studies Protocols

Category: BACs Bacterial Artificial Chromosome Protocols

Protocol presents the amplification of insert end sequences from bacterial artificial chromosome clones using TAIL-PCR. The amplified products are suitable as probes for chromosome walking and genome mapping and as templates for direct sequencing. The protocol has been used in rice genome studies. - [Read Amplification of Insert End Sequences from BACs Clones by Thermal Asymmetric Interlaced PCR]

Category: Protein Protocols: Protein Trafficking

Protocol is based on methods for the resolution of GLUT4 containing vesicles and the identification of phosphoinositide kinase containing vesicles in 3T3-L1 adipocytes. They may have a wider application to any low-medium density membranes. Protocol incorporates the strategy of using a low density microsome fraction as the gradient input, commonly used in GLUT 4 studies that may have a wider application to other investigations. - [Read Analysis of Membrane Trafficking and Intracellular Signaling in Self-Generated Iodixanol Gradients]

Category: Protein Protocols: Protein Electrophoresis

An excellent guide on the analysis of proteins on SDS-PAGE gels, through staining with coomassie blue dye and western blot analysis. Analysis of Protein Gels (SDS-PAGE). David R. Caprette, Rice University. - [Read Analysis of Protein Gels (SDS-PAGE)]

Category: Bacterial Cell Culture Protocols: Bacterial Stocks Protocols

4 strains of E. coli are used in these studies: JM101 for M13 infection and isolation, XL1BMRF'for M13 or pUC-based DNA transformation, and ED8767 for cosmid DNA transformation. To maintain their respective F' episomes necessary for M13 viral infection, JM101 is streaked onto a M9 minimal media plate and XL1BMRF' is streaked onto an LB plate containing tetracycline. ED8767 is streaked onto an LB plate. These plates are incubated at 37degC overnight. For each strain, 3 ml. of appropriate liquid.. - [Read Bacterial Cell Maintenance Protocol]

Category: Cell Culture Protocols: Sterile Technique Protocols

Cultured mammalian cells are used extensively in cell biology studies; it requires a number of special skills in order to be able to preserve the structure, function, behavior and biology of the cells. This unit describes the basic skills required to maintain and preserve cell cultures: aseptic technique, medium characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. - [Read Basic Techniques for Mammalian Cell Tissue Culture Protocol]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

Cultured mammalian cells are used extensively in cell biology studies; it requires a number of special skills in order to be able to preserve the structure, function, behavior and biology of the cells. This unit describes the basic skills required to maintain and preserve cell cultures: aseptic technique, medium characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. - [Read Basic Techniques for Mammalian Cell Tissue Culture Protocol]

Category: Fungi Protocols: Neurospora Protocols

Protocol for cryofixation and freeze substitution for ultrastructural studies of Neurospora crassa hyphae. - [Read Cryofixation and Freeze Substitution for Ultrastructural Studies of Neurospora crassa hyphae]

Category: Cell Culture Protocols

Protocol used to study secretion of proteins and prostaglandins by endometrium from the cow, ewe, mare, bitch and other species. The technique is also useful for culture of peri-implantation conceptuses and placental tissues for metabolic labelling studies and to obtain conceptus secretory proteins for biological studies.The medium used is called Pig MEM, which is a modified minimum essential medium supplemented with non-essential amino acids, vitamins, insulin and additional glucose. - [Read Culture of Endometrial Explants and Peri-implantation Conceptuses to Monitor Synthesis and Secretion]

Category: Cell Culture Protocols: Insect Cell Culture Protocols

The use of Ferbach flasks is an ideal method to amplify the cells and virus inoculum for the bioreactor. The method is also very practical for producing sufficient amounts of various proteins for feasibility studies using HyQ© SFX-Insect™ medium. The large surface area in this vessel enables sufficient aeration and, therefore, the cells, when they reach high density, are not oxygen depleted. - [Read Culturing of Insect Cells and Production of Baculovirus Expressed Recombinant Proteins in 2.8 Liter]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

When choosing a particular molecule for photoactivation studies, it is necessary to have some structural knowledge of the molecule in order to design an appropriately caged species that will retain its biological inactivity until uncaging is effected. Includes synthesis of caged peptides or proteins. - [Read Design, Synthesis, and Characterization of Caged Compounds]

Category: Microinjection Protocols: Microinjection of Proteins Protocols

When choosing a particular molecule for photoactivation studies, it is necessary to have some structural knowledge of the molecule in order to design an appropriately caged species that will retain its biological inactivity until uncaging is effected. - [Read Design, Synthesis, and Characterization of Caged Compounds Protocol]

Category: Mouse Protocols: Mouse Genetics Protocols

Protocol describes a method for electroporating DNA into ES cells, as well as selection methods. Pilot studies should be performed to optimize the conditions for each DNA construct. The selection method described here is one of the most complex. It involves targeting constructs in which the bacterial neomycin-resistance gene disrupts the coding sequence of the mouse gene. - [Read Electroporating DNA into Embryonic Stem (ES) Cells and Selection Methods Protocol]

Category: Cell Culture Protocols

Glass is an excellent substrate for most tissue-culture-adapted cells and is compatible with all fixing and staining solutions. Glass coverslips in tissue-culture dishes or in 24-well multiwell plates are suitable carriers, as are multiwell slides. For high-resolution studies, choose glass coverslips of the highest available grade; #1 or #1.5 coverslips are the appropriate thickness. - [Read Growing Adherent Cells on Coverslips or Multiwell Slides Protocol]

Category: Immunology: T Cell Protocols

Protocol for the Stimulation of human peripheral blood mononuclear cells with anti-human CD3 monoclonal antibody; MTT assay for detection of cellular proliferation. Human PBMCs can be activated in vitro by soluble anti-human CD3 antibodies. Performed titration studies with these antibodies and established the following protocol for stimulation of PBMC. - [Read Human T Cell Activation Protocol]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

This protocol describes the use of SEMV cells (a cell line derived from ram seminal vesicles) in studies into prostaglandin H synthase-mediated metabolism of xenobiotics in intact cells. - [Read In Vitro Model For Studies Of Prostagland H Synthase (PHS)- Mediated Genotoxicity Of Xenobiotics]

Category: Immunology: T Cell Protocols

Describes methods for labeling high or low numbers of lymphocytes with CSFE. Protocols are provided to use CSFE-labeled cells in cell transfer studies or as cells to be cultured in vitro. Detailed guidelines for positioning of CSFE-labeled lymphocytes in lymphoid organs or other tissues are included for those wishing to use this approach to study lymphocyte migration. - [Read Intracellular Fluorescent Dye CFSE to Monitor Lymphocyte Migration and Proliferation]

Category: Protein Protocols: Protein Electrophoresis

A great introduction to SDS-PAGE. David R. Caprette, Rice University. - [Read Introduction to SDS-PAGE]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols

Protocol describes the use of SEMV cells (a cell line derived from ram seminal vesicles) in studies into prostaglandin H synthase-mediated metabolism of xenobiotics in intact cells. - [Read Invitro Model for Prostoglandin H Synthase (PHS) Mediated Genotoxicity of Xenobiotics]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols

Mammalian toxicology for acute toxicity studies.  Includes: M101 Acute Limit Test (Rodents), M102 Acute Toxicity Study (Rats or Mice), M120 Dose Escalation Study (Dog), M103 Acute Dermal Limit Test, M104 Acute Dermal (LD50), Study M105 Primary Dermal Irritation, M106 Primary Eye Irritation Study, M106-A, M110 Local Lymph Node Assay in Mouse, M115 Rabbit Vaginal Irritation Assay. - [Read Mammalian Toxicology Acute Toxicity Studies]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols

Mammalian toxicology repeat dose toxicity studies. Includes: M204 14-Day Oral Toxicity Study (Rodents), M204-H, M204-RH, M205 14-Day Oral Toxicity Study (Dogs), M205-H, M205-RH, M201 28-Day Oral Toxicity Study, M201-R, M202 28-Day Oral Toxicity Study, M202-R, M211 90-Day Oral Toxicity Study (Rodent), M211-20, M212 90-Day Oral Toxicity Study (Dog), M215 90-Day Dermal Toxicity Study, M213 Two-Year Chronic Toxicity/Carcinogenicity Study, M214 6-Month Carcinogenicity Study in p53+/Transgenic Mice. - [Read Mammalian Toxicology Repeat-Dose Toxicity Studies]

Category: Neuroscience Protocols

Methods for structure function studies of neurotrophic factors. - [Read Methods for Structure Function Studies of Neurotrophic Factors]

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

Human tissues are comprised of multiple interacting cell populations in a complex three dimensional arrangement with each cellular phenotype determined by a unique profile of mRNA and protein expression. Before microdissection techniques were developed, the only analysis tools for phenotypic studies were primarily immunohistochemistry and in-situ hybridization. While useful, these tools are limited to single gene analysis and, in general, do not allow qualitative studies. - [Read Microdissection Overview]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols

Molecular and genetic toxicology studies for in vitro cytogenetics with CHO or human lymphocytes. Includes: G401 Chromosomal Aberrations in Chinese Hamster Ovary (CHO) Cells, G401-2, G401-R, G401-R2, G401-R2H, G402 Chromosomal Aberrations in Human Lymphocytes, G402-2, G402-R, G402-R2, G402-R2H, G403 Sister Chromatid Exchanges in Chinese Hamster Ovary (CHO) Cells. - [Read Molecular and Genetic Toxicology Studies for In Vitro Cytogenetics with CHO or human lymphocytes]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols

Molecular and genetic toxicology studies for in vivo cytogenetics. Includes: G501 Chromosomal Aberrations in Rodent Bone Marrow Cells, G502 Single-Exposure Bone Marrow Cytotoxicity Assay. - [Read Molecular and Genetic Toxicology Studies for In Vivo Cytogenetics]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols

Molecular and genetic toxicology studies on gene mutation in mammalian cells In vitro. Includes: G201 Mouse Lymphoma L5178Y/tk+/Cell Gene Mutation Assay; G201-R; G202 Mouse Lymphoma L5178Y/tk+/Cell Gene Mutation Screening Assay; G203 Chinese Hamster Ovary or V79 Cell Mutation Assay at the hprt Locus; G203R. - [Read Molecular and Genetic Toxicology Studies on Gene Mutation in Mammalian Cells In Vitro]