living Protocols

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants. - [Read A Novel Fluorescent pH Probe for Expression in Plants]

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: Immunology: Immunoprecipitation Protocols: Chromatin Immunoprecipitation Protocols

Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. - [Read Chromatin Immunoprecipitation and Microarray-Based Analysis of Protein Location 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: Protein Protocols: Protein Phosphorylation Protocols

Fluorescent indicators for imaging protein phosphorylation in single living cells. Using Phocuses, genetically encoded fluorescent indicators, one can visualize signal transduction based on protein phosphorylation in living cells. Moritoshi Sato1, Takeaki Ozawa1, Kouichi Inukai2, Tomoichiro Asano2 & Yoshio Umezawa1. Nature Biotechnology - [Read Fluorescent indicators for imaging protein phosphorylation in single living 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: Microscopy Protocols

FM 4-64 is a lipophilic styryl dye and a vital stain: it fluoresces only in living cells, so cells cannot be fixed then stained nor stained then fixed. You must stain and observe living cells. FM 4-64 does not permeate cell membranes but, instead, intercalates into the plasma membrane is then taken into the cells by endocytosis. - [Read FM 4-64 Labeling of Yeast Vacuole Membranes Protocol]

Category: Reporter Gene Assays: GFP Assay Protocols

Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. These methods should be followed in the order presented, but any of them can be omitted when not needed. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]

Category: Microinjection Protocols

Fluorescence microscopy provides a powerful tool for imaging molecular components in living cells. Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Using confocal laser-scanning microscope & GFP fusion proteins in time-lapse imaging to visualize the behavior of organelles and to track membrane-bound transport intermediates that bud off from organelles. Practical issues related to construction & expression of GFP fusion proteins are discussed. Essential for optimizing the brightness and expression levels of GFP fusion proteins so that intracellular membrane-bound structures containing these fusion proteins can be readily visualized. - [Read Imaging of Organelle Membrane Systems and Membrane Traffic in Living Cells]

Category: Histology Protocols: Laser Capture and Microdissection

Laser Capture Microdissection of Living in vitro Cells. This PDF describes a precise, rapid and convenient Laser Capture Microdissection (LCM) method for the positive selection of living adherent cells and the successful subsequent re-cultivation of homogenous sub-populations. Arcturus. - [Read Laser Capture Microdissection of Living in vitro Cells PDF]

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: 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: Microscopy Protocols: Live-Cell Imaging Protocols

This protocol describes a method for observing and measuring the movement of RNA molecules in the nucleus of living mammalian cells. Caged fluorescein-labeled DNA oligonucleotides are introduced into living mammalian cells, where they demonstrably hybridize to complementary RNA. After site-specific photoactivation at desired sites within the cell, the RNA movements away from those sites are followed and digitally recorded using a rapid acquisition microscopy system. - [Read Photoactivation-Based Labeling and In Vivo Tracking of RNA Molecules in the Nucleus]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Protocol describes a split luciferase complementation assay that can be used to repetitively and noninvasively study the interaction of proteins in small living animals. After the expression of the appropriate vectors has been checked in cell culture in vivo, studies can be performed either by implanting transiently transfected cells for short-term analysis (maximum of 7 days), or with tumor models grown from tumor cells stably expressing the complete reporter system. - [Read Split Luciferase Complementation Assay for Studying Interaction of Proteins X and Y in Living Mice]

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

Protocol describes methods for maintaining healthy, dividing mammalian cells in culture and during imaging, when mitosis can be examined. Rose chambers are preferable for observation and microinjection of living mitotic cells, but slide/coverslip preparations are easy to make and do not require any special equipment. - [Read Studying Mitosis in Cultured Mammalian Cells Protocol]

Category: Cytogenetics Protocols

Protocol describes methods for maintaining healthy, dividing mammalian cells in culture and during imaging, when mitosis can be examined. Rose chambers are preferable for observation and microinjection of living mitotic cells, but slide/coverslip preparations are easy to make and do not require any special equipment. Another inexpensive and easy-to-use alternative is to grow cells in a culture dish with a glass bottom. Such dishes are suitable for microinjection experiments. - [Read Studying Mitosis in Cultured Mammalian Cells Protocol]

Category: Cell Biology and Cell Culture: Cell Cycle Protocols: Mitosis Protocols

Protocol describes methods for maintaining healthy, dividing mammalian cells in culture and during imaging, when mitosis can be examined. Rose chambers are preferable for observation and microinjection of living mitotic cells, but slide/coverslip preparations are easy to make and do not require any special equipment. Another inexpensive and easy-to-use alternative is to grow cells in a culture dish with a glass bottom. Such dishes are suitable for microinjection experiments. - [Read Studying Mitosis in Cultured Mammalian Cells Prtocol]

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

GUS is used as a tag to address nuclear localization whereas GFP is more versatile. GFP is detectable directly in living cells, GUS is only detected indirectly by staining of fixed tissue which may lead to artifacts or may obscure problems with protein solubility. In this protocol, protein localization is routinely assayed after particle-mediated transient transformation of onion epidermal cells. With this method it can be determined rapidly whether a given fusion protein is active and.... - [Read Subcellular Localization of GUS- and GFP-Tagged Proteins in Onion Epidermal Cells]

Category: Microscopy Protocols: In Vivo Imaging Protocols

Sophisticated fluorescence microscopy methods & equipment, now allow cellular events to be studied at high resolution in living material. The studying of living fly tissues presents unique difficulties in keeping the cells alive, introducing fluorescent probes, & imaging through thick hazy cytoplasm. This protocol outlines the preparation of major tissue types amenable to study by time-lapse cinematography and different methods for keeping them alive. - [Read Time-Lapse Cinematography in Living Drosophila Tissues: Preparation of Material]

Category: Drosophila Protocols

Protocol outlines the preparation of major tissue types amenable to study by time-lapse cinematography and different methods for keeping them alive. - [Read Time-Lapse Cinematography in Living Drosophila Tissues: Preparation of Material Protocol]