signaling Protocols

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: 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: 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]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Protocol uses specific antibodies coupled to one of four fluorochromes: fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), peridinin chlorophyll-a (PcP), and allophycocyanin (APC). These fluorochromes can be used simultaneously to stain and analyze the expression patterns of four different proteins in the same sample. The fluorochrome stained cell populations are analyzed using a FACSCalibur dual-laser flow cytometer. - [Read Characterization of Cells by Flow Cytometry Protocol]

Category: Immunology: B Cell Protocols

Fluorochromes can be used simultaneously to stain and analyze the expression patterns of four different proteins in the same sample. The fluorochrome stained cell populations are analyzed using a FACSCalibur dual-laser flow cytometer. - [Read Characterization of Cells by Flow Cytometry Protocol]

Category: Neuroscience Protocols: Neuronal Cell Culture Protocols: Rat Neuronal Cell Culture Protocols

Conditional ablation of stat3/socs3 discloses the dual role for reactive astrocytes after spinal cord injury. The current protocol demonstrates that reactive astrocytes play a crucial role in wound healing and functional recovery by using mice with a selective deletion of the signal transducer and activator of transcription-3 (STAT3) or suppression of cytokine signaling-3 (SOCS3) under the control of Nestin gene promoter/enhancer (STAT3N–/–, SOCS3N–/–). - [Read Conditional Ablation of Stat3 Socs3 Discloses the Dual Role for Reactive Astrocytes]

Category: Fluorescent Dyes Protocols

Cyanine dye reagents are useful as fluorescent labels for proteins. This protocol has been designed to label the thiol group on cysteine using Cy3 or Cy5 minimal maleimide labeling dyes. - [Read Cyanine Dye (Maleimide) Protein Labeling Protocol]

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

This procedure describes the isolation and culture of adult mouse cardiac myocytes from one heart. The isolation routinely yields approximately 1 million rodshaped myocytes per heart. - [Read Isolation of Adult Mouse Cardiac Myocytes from One Heart Protocol]

Category: Mouse Protocols: Mouse Cell Culture Protocols

Protocol describes the isolation and culture of adult mouse cardiac myocytes from one heart. The isolation routinely yields approximately 1 million rodshaped myocytes per heart. - [Read Isolation of Adult Mouse Cardiac Myocytes from One Heart Protocol]

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

This procedure describes the isolation and culture of adult mouse cardiac myocytes from two or more hearts. Includes modifications for the digestion of two or more hearts in the same procedure and subsequent pooling of myocytes derived from the multiple hearts. The isolation procedure is performed by one or more technicians and routinely yields approximately 1 million rod-shaped myocytes per heart. - [Read Isolation of Adult Mouse Cardiac Myocytes from Two or More Hearts Protocol]

Category: Immunology: B Cell Protocols

Describes the isolation of resting B lymphocytes (B cells) from mouse spleens by using negative selection with anti-CD43 and anti-Mac-1/CD11b monoclonal antibodies coupled to magnetic microbeads. This strategy depletes non-B cells from a mixed population of splenocytes and relies on the fact that most mature leukocytes, with the exception of resting splenic B cells, express CD43. - [Read Isolation of Resting B Lymphocytes from One or More Groups of Four Mouse Spleens Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: B Lymphocyte Isolation Protocols

Protocol describes the isolation of resting B lymphocytes (B cells) from mouse spleens by using negative selection with anti-CD43 and anti-Mac-1/CD11b monoclonal antibodies coupled to magnetic microbeads. This strategy depletes non-B cells from a mixed population of splenocytes and relies on the fact that most mature leukocytes, with the exception of resting splenic B cells, express CD43. - [Read Isolation of Resting B Lymphocytes from Sixteen Mouse Spleens Protocol]