potential Protocols

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cytotoxicity Assays in Cell Culture Protocols

Protocol descibes the use of L929 mouse fibroblast cells cultured in vitro in an agarose overlay assay to assess the toxicity of test substances.  The assay may be useful in assessing the irritation potential of test substances (e.g. surfactant-based products) as an alternative to the Draize rabbit eye test. - [Read Agarose Overlay Assay Protocol]

Category: Cell Organelle Protocols: Mitochondria Protocols

The technique of JC-1 staining has been developed with the intent to detect DY in intact, viable cells. For this purpose JC-1 acts as a marker of mitochondrial activity, since the formation of J-aggregates, which give red emission, is reversible. Cells with high DY are those forming J-aggregates, thus showing high red fluorescence. On the other hand, cells with low DY are those in which JC-1 maintains (or re-acquire) monomeric form, thus showing only green fluorescence. - [Read Analysis of Mitochondrial Membrane Potential with the Sensitive Fluorescent Probe JC-1]

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

BN-PAGE has become the method of choice for the investigation of the respiratory protein complexes of the electron transfer chains of a range of organisms. It allows the separation in two dimensions of extremely hydrophobic protein sets for analysis and also provides information on their native interactions. In this review we discuss the capabilities of BN-PAGE in proteomics and the wider investigation of protein:protein interactions with a focus on its use and potential in plant science. - [Read Blue-Native PAGE in Plants: A Tool in Analysis of Protein-Protein Interactions]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cytotoxicity Assays in Cell Culture Protocols

The potential cytotoxicity of compounds under hypoxic conditions is determined by exposing cell cultures to test compounds in a low oxygen atmosphere.  Subsequent cell survival is determined by the MTT and methylene blue colorimetric assays. - [Read Colorimetric Cytotoxcity Assays for Anchorage Dependent Cells Protocol]

Category: Cell Organelle Protocols: Mitochondria Protocols

Protocol for the detection of changes in mitochondrial membrane potential on the FACSCalibur. - [Read Detection of Changes in Mitochondrial Membrane Potential on the FACSCalibur]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

Cell-based assays are important tools for contemporary biology and drug discovery because of their predictive potential for in vivo applications.This assay gives ratiometric, inversely proportional values of viability and cytotoxicity (Figure 4.15) that are useful for normalizing data to cell number. Also, this reagent is compatible with additional fluorescent and luminescent chemistries. - [Read Determining Number of Live and Dead Cells in Cell Population: Cytotoxicity Assay Protocol]

Category: ELISA Protocols

The cyclooxygenase (COX) reaction can be monitored by measurement of oxygen consumption, peroxidase co-substrate oxidation or prostaglandin (PG) detection. This protocol describes a procedure measuring cyclooxygenase activity by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors. - [Read ELISA Method to Measure Inhibition of the COX Enzymes Protocol]

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture: Embryotoxic Potential Testing of Chemicals

EMBRYONIC STEM CELL TEST (EST). The embryotoxic potential of chemicals is determined by the evaluation of the inhibition of differentiation of embryonic stem cells (ES) and the inhibition of growth of ES and 3T3 cells. Scientific Information Service - [Read EMBRYONIC STEM CELL TEST (EST)]

Category: Cell Organelle Protocols: Mitochondria Protocols

Protocol describes a method for evaluation of mitochondrial function using the fluorochrome CMXRos. CMXRos is sequestered by actively respiring mitochondria, but washed out when the mitochondrial membrane potential is lost. This analysis can be combined with the TUNEL technique or immunocytochemistry. - [Read Flow Cytometric Analysis of Mitochondrial Transmembrane Potential ({Delta}{Psi}m)]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

Acidocalcisomes, the dense acidic calcium-storing organelles, which were originally identified in Trypanosoma cruzi, have no parallels in mammalian cells. They thus represent a unique functional characteristic, not shared by the host and hence offer an important potential target for chemotherapy of Chagas disease. - [Read Fractionation of Acidocalcisomes and Other Organelles from Trypanosoma, Leishmania, Chlamydomonas]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cell Specific Cytotoxicity Assays Protocols

Accumulation of lipophilic substances in the plasma membrane may affect the membrane lipid order and consequently affect the function of these proteins.  Changes in the activity of the Na+/K+ -ATPase, which is the major active transport system responsible for the electrochemical potential in mammalian cells, can therefore be an indication of the effect that a chemical may have on the viability of the cell membrane and possibly the whole cell. - [Read Hamster Ovary Cell NA+/K+ -ATPase Test]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cell Specific Cytotoxicity Assays Protocols

Potential embryotoxicity is assessed by monitoring the effect of the test compound on total protein synthesis, and DNA synthesis in cultured human foetal lung fibroblasts.  Rat lung epithelial cells can be used to determine cytotoxicity of select compounds because of their ability to metabolise xenobiotics. - [Read Lung Cell Assay Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Protocol for membrane potential analysis by flow cytometry. Includes: Preparation of the dyes; Loading of the dyes; Flow Cytometric Analysis. - [Read Membrane Potential Analysis by Flow Cytometry Protoocol]

Category: Immunology

A method for ameliorating autoimmune disease by passive transfer of IVIg-primed leukocytes. Developed a method for ‘priming’ specific leukocyte populations with IVIg to study their potential role in ameliorating ITP upon passive transfer, which we describe herein. Using this technique, found that splenic CD11c+ dendritic cells reacted with IVIg, or several IVIg mimetic regimes, were primed to ameliorate ITP upon transfer to thrombocytopenic mice. - [Read Method for Ameliorating Autoimmune Disease by Passive Transfer of IVIg-Primed Leukocytes]

Category: DNA Microarray Protocols: DNA Methylation Microarray CpG

Kimura et al., NAR 2005. "easy-handling technology provided reproducible and precise measurement of methylated CpGs in MGMT promoter and, thus, our method may bring about a potential evolution in the handling of a variety of high-throughput DNA methylatio - [Read Methylation profiles of genes utilizing newly developed CpG island methylation microarray on colorec]

Category: Cell Organelle Protocols: Mitochondria Protocols

Protocol describes a method for the evaluation of mitochondrial function using the fluorochrome CMXRos. CMXRos is sequestered by actively respiring mitochondria, but washed out when the mitochondrial membrane potential is lost. This analysis can be combined with the TUNEL technique or immunocytochemistry. - [Read Microscopic Analysis of Mitochondrial Transmembrane Potential Protocol]

Category: Cell Organelle Protocols: Mitochondria Protocols

Kit for detecting the mitochondrial membrane potential. - [Read Mitochondrial Membrane Potential Detection Kit]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Flow Cytometry Immunofluorescence Staining Protocols

Provides a brief historical perspective to illustrate the demands for this technology and to lay the foundation for its application; Explains the hurdles that were surmounted to achieve the current level of multiparametric analysis which serves to alert researchers to potential problems they may encounter when they either bring this technology to their own laboratories, or when they use extant instrumentation in another laboratory; Illustrates some of the complexities that arise. - [Read Multiparameter FACS Analysis]

Category: Peptide Protocols: Dissolving Handling Peptides

Peptide Handling Guides. Storage Guidelines for Lyophilized Peptides, Strategy for Dissolving Single Peptides, Determining Solubility Characteristics of peptides, Dissolving Approach for Charged Peptides, Dissolving Approach for Hydrophobic/Uncharged Peptides, Guidelines for Dissolving Several Peptides, Peptide Stability and Potential Degradation Pathways, and storage of peptides. Sigma Aldrich. PDF. - [Read Peptide Handling Guides]

Category: Signalling Signal Transduction Protocols

Detection of phosphorylated tyrosine residues can be performed using anti-P-TYR Ab and Western Analysis.Includes 2nd method,which uses phosphotyrosine in conjunction with anti-P-TYR Ab to "unlabel" potential proteins.By comparing Westerns developed with the 1st method(reveals phosphorylated protein) and the 2nd method(reveals non-specific labeling), a more accurate picture of those proteins phosphorylated on tyrosine can be seen. Includes: Protein Preparation, Electrophoresis and Transfer. - [Read Protocol for Antiphosphotyrosine Western Blot Analysis]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cell Specific Cytotoxicity Assays Protocols

An in vitro red blood cell assay is presented which allows the estimation of the irritation potential of tensides and tenside containing materials such as shampoos, shower gels, cleaning products, etc. The estimation is based on the fact that surfactants interact strongly with cellular membranes and proteins.  Both effects are measured photometrically by use of the inherent native dye, oxyhemoglobin. - [Read Red Blood Cell Test System Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cytotoxicity Assays in Cell Culture Protocols

Rabbit-derived corneal cells are cultured in the presence of test compounds, the toxicity of which are determined by their effect upon cell viability.  A decrease in cell number, as measured by uptake of the dye Neutral Red, serves as an indicator of potential cytotoxicity.  This test has been proposed as a potential replacement alternative for the Draize Eye Irritation test. - [Read SIRC Cytotoxcitiy Test]

Category: Mouse Protocols: Mouse Surgical Procedures Protocols

Protocol describes a method for transplanting tissue under the kidney capsule. The in vivo developmental potential of transplanted adult or embryonic tissues can then be determined. - [Read Transplantation of Tissues Under the Kidney Capsule Protocol]