...Electron Microscopy Protocols. UBC BioImaging Facility - Bio-Rad Radiance Plus Confocal Microscope - Starting Up, page 1 - [Read Electron Microscopy Protocols]

Enrichment of PBMCs with Monocytes Protocol - http://www.methods.info/Methods/Cell_biology/monocyte_enrichment.pdf

Category: Immunology: Mononuclear Cell Isolation Protocols

Protocol for enrichment of PBMCs with monocytes. The cell suspension obtained after this protocol contains 40-70% monocytes. This cell suspension is than used for positive or negative MACS separation. - [Read Enrichment of PBMCs with Monocytes Protocol]

Fixing Suspension Cells with Paraformaldehyde Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4295

Category: Cell Culture Protocols: Cell Fixing Protocols

Treating cells with paraformaldehyde leads to the establishment of chemical cross-links between free amino groups. When the cross-links join different molecules, a latticework of interactions occurs that holds the overall architecture of the cell together. - [Read Fixing Suspension Cells with Paraformaldehyde Protocol]

Fluorescence In Situ Hybridization of a Repetitive DNA Probe to Human Chromosomes in Suspension - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_104-107.pdf

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Protocol for fluorescence in situ hybridization of a repetitive DNA probe to human chromosomes in suspension. Hybridization technique which does not need formamide and dextran sulfate. As a model system, we used the repetitive specific human DNA probe pUC 1.77, labeled it with digoxigenin-11-dUTP by nick-translation, and hybridized it to metaphase chromosomes in suspension. These chromosomes were isolated by standard techniques from human lymphocytes. - [Read Fluorescence In Situ Hybridization of a Repetitive DNA Probe to Human Chromosomes in Suspension]

Fluorescence Protocol Immunocytochemistry - http://www.rndsystems.com/ihc_detail_objectname_FluorescenceProtocol20.aspx

Category: Immunology: Immunocytochemistry Protocols

Staining Cells in Suspension, Staining Cells Smeared on Slides. Immunofluorescent Technique. R & D Systems. - [Read Fluorescence Protocol Immunocytochemistry]

G1 Synchronization of CHO Cells by Isoleucine Deprivation Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4486

Category: Cell Biology and Cell Culture: Cell Cycle Protocols: G Phase Protocols

This protocol provides a method for the synchronization of a monolayer culture of CHO cells in G1 using isoleucine deprivation. Since CHO cells can also be adapted to grow in suspension culture, this procedure can be used to obtain larger quantities of cells. When isoleucine is replaced, the cells resume growth and begin to enter S phase ~4 hours later. This method arrests almost 100% of the CHO cells in G1, and upon reversal, leads to rapid recovery of cell growth and very high cell viability. - [Read G1 Synchronization of CHO Cells by Isoleucine Deprivation Protocol]

G1 Synchronization of CHO Cells by Isoleucine Deprivation Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4486

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Cycle Analysis Protocols

Immunofluorescence / confocal microscopy - http://depts.washington.edu/keck/susan.htm

Category: Molecular Imaging: Immunofluorescence Microscopy

B or T cells in suspension, adherent cells on chambered coverglass or chamberslides, cryostat sections of unfixed, OCT embedded tissue. Susan Anderson. - [Read Immunofluorescence / confocal microscopy]

Immunohistochemistry using Anti-Ganglioside Antibodies - http://www.glycotech.com/protocols/Proto2.html

Category: Molecular Biology Protocols: Carbohydrate Protocols

Indirect method measuring immunofluorescence coupled to second antibody. Best for membrane antigens in addition to intra- and extracellular antigens, may be applied to frozen tissue sections, to cells in suspension, and to cells attached to glass slides or coverslips. Tadashi Tai~Head, Department of Tumor Immunology, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan - [Read Immunohistochemistry using Anti-Ganglioside Antibodies]

Isolation of Rat Hepatocytes Protocol - http://embryo.ib.amwaw.edu.pl/invittox/prot/20.htm

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Liver Toxicity Assays Protocols

Collagenase perfusion of rat liver yields a hepatocyte suspension which may be exposed to test compounds in order to assess their effects on cell viability and enzyme leakage. - [Read Isolation of Rat Hepatocytes Protocol]

Leukostat Staining of Cytospin Preparations to Detect Apoptosis - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4491

Category: Cell Biology and Cell Culture: Apoptosis Protocols: Apoptosis Analysis

Leukostat Staining of Cytospin Preparations to Detect Apoptosis. Shailaja Kasibhatla et al. Leukostat staining is used to visualize nuclear changes and apoptotic body formation that are characteristic of apoptosis. Cells are viewed under a light microscope and counted to quantify apoptosis. This protocol can be used both for cells that grow in suspension and for adherent cells. - [Read Leukostat Staining of Cytospin Preparations to Detect Apoptosis]

Lipoplex and LPD Nanoparticles for In Vivo Gene Delivery Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/1/pdb.prot4448

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

Lipoplex (cationic liposome-DNA complex) is formed via electrostatic interaction of anionic nucleic acids with cationic liposomes. A thin film of lipids is dried on the bottom of a glass tube and rehydrated in an aqueous solution. The resulting liposome suspension is passed through polycarbonate filters of desired pore size. This protocol also describes the preparation, physical properties, and biological activity of liposome-polycation-DNA (LPD) nanoparticles. - [Read Lipoplex and LPD Nanoparticles for In Vivo Gene Delivery Protocol]

Measuring Volume Protocol - http://homepages.gac.edu/~cellab/chpts/chpt1/ex1-6.html

Category: Microscopy Protocols

Protocol for measuring volume. Material required includes: Microscope, Hemacytometer and coverslip, Suspension of yeast. - [Read Measuring Volume Protocol]

Mitotic Chromosome Preparations from Mammalian Suspension Cell Cultures Protocol - http://www.le.ac.uk/bl/phh4/promitotic.htm

Category: Cytogenetics Protocols

Protocol for mitotic chromosome preparations from mammalian suspension cell cultures. - [Read Mitotic Chromosome Preparations from Mammalian Suspension Cell Cultures Protocol]

Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/1/pdb.prot4449

Category: Transfection Protocols

Protocol describes transfection of plasmid DNA into mammalian cell lines using electroporation, a process whereby external application of electric pulses induce cell membrane permeability. Cells in suspension and small volume cells are difficult to transfect, whereas adherent cells and large volume cells are relatively easy. Regardless of cell size or phenotype, transfection efficiency increases with a high concentration of cells in a small volume. - [Read Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol]

Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/1/pdb.prot4449

Category: Transfection Protocols: Electroporation Sonoporation Protocols

Protocol describes transfection of plasmid DNA into mammaliancell lines using electroporation, a process whereby externalapplication of electric pulses induce cell membrane permeability.A number of factors can affect electrotransfection efficiency.In general, cells in suspension and small volume cells are difficultto transfect, whereas adherent cells and large volume cellsare relatively easy. - [Read Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol]

Preparation of cytospin from single cell suspension - http://icg.cpmc.columbia.edu/cattoretti/Protocol/Immunohistochemistry/CytospinPreparation.html

Category: Molecular Imaging: Immunofluorescence Microscopy

Preparation of cytospin from single cell suspension. Columbia University. Cattoretti Lab. - [Read Preparation of cytospin from single cell suspension]

Selection of Transfected Suspension Cells - http://www.musc.edu/BCMB/ceramide/protocols/0008.html

Category: Cell Biology and Cell Culture: Cell Transfection Protocol

hygromycin, neomycin selection of stable cells in suspension. Suprya Jayadev / Dr.Hannun Dr.Obeid Lab, BCMB, South Carolina. - [Read Selection of Transfected Suspension Cells]

Articles (1-4 of 4)

Tubulin Polymerization Protocol using GTP and GMPCPP

Tubulin is polymerized into microtubules by incubating tubulin at 37°C with GTP. A nucleation seed is added when the purpose is to assay microtubule elongation. Tubulin can also be polymerized for the purposes of recycling the tubulin or labeling the microtubules with fluorescently labeled tubulin. Based on the protocol by Timothy Mitchison of Harvard University.

Acid Guanidinium Thiocyanate RNA Isolation Phenol Chloroform Extraction

A single step RNA isolation protocol using Phenol Chloroform Extraction and Acid Guanidinium Thiocyanate. This RNA isolation method uses the fact that guanidinium thiocyanate can simultaneously lyse the cells and inactive cellular RNAses during the initial RNA isolation step allow a single step in the method.

In Vitro Translated Xenopus Mos Kinase Assay Protocol

In Vitro Translated Xenopus Mos Kinase Assay Protocol. In response to progesterone, immature Xenopus oocytes mature to eggs that can be fertilized. The Mos protein kinase is essential for oocyte maturation, most likely due to its ability to activate the MAP kinase cascade. This MAP kinase cascade eventually leads to the activation of Cdc2/cyclin B and entry into M phase. In this protocol, tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay.

Electrotransformation of BMH 81-17mut S for Isolating Site-Directed dsDNA Mutants

This protocol describes the electroporation of the BMH 81-17 mut S strain that is recommended for tranformation of the site directed mutagenesis of dsDNA (See Protocol on Site-Directed Mutagenesis on Double Stranded DNA). BMH 81-17 mut S are a mismatch repair defective (mut S) Escherichia coli strain. The probability that the two mutations will cosegregate during the first round of DNA replication is increased in this strain.

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