level Protocols

Category: Transfection Protocols: Stable Transfection Protocols

The AfCS is utilizing antisense technology to manipulate signaling protein expression in the RAW 264.7 macrophage-like cell line. This can be achieved by the transfection of gene-specific antisense oligonucleotides (ASOs). The following procedure involves the transfection of ASOs into RAW 264.7 cells using FuGENE 6 transfection reagent. Subsequently, the isolated total RNA or protein from these transfected cells can be used to assess the level of mRNA or protein knockdown, respectively. - [Read Antisense Oligonucleotide Transfection of RAW 264.7 Cells with FuGENE 6 in a 24-Well Dish]

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]

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]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Protocol for estimating the number of CD38 molecules on the CD8+ T lymphocytes of HIV-infected individuals. Includes: RECOMMENDATION OF VENDOR FOR PE-CD38 AND PE-CD4; VALIDATION OF LOGARITHMIC AMPLIFIER LINEARITY AND SENSITIVITY; CONSERVATION OF THE LEVEL OF CD4 ANTIGEN EXPRESSION ON CD4+ LYMPHOCYTES AND ITS USE AS A BIOLOGIC STANDARD FOR FLOW CYTOMETER INSTRUMENT CHARACTERIZATION; DETERMINATION OF THE NUMBER OF CD38 MOLECULES PER CD8+ CELL; etc.. - [Read Estimating the Number of CD38 Molecules on the CD8+ T Lymphocytes of HIV-Infected Individuals]

Category: Yeast Protocols: Yeast Genetics Protocols

Protocol describes mutagenesis of yeast with ethyl methane sulfonate (EMS). It causes approximately 40-70% cell death in most haploid laboratory strains, a level of cell killing that is commonly used in mutant hunts with haploid strains. - [Read Ethyl Methane Sulfonate (EMS) Mutagenesis Protocol]

Category: Cytoskeleton Protocols: Actin Myosin Protocols

The same GFP-tagged actin construct used in cell transfection experiments has been used to produce transgenic mice. Transgenic animals allow the imaging of brain tissue in the intact animal, as acutely cut slices or as organotypic slice cultures. They also serve as a source of cells for imaging neurons at high resolution in dispersed low-density cell culture. In contrast to cells transfected in culture, where the level of actin-GFP expression in neurons varies considerably, transgenic mice... - [Read Imaging Actin in Tissue Slices from Transgenic Mouse Brain Protocol]

Category: Immunohistochemistry Protocols

Describes two methods for using the immunoperoxidase reaction to localize antigens at the electron microscope level; one for adherent cultured cells and one for tissue sections. The reaction conditions are first optimized at the light microscope level and then adapted for EM level observation. These methods allow for reliable detection of antigens at the cell surface, within the cell, and especially in membrane bounded organelles. - [Read Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues]

Category: Immunology: Immunofluorescence Protocols

The study of the activation of DNA damage response factors at the single cell level relies on the observation of their accumulation (or the accumulation of their activated forms) in nuclear foci by plain immunofluorescence techniques. - [Read Indirect Immunofluorescence with Preextraction (In Situ Cell Fractionation) Protocol]

Category: Signalling Signal Transduction Protocols: Cytokine Protocols

Intracellular Cytokine Staining Protocol. Analysis of surface molecules and intracellular cytokines at the single-cell level using FACS or flow cytometry. - [Read Intracellular Staining for Flow Cytometric Analysis Protocols]

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

During incubation period and the course of disease, SARS virus replicates and releases from cells. The level of virus varies greatly from sample to sample. At early stage of incubation period or during the late convalescent phase, the concentration of virus is very low in all kinds of samples. Virus is also detected at extremely low concentrations in plasma during... - [Read Kit for Rapid Enrichment of SARS Virus]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

GFP serves as a molecular marker that can be imaged dynamically in living cells, both in its native form & as a fusion to other proteins. For GFP imaging, plants present the challenge of autofluorescence from chlorophyll, lignified cell walls, vacuolar contents, and other cell materials, all of which can obscure the GFP signal. Maximizing the signal-to-noise ratio is a major concern, and careful consideration should be given to the choice of tissue imaged, GFP expression level, etc. - [Read Live-Cell Imaging of GFP in Plants]

Category: PCR Protocols

Protocol illustrates the rules of successful long PCR: No more than 1 ng of template DNA is used per microliter of PCR in a 100-µl reaction; approximately 0.1 µl of KlentaqLA (not plain Taq) is used per kilobase of target (for targets >10 kb, 1-1.3 µl of enzyme should be used); the Mg++ concentration is considered as the excess over the level of dNTPs. - [Read Long and Accurate PCR Protocol]

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: Cytoskeleton Protocols: Microtubules Protocols

Negative staining is a rapid, qualitative method for analyzing microtubule structure at the EM level. Because negative staining involves deposition of heavy atom stains, structural artifacts such as flattening of the cylindrical microtubule and opening up of microtubules into flat sheets are common. Negative staining is very useful because of its ease, rapidity and lack of requirement for specialized equipment other than that found in a regular EM facility. - [Read Negative Stain Electron Microscopy of Microtubules Protocol]

Category: Chromatography Protocols: Affinity Chromatography Protocols

Protein complexes can be isolated by several different approaches.  For example, a protein can be tagged with an epitope such as Flag or TAP and then overexpressed in a target cell, allowing the interacting proteins to be purified.  Similarly, epitope tags can be homologously recombined into the endogenous locus ("knocked-in"), allowing protein complexes containing the tagged proteins to be isolated at their natural expression level. - [Read Overview of Affinity Purification in Combination with Mass Spectrometry Protocol]

Category: Signalling Signal Transduction Protocols

CHIESA et al 2001. J Biochem. Calcium-sensitive photoprotein aequorin used to analyse Ca2+ homoeostasis at the subcellular level. GFP chimaeras for studying the recruitment of protein kinase C isoforms and the activity of intracellular proteases. - [Read Recombinant aequorin and green fluorescent protein as valuable tools in the study of cell signalling]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Contamination of plasmid DNA by fragments of DNA and RNA is reduced to an acceptable level by centrifugation through 1 M sodium chloride.  This method was devised by Brian Seed when he was a graduate student at Harvard University. - [Read Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Centrifugation]

Category: Transgenic Mouse Protocols: Mouse Genotyping Protocols

PCR screens must be designed to detect transgene DNA at the single copy level.Copy standards are prepared by mixing non-transgenic tail DNA with a known amount of transgene DNA to produce transgene copy standards. University of Michigan Transgenic Animal - [Read reparation of Copy Standards for Southern Blot Copy Number Determination]