live Protocols

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

Simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and proteolytic treatment to permeabilize cells. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells for subsequent culturing based on DNA-content differences, is also described. Also presented are methods for staining of cell nuclei isolated from paraffin-embedded tissues, and deconvolution of DNA-content-frequency... - [Read Analysis of Cellular DNA Content by Flow Cytometry Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Manual measurement and manipulation of the cell surface requires access to the cells, usually in an open chamber. Temperature-controlled chambers or stage inserts are preferred for maintaining physiological activity during the experiment. For example, heated culture dishes with coverslip glass bottoms (Bioptechs) permit high-resolution fluorescence microscopy of living cells during force application. - [Read Chambers for Examination of Live Cells under Mechanical Stress Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Specimen chambers have had many designs published over the years describing systems that offer excellent optical properties while allowing specimens to be maintained for varying amounts of time. Ranging in complexity from the simple preparation of a sealed coverslip on a microscope slide to sophisticated perfusion chambers that enable tight control of virtually all environmental variables culture chambers are designed to to allow living specimens to be observed with minimal invasion at high res. - [Read Culture Chambers for Live-Cell Imaging]

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: Microscopy Protocols: Electron Microscopy Protocols

Article describe the preparation of cells for correlative electron microscopy after live light microscopic observation of fluorescently labeled cytoskeletal proteins microinjected into the same cells. Since identification of cytoskeletal elements in electron microscopic preparations is an essential part of any correlative study, procedures for immunogold labeling of cytoskeletal components and for myosin S1 decoration of actin filaments are also described. - [Read Electron Microscopy of the Cytoskeleton of Cultured Cells]

Category: Microscopy Protocols: Fluorescence Microscopy Protocols

Most biological specimens are relatively transparent, so details of internal and intracellular morphology are difficult to image in untreated living specimens using simple bright-field techniques. Fluorescence microscopy offers greater advantages and possibilities for increasing contrast and determining the specific localization of molecules in cells. Article outlines the three methods most commonly used to introduce an appropriate label into Drosophila tissue without perturbing the process. - [Read Fluorescent Reagents for Live Cell Imaging and Their Introduction into Cells]

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

The procedures involve the isolation and growth of primary cell cultures from rodent and human tissue as well as the use of viral vectors for the introduction and expression of mammalian genes in cells in culture and in live rodents. - [Read Growth of Primary Cell Culture and Viral Vector Handling Protocols]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Live-cell imaging techniques provide critical insight into the fundamental nature of cellular & tissue function, especially due to the rapid advances that are currently being witnessed in fluorescent protein & synthetic fluorophore technology. Because of these advances, live-cell imaging has become a requisite analytical tool in most cell biology labs. Includes: Maintaining Live Cells on the Microscope Stage; Live-Cell Imaging Culture Chambers; Optical System and Detector Requirements etc. - [Read Introduction to Live-Cell Imaging Techniques]

Category: Mouse Protocols: Mouse Cell Culture Protocols

The procedures involve the isolation and growth of primary cell cultures from rodent and human tissue as well as the use of viral vectors for the introduction and expression of mammalian genes in cells in culture and in live rodents. - [Read Isolation and Growth of Primary Cell Cultures from Mouse Protocol]

Category: Bacterial Protocols

Protocol was developed to isolate Wolbachia from adult Drosophila, but it can be adapted for other insects. In some insects leg removal prior to isolation facilitates hemolymph extrusion. - [Read Isolation of Live Bacteria from Adult Insects Protocol]

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

Protocol for live cell prep for sorting. - [Read Live Cell Prep for Sorting Protocol]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent and bright field images of live cells, expressing cyan fluorescent protein(CFP) and/or yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope. This procedure is used to help determine if N- or Cterminal tagging of signaling molecules alters the steady state localization pattern of the signaling protein in question. - [Read Live Cell Spinning Disk Confocal Fluore Imaging of Cells- Colocalization of Fluorescent Protein Tags]

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: C. elegans Protocols

To image early cleavages and chromatin dynamics, it is convenient to use histone H2B fused to GFP or lamin::GFP. Time-lapse movies can be obtained using conventional confocal microscope systems and their included software. Early embryos dissected from transgenic hermaphrodites are placed with egg salts on agar pads. - [Read Live Imaging of Caenorhabditis elegans: Examples]

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: In Vivo Imaging Protocols

Caenorhabditis elegans, a small (adults are ~1 mm long), free-living soil nematode that feeds on bacteria, is an ideal organism for applying various live microscopy techniques. This protocol describes useful techniques for preparing C. elegans for live microscopic analysis. Details of sample preparation depend on the developmental stage of the worm to be studied. - [Read Live Imaging of Caenorhabditis elegans: Preparation of Samples]

Category: C. elegans Protocols

Protocol describes useful techniques for preparing C. elegans for live microscopic analysis. Details of sample preparation depend on the developmental stage of the worm to be studied. - [Read Live Imaging of Caenorhabditis elegans: Preparation of Samples]

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: 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]