data Protocols

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent and bright field images of live cells expressing yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope when three planes along the z-axis of the cell are acquired. Protocol includes: Description of Microscope and Imaging Setup; Description of Acquisition Parameters; Image Processing. - [Read Live Cell Spinning Disk Confocal Fluorescence Imaging of Cells- YFP & Bright Field—Three Z Axis]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent and bright field images of live cells expressing yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope. Protocol includes: Description of Microscope and Imaging Setup; Description of Acquisition Parameters; Image Processing. - [Read Live Cell Spinning Disk Confocal Fluorescence Imaging of Cells- YFP and Bright Field Images]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent images of live cells expressing yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope. Protocol includes: Description of Microscope and Imaging Setup; Description of Acquisition Parameters; Movie Processing. - [Read Live Cell Spinning Disk Confocal Fluorescence Imaging of Cells- YFP Time Series for Markers]

Category: Immunology: B Cell Protocols

This assay is used to measure cell viability. It is a two-color fluorescence assay that simultaneously determines: Live cell number and Dead cell number. - [Read Live Dead Assay for Cell Viability Protocol]

Category: Microscopy Protocols: In Vivo Imaging Protocols

Live nematodes can be studied at all levels of microscopic resolution. Data collection includes information on: Photographic Records, Video Microscopy,Multiple-Focal-Plane Time-Lapse Recording Systems and Digital Imaging. - [Read Live Imaging of Caenorhabditis elegans: Observation of Nematodes and Data Collection]

Category: C. elegans Protocols

At low magnification, dissection microscopes are useful, whereas at higher magnification and resolution, a wide range of optical methods can be used, including differential interference contrast (DIC) optics, phase contrast, fluorescence, polarized light, confocal, and dark field. - [Read Live Imaging of Caenorhabditis elegans: Observation of Nematodes and Data Collection Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+]i, for cultured adherent RAW 264.7 cells in an 8-well coverglass. This objective is accomplished using the Ca2+-sensitive fluorescent dye, fura-2 acetoxymethyl (AM), 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 with cells that have been washed free of extracellular dye. - [Read Live Single-Cell Fura-2 Measurements to Determine the Intracellular Free Calcium]

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+], for cultured adherent RAW 264.7 cells in an 8-well coverglass. This objective is accomplished using the Ca2+-sensitive fluorescent dye, fura-2 acetoxymethyl (AM), which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. - [Read Live Single-Cell Fura-2 Measurements to Determine the 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+]i, for cultured adherent RAW 264.7 cells in an 8-well coverglass. This objective is accomplished using the Ca2+-sensitive fluorescent dye, fura-2 acetoxymethyl (AM), which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. - [Read Live Single-Cell Fura-2 Measurements to Determine the Intracellular Free Calcium Protocol]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

Assay measures cell viability. It is a two-color fluorescence assay that simultaneously determines Live cell number and Dead cell number. This protocol is designed for use with the GEMINI XS Microplate Spectrofluorometer, a multi-well plate scanner with dual excitation/emission capabilities, but the assay is also adaptable for flow cytometry and fluorescence microscopy. Includes: Cell Culture; Preparation for the Assay; Live/Dead Assay; Reading the Plate; Data Analysis; Alternative protocol. - [Read Live/Dead Assay for Cell Viability Protoco]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Luciferase assay using a 24 well plate. Includes: Cell Lysis; Reagent Preparation; Luminescence measurement; Wash injector; Protein quantitation; Data Analysis. - [Read Luciferase Assay 24 Well Plate (Promega System)]

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol is the first step in a three-step process for the preparation and enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC) for the identification of the phosphopeptides by LC-MS/MS.  This procedure is used to prepare protein extracts from WEHI-231 cells.  This preparation method provides total cellular protein samples that are free of contaminating nucleic acids. - [Read Lysis and Protein Extraction from WEHI-231 Cells with TriPure Isolation Reagent Protocol]

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

This protocol has been established to maintain the efficient use of the AutoMACS cell sorter during periods of heavy use (e.g., three preparations of splenocytes from 16 spleens per week). Includes: Running the SAFE Clean Program; Clearing Debris. - [Read Maintenance of the AutoMACS Cell Sorter Protocol]

Category: Microarray Protocols: Microarray Hybridization Protocols

Protocol details the preparation of biotin-labeled target samples and hybridization of these samples to an Affymetrix in situ synthesized oligonucleotide GeneChip array.  The procedure requires a minimum of 5 µg of purified total RNA as starting material. - [Read Microarray Protocol for Affymetrix In Situ Synthesized Oligo Arrays]

Category: Microarray Protocols: Amino-allyl Microarray Methods

Protocol details the preparation of fluorescently labeled target samples (aminoallyl method) and hybridization of these samples to a microarray of Agilent inkjet-deposited presynthesized oligonucleotides. The procedure requires a minimum of 3 µg of purified total RNA as starting material. - [Read Microarray Protocol for Agilent Inkjet-Deposited Presynthesized]

Category: Microarray Protocols: Microarray DNA Labeling Protocols

Protocol details the preparation of fluorescently labeled target samples and hybridization of these samples to a microarray of Agilent inkjet-deposited presynthesized oligonucleotides. The procedure requires a minimum of 3 µg of purified total RNA as starting material. - [Read Microarray Protocol for Agilent Inkjet-Deposited Presynthesized Oligo Array]

Category: Microarray Protocols: Microarray Hybridization Protocols

Protocol details the preparation of fluorescently labeled target samples and hybridization of these samples to a microarray of Agilent inkjet-deposited presynthesized oligonucleotides.  The procedure requires a minimum of 3 µg of purified total RNA as starting material. Includes: cDNA Synthesis; Fluorescent cRNA Synthesis; cRNA Precipitation and Cleanup; cRNA Quantification; Hybridization; Washing. - [Read Microarray Protocol for Agilent Inkjet-Deposited Presynthesized Oligo Arrays]

Category: Mass Spectrometry Protocols

Millipore's ZipTip Protocol. Intended for purifying and concentrating femtomoles to picomoles of protein, peptide or oligonucleotide samples prior to analysis, providing better data quality. Millipore. - [Read Millipore's ZipTip Protocol]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

The MTT Cell Proliferation Assay measures the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. The number of assay steps has been minimized as much as possible to expedite sample processing. The MTT Reagent yields low background absorbance values in the absence of cells. Includes: Determining optimal cell counts, performing an assay, data interpretations and troubleshooting. - [Read MTT Cell Proliferation Assay Protocol]

Category: Pharmacology and Toxicology Protocols: Pharmacokinetics Protocols

Non-linear Regression Programs for the Analysis of Pharmacokinetic and Pharmacodynamic Data - [Read Non-linear Regression Programs for the Analysis of Pharmacokinetic and Pharmacodynamic Data]

Category: Cell Culture Protocols: Cell Lines Protocols

RAW 264.7 cells are a macrophage-like, Abelson leukemia virus transformed cell line derived from BALB/c mice. For routine maintenance in culture (passage), cells are seeded at a confluence of approximately 10% (1 x 106 and 3 x 106 cells in 100-mm and 150-mm plates, respectively) and grown to a confluence of approximately 80%. This procedure requires the cells to be split every two days. - [Read Passage Procedure for RAW 264.7 Cells]

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol is specifically for the further enrichment of phosphopeptides from a phosphotyrosine pull-down.  This is the final step for the preparation and enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC) for the identification of the phosphopeptides by liquid chromatography tandem mass spectrometry (LC-MS/MS). - [Read Preparation and Enrichment of Phosphopeptides from Phosphotyrosine Protocol]

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol is the third step in a three-step process for the preparation and enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC) for the identification of the phosphopeptides by LCMS/ MS. - [Read Preparation and Enrichment of Phosphopeptides Using IMAC and LC-MS/MS]

Category: Signalling Signal Transduction Protocols

This protocol provides a sufficient sample for several determinations of cAMP using the acetylation protocol. The method chosen for measuring the content of cyclic adenosine 3',5'-monophosphate (cyclic AMP or cAMP) in splenic B lymphocytes (B cells) is an enzyme linked immunoassay system. Protocol includes information on: Treatment of Cells and Preparation of Extracts; Reagents and Materials. - [Read Preparation of B-Lymphocyte Lysates for Cyclic AMP Determination]

Category: Signalling Signal Transduction Protocols

Protocol provides a method of achieving a sufficient sample for several determinations of cAMP using Amersham’s acetylation protocol. Protocol includes information on: Treatment of Cells and Preparation of Extracts; Reagents and Materials. - [Read Preparation of B-Lymphocyte Lysates for Cyclic AMP Determination for Dual Ligand Screen]