data Protocols

Category: DNA Microarray Protocols

Hegde et al., Array Fabrication, microarray Printing, Probe Preparation and Hybridization, RNA Extraction, RNA labeling, Data Collection, Normalization, and Analysis.Institute for Genomic Research, Rockville, MD - [Read A Concise Guide to cArray Hybridization Procedures - DNA Microarray Analysis]

Category: DNA Microarray Protocols

Micro Array Fabrication, Probe Preparation and Hybridization, and Data Collection, Normalization and Analysis. Hegde, et al. biotechniques 2000. - [Read A Concise Guide to cDNA Microarray Analysis – II PDF.]

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

Advanced Methodologies in Flow Cytometry. Andrea Cossarizza et al.. Materials from a workshop held March, 1997 in Modena, Italy. - [Read Advanced Methodologies in Flow Cytometry]

Category: PCR Protocols: AFLP PCR

Restriction and Ligation reactions, Setting up the reactions, AFLP PCR reactions protocols. Analyzing the data using Genescan. Paul G. Wolf, Utah State Univ. - [Read AFLP protocol Wolf Lab]

Category: DNA Microarray Protocols: DNA Microarray Software

DNA Microarray Software. Data Analysis, Another Microarray Database AMAD, Cluster, Gal File Maker, ScanAlyze. DeRisi Lab. - [Read AMAD: Another Microarray Database.]

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

Provides several approaches to flow cytometry data analysis. Frequency determinations based on analysis of single-parameter fluorescence histograms and dual-parameter contour plots are presented. Steps are described for calculating values for signal-to-noise ratios when logarithmic amplification is used for data collection. - [Read Analysis of Flow Cytometry Data 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: RNAi Technology Protocols

Protocol describes the preparation of an shRNA-expressing Gateway entry vector.  This process is preceded by the design and synthesis of target gene-specific sense and antisense oligonucleotides which, when annealed, will constitute an shRNA cassette. - [Read Annealing, Cloning, and Sequencing of shRNA Linkers into pEN_H1 Plasmids Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Apoptosis Cytometry Protocols

Annexin V, belonging to a recently discovered family of proteins, the annexins, with anticoagulant properties has proven to be a useful tool in detecting apoptotic cells since it preferentially binds to negatively charged phospholipids like PS in the presence of Ca2+ and shows minimal binding to phosphatidylcholine and sphingomyeline. Changes in PS asymmetry, which is analyzed by measuring Annexin V binding to the cell membrane, were detected before morphological changes associated with... - [Read Annexin V Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Annexin V Staining Flow Cytometry Protocol  - a general protocol for staining cell for cytometry analysis. BD PharMingen. - [Read Annexin V Staining Flow Cytometry Protocol]

Category: Antibody Microarray Protocols

Antibody Array Production and Data Analysis. Probing Antigen Arrays and washing/ Blocking. Great Protocols and information. Brian Kidd. Stanford. - [Read Antibody Array Production and Analysis]

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

Protocol allows you to measure the content of cyclic adenosine 3',5'-monophosphate (cyclic AMP or cAMP) in splenic B lymphocytes (B cells) in an enzyme-linked immunoassay. This protocol utilizes acetylation of cAMP to improve sensitivity and reduce interference. Protocol includes information on: how to determine cAMP, calculations and reagents and materials. - [Read Assay of Cyclic AMP in Lysates of Cells]

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: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+]i, in cultured adherent RAW 264.7 cells, using a 96-well plate format. This objective is accomplished by using the Ca2+-sensitive fluorescent dye, fluo-3, which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. - [Read Assay of Intracellular Free Calcium in RAW 264.7 Cells]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Intracellular Ion Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium (Ca2+) in cultured RAW 264.7 cells. This objective is accomplished with the Ca2+-sensitive fluorescent dye, fluo-3, which permeates cells as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. Fluorescence is measured over time with adherent cells that have been washed free of extracellular dye. - [Read Assay of Intracellular Free Calcium in RAW 264.7 Cells for Ligand Screen Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Intracellular Ion Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+]i, in cultured adherent RAW 264.7 cells, using a 96- well plate format. This objective is accomplished by using the Ca2+-sensitive fluorescent dye, fluo-3, which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. Fluorescence for the adherent cells is measured over time by using a bottom read of a 96-well plate, with cells that have been washed. - [Read Assay of Intracellular Free Calcium in RAW 264.7 Cells Loaded with Fluo-3 Protocol]

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+], in cultured adherent RAW 264.7 cells, using a 96-well plate format. This objective is accomplished by using the Ca2+-sensitive fluorescent dye, fura-2, which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. - [Read Assay of Intracellular Free Calcium in RAW 264.7 Cells Loaded with Fura-2 (with FLEXstation)]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Intracellular Ion Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+]i , in cultured adherent RAW 264.7 cells, using a 96- well plate format. This objective is accomplished by using the Ca2+-sensitive fluorescent dye, fura-2, which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. - [Read Assay of Intracellular Free Calcium in RAW 264.7 Cells Loaded with Fura-2 Protocol]

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium (Ca2+) in mouse splenic B cells in the absence and presence of ligands for cell surface receptors. This objective is accomplished with the Ca2+-sensitive fluorescent dye, Fluo-3, which permeates cells as an ester and is hydrolyzed in the cell to its Ca2+ sensitive acidic form. - [Read Assay of Intracellular Free Calcium in Suspended B Cells]

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

The following protocol is a method to measure concentrations of free cytoplasmic calcium (Ca2+) in mouse splenic B cells in the presence of two ligands. Signalling Gateway - [Read Assay of Intracellular Free Calcium in Suspended B Cells Dual Ligand PDF]

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

Protocol for a method which assesses concentrations of free cytoplasmic calcium (Ca2+) in mouse splenic B cells in the absence and presence of ligands for cell surface receptors. Signalling Gateway - [Read Assay of Intracellular Free Calcium in Suspended B Cells PDF]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Intracellular Ion Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium (Ca2+) in mouse splenic B cells in the absence and presence of ligands for cell surface receptors. This objective is accomplished with the Ca2+-sensitive fluorescent dye, Fluo-3, which permeates cells as an ester and is hydrolyzed in the cell to its Ca2+- sensitive acidic form. - [Read Assay of Intracellular Free Calcium in Suspended B Cells Protocol]

Category: Immunology: B Cell Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium (Ca2+) in mouse splenic B cells in the absence and presence of ligands for cell surface receptors. This objective is accomplished with the Ca2+-sensitive fluorescent dye, Fluo-3, which permeates cells as an ester and is hydrolyzed in the cell to its Ca2+- sensitive acidic form. - [Read Assay of Intracellular Free Calcium in Suspended B Cells Protocol]