Brad Chazotte

Labeling Mitochondria with MitoTracker Dyes

INTRODUCTION Membrane-potential-dependent dyes such as Rhodamine 123, tetramethylrhodamine methyl ester (TMRM), and tetramethylrhodamine ethyl ester (TMRE) are useful as long as the mitochondrion maintains its negative membrane potential. MitoTracker is a commercially available fluorescent dye (Invitrogen/Molecular Probes) that, like the aforementioned dyes, labels mitochondria within live cells utilizing the mitochondrial membrane potential. However, MitoTracker is chemically reactive, linking to thiol groups in the mitochondria. The dye becomes permanently bound to the mitochondria, and thus remains after the cell dies or is fixed. In addition, it can be used in experiments in which multiple labeling diminishes mitochondrial function. This protocol describes the labeling of mitochondria in live and fixed cells with MitoTracker dyes.

Labeling Nuclear DNA with Hoechst 33342

A number of fluorescent stains are available that label DNA and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells. One advantage of Hoechst 33342 is that it is membrane permeant and, thus, can stain live cells. Hoechst 33342 binds to adenine-thymine-rich regions of DNA in the minor groove. On binding to DNA, the fluorescence greatly increases. This protocol describes the use of Hoechst 33342 to label nuclear DNA of cells grown in culture.

Labeling Membrane Glycoproteins or Glycolipids with Fluorescent Wheat Germ Agglutinin

INTRODUCTION Glycoprotein and glycolipids are found as part of the outer leaflet of cellular plasma membranes. Those glycoproteins or glycolipids that contain sialic acid and N-acetylglucosamine residues can be labeled with wheat germ agglutinin (WGA), a plant lectin that exists as a dimer (mwt ~36,000) and is normally cationic. Fluorescently labeled WGA is commercially available with fluorescein, Alexa Fluor, or rhodamine moieties. Fluorescent WGA can also be used to stain the Golgi (the trans-Golgi) in fixed cells. Fluorescently labeled concanavalin A (Con A), which selectively binds to α-mannopyranosyl and α-glucopyranosyl residues, can also be used to label plasma membranes. This protocol describes the labeling of membrane glycoproteins or glycolipids with fluorescent WGA.

Labeling cytoskeletal F-actin with rhodamine phalloidin or fluorescein phalloidin for imaging.

The eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, in cell biology, the study of organization and structure/function relationships is of great importance. The cytoskeleton is composed of a series of filamentous structures, including intermediate filaments, actin filaments, and microtubules. Immunofluorescent staining has been most frequently used to study cytoskeletal components. However, it is also possible to fluorescently label isolated cytoskeletal proteins and either microinject them back into the cell or add them to fixed, permeabilized cells. Alternatively, it is possible to use the mushroom-derived fluorescinated toxins, phalloidin or phallacidin, to label F-actin of the cytoskeleton, as is described in this article. Phalloidin is available labeled with different fluorophores. The choice of the specific fluorophore should depend on whether phalloidin labeling for actin is part of a double-label experiment. In most cells, the abundance of actin filaments should provide a very strong signal. In double-label experiments, the fluorophore should be chosen to take this into account. In general, rhodamine labels are more resistant to photobleaching and can be subjected to the longer exposures required for finer structures.

Mounting Live Cells onto Microscope Slides

This protocol describes the mounting of cells which have been grown on cleaned coverslips in cell culture dishes containing the appropriate medium and supplements. Labeling is performed with the coverslips placed in culture dishes. After cells are labeled, the coverslips can be mounted onto microscope slides to be viewed for short-term observation of live cells or longer-term observation of fixed cells. For viewing live cells for longer periods of time, specialized chambers should be used in which suitable growth conditions can be maintained.

Labeling Mitochondria with Fluorescent Dyes for Imaging

Cold Spring Harb Protoc Brad Chazotte Labeling Mitochondria with Fluorescent Dyes for Imaging Service Email Alerting click here. Receive free email alerts when new articles cite this article Categories Subject Cold Spring Harbor Protocols. Browse articles on similar topics from (69 articles) Visualization of Organelles (456 articles) Visualization (226 articles) Live Cell Imaging (269 articles) Labeling for Imaging (222 articles) In Vivo Imaging (507 articles) Imaging/Microscopy, general (210 articles) Fluorescence, general (322 articles) Fluorescence (425 articles) Cell Imaging (238 articles) Cell Culture (983 articles) Cell Biology, general

Labeling Golgi with Fluorescent Ceramides

The Golgi may be considered a principal organizer of macromolecular traffic in the cell because many molecules, such as secreted proteins, glycoproteins, glycolipids, and plasma membrane glycoproteins, pass through the Golgi during their maturation. The fluorescent probes used to tag the Golgi make use of this function for labeling by using a fluorescent ceramide. In this protocol, two probes for labeling the Golgi are presented: a classic NBD-ceramide and a BODIPY-ceramide probe that is more resistant to photobleaching.

Labeling Membranes with Fluorescent Phosphatidylethanolamine

INTRODUCTION The phospholipid phosphatidylethanolamine (PE) can be conjugated via its head group to a number of fluorophores, including rhodamine, BODIPY (boron-dipyrromethene; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), and NBD (N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)). These probes can be used to label biological membranes and to study phospholipids within membranes. Rhodamine-PE, which does not readily exchange between lipid bilayers, has also been used in membrane fusion assays. Rhodamine-DHPE (lissamine rhodamine B 1,2-dihexyldecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) and NBD-PE have been used together in resonance energy transfer studies in membranes. This protocol describes the labeling of membranes with fluorescent PE.

Labeling the Plasma Membrane with TMA-DPH

INTRODUCTION TMA-DPH (trimethylamine-diphenylhexatriene) is a fluorescent membrane probe that has classically been used to label the outer leaflet of a membrane bilayer, to label the outer leaflet of the plasma membrane in cells, and to report on membrane dynamics using the techniques of fluorescence polarization and/or fluorescence lifetime. This probe has also been used to follow exocytosis and endocytosis of labeled plasma membranes. The interaction of the aqueous environment with mitochondrial inner membrane dynamics has also been studied following the fluorescence polarization and the lifetime of TMA-DPH. This protocol describes the use of TMA-DPH to label the plasma membrane.

Labeling lysosomes in live cells with fluorescent dyes for imaging.

INTRODUCTIONThe eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, in cell biology, the study of organization and structure/function relationships is of great importance. Lysosomes are membranous sacs--diverse in shape and size--containing more than 40 different acid hydrolases. The enzymes operate optimally at the acidic pH (~5) within the lysosome to break down various substances. It is thought that the highly glycosylated nature of the proteins of the Golgi membrane helps to protect them from degradation. A number of the fluorescent approaches to visualizing lysosomes make use of their acidic pH. Commonly used probes include neutral red, N-(3-[2,4-dinitrophenyl amino] propyl)-N-(3-aminopropyl)methylamine (DAMP), and acridine orange (a DNA stain). This protocol describes the labeling of lysosomes in live cells with neutral red.