<ul><li> Kai Johnsson </li><li> Iván Correa </li><li> Andreas Brecht </li><li> Katrin Müentener </li><li> Chris Noren </li><li> Luo dom </li><li> Xu Ming </li><li> Theodore Davis </li><li> Salvatore Russello </li><li> Aihua Zhang </li><li> Inca Ghosh </li><li> Elissa Maunus </li><li> Nicholas Labarthe </li><li> James MacFarland </li><li> John Buswell </li><li> Brenda Desmond </li><li> James Ellard </li><li> Don Peine </li></ul>

Revestimiento de células CHO-K1: <ol><li> Agregar 50-100μl de células CHO-K1 que han sido trypsinized acuerdo con los protocolos estándar de 6 ml de completar F-12K. </li><li> Mezcle la suspensión de células pipeteando arriba y abajo varias veces, entonces alícuota de 300 l de suspensión celular con tripsina a cada pocillo de una solución estéril de 8 de cámara Lab-Tek II cubreobjetos cámaras con # 1.5 borosilicato cubreobjetos cámaras (Nalgene Nunc Int.;. Product # 155409; Lote: 100808-8-0). </li><li> Incubar las muestras a 37 ° C, 5% de CO 2 durante la noche. </li></ol> La transfección transitoria de células CHO-K1 con pSNAP-ADRB2 y H2B-pSNAP: <ol><li> Compruebe las cámaras de cultivo de células sembradas en el día anterior en busca de la densidad celular y la salud. </li><li> Etiqueta el número apropiado de tubos de microcentrífuga. </li><li> En una campana de flujo laminar, creado mezclas complejas de transfección de acuerdo a la tabla de ejemplo siguiente, utilizando 0,3 g pSNAP-ADBR2 ADN plásmido de control o de 0,3 mg de ADN de control pSNAP-H2B plásmido, 1 l de TransPass D2 y 1 l de TransPass V por reacción. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> plásmido </td><td> número de reacciones </td><td> l de medio libre de suero </td><td> l de D2 TransPass </td><td> l de TransPass V </td><td> l de ADN plásmido </td><td> l de D2/reaction TransPass </td><td> l de TransPass V / reacción </td><td> ng del ADN / reacción </td><td> ADN conc. (Ng / l) </td><td> l de ADN de plásmido / reacción </td></tr><tr><td> SNAP-ADRB2 </td><td> 5 </td><td> 236.5 </td><td> 5 </td><td> 5 </td><td> 3.5 </td><td> 1 </td><td> 1 </td><td> 300 </td><td> 472,34 </td><td> 0.7 </td></tr><tr><td> H2B-SNAP </td><td> 5 </td><td> 237 </td><td> 5 </td><td> 5 </td><td> 3 </td><td> 1 </td><td> 1 </td><td> 300 </td><td> 515,89 </td><td> 0.6 </td></tr><tr><td> Burlarse de </td><td> 5 </td><td> 240 </td><td> 5 </td><td> 5 </td><td> 0 </td><td> 1 </td><td> 1 </td><td> 0 </td><td> 0 </td><td> 0 </td></tr><tr><td> Untransfected </td><td> 5 </td><td> 250 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td><td> 0 </td></tr></tbody></table></li><li> Primero, mezclar el suero libre de F-12K con el ADN del plásmido apropiado, a continuación, añadir el reactivo de transfección TransPass D2, a continuación, añadir el reactivo de transfección TransPass V. Mezclar los componentes, así pipeteando arriba y hacia abajo con suavidad varias veces después de la adición de cada reactivo. </li><li> Incubar la reacción a temperatura ambiente durante 30 minutos. </li><li> Durante la incubación, lavar las células una vez con 600 l de DMEM completo y añadir 450 l de DMEM completo a cada muestra. </li><li> Añadir 50 l de la mezcla de transfección complejos para cada muestra, lentamente y de forma gota a gota. </li><li> Incubar las muestras de la noche a 37 ° C, 5% de CO 2. </li></ol> SNAP-celular TMR estrella Etiquetado de células CHO-K1 transfectadas transitoriamente con pSNAP-ADRB2 y H2B-pSNAP: <ol><li> Retire con cuidado los medios de comunicación complejos de transfección de cada muestra por medio de succión al vacío. Lavar las células dos veces con 600 l de completar F-12K y reemplazar los medios de comunicación en cada pozo con 600 l de completar F-12K. </li><li> Mix de medios tinte etiquetado mediante la adición de 995 l de completar F-12K y 51 l 0,6 mM SNAP-sustrato celular TMR-Star. La concentración final de SNAP-celular TMR-Star debe ser de 3 mm. Pipeta hacia arriba y abajo varias veces para mezclar después de la adición del substrato. </li><li> Retire con cuidado los medios de cultivo de las muestras y añadir 200 l de los medios de comunicación tinte de etiquetado a cada pocillo. Incubar las muestras a 37 ° C, 5% de CO 2 durante 30 minutos. </li><li> Mientras que las muestras se incuba, prepare la solución de Hoechst 33342 para la tinción nuclear de las muestras mediante la mezcla de 1 l de Hoechst 33342, trihidrocloruro, trihidrato (10 mg / ml de solución en agua, 16,2 mM solución; Invitrogen sondas moleculares) en 10 ml de completar F-12K . </li><li> Retire el papel de todas las muestras y añadir 600 l de la solución diluida de Hoechst para cada muestra. Incubar las muestras a 37 ° C, 5% de CO 2 durante 5 minutos. </li><li> Lávese las muestras tres veces con 600 l de completar F-12K. </li><li> Incubar las muestras a 37 ° C, 5% de CO 2 durante 30 minutos para permitir que fluoróforo no incorporadas a difundir fuera de las células. </li><li> Después de 30 minutos, cambiar los medios de comunicación en las muestras de células SNAP-una vez más y proceder a la imagen. </li><li> Imagen de las células utilizando un microscopio Zeiss Axiovert 200M ApoTome fluorescentes. </li></ol> Resultados representante <img src="/files/ftp_upload/1876/1876fig1.jpg" alt="Figura 1" /> Figura 1. A continuación sealgunos resultados representativos de las imágenes fluorescentes por microscopía fluorescente estándar de vida COS-7 en células que expresan etiqueta SNAP-fusiones. Esta primera imagen muestra en vivo COS-7 células que expresan pSNAP-H2B (nuclear histonas) marcado con SNAP-celular TMR-Star. La construcción pSNAP-H2B se ha generado mediante la pSNAP-tag (m) del vector. Las células se marcaron con SNAP-celular TMR-Star durante 30 minutos y contrastados con Hoechst 33342 (azul) de los núcleos. La fluorescencia de color rosa muestra la superposición de fluorescencia de color rojo donde la proteína histona está presente, además de la fluorescencia azul que indica el núcleo. La expresión de la proteína H-2B es clara y fácilmente identificables. <img src="/files/ftp_upload/1876/1876fig2.jpg" alt="Figura 2" /> <img src="/files/ftp_upload/1876/1876fig3.jpg" alt="Figura 3" /> Figuras 2 y 3. Estas dos imágenes siguientes show en vivo células COS-7 transfectadas transitoriamente con pSNAP-ADRβ2. Las células fueron marcadas con SNAP-celular TMR-Star (rojo) y de contraste con Hoechst 33342 (azul). La construcción pSNAP-ADRβ2 se ha generado mediante la pSNAP-tag (m) del vector. Las células se marcaron con SNAP-celular TMR-Star durante 30 minutos y contrastados con Hoechst 33342 (azul) de los núcleos. La fluorescencia de color rojo indica la presencia de ADRB2 de la superficie celular receptor de la proteína de fluorescencia azul, mientras que identifica el núcleo.

<ol>
	<li>Johnsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+biochemical+activities+in+living+cells.">Visualizing biochemical activities in living cells.</a> Nat Chem Biol. 5, 63-65 (2009).</li><li>Johnsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNAP-tag+Technologies:+Novel+Tools+to+Study+Protein+Function.">SNAP-tag Technologies: Novel Tools to Study Protein Function.</a> NEB Expressions. 3.3, 1-3 (2008).</li></ol>

Los autores son empleados de New England Biolabs que produce los reactivos y los instrumentos utilizados en este artículo.

Hay algunas variables que afectan los resultados observados, tales como la intensidad del láser se centran, las capacidades del equipo, la lente utilizada, etc fotoestabilidad del fluoróforo determina la rapidez con la fluorescencia debe ser capturado antes de desaparecer. Este método de etiquetado de proteínas es muy versátil y útil para muchas aplicaciones. Tecnologías de la etiqueta de SNAP-y CLIP etiquetas para el etiquetado específico de proteínas de fusión con sondas sintéticas permiten diversos aspectos de la función de las proteínas, incluyendo una variedad de procesos dinámicos en células vivas y en lisados ​​celulares. SNAP-, CLIP-, MCP y ACP-tag tecnologías proporcionan una versatilidad sencillez y extraordinaria a la imagen de las proteínas en células vivas y fijos y al estudio de las proteínas in vitro. La creación de una única construcción génica produce una proteína de fusión etiquetados capaz de formar un enlace covalente a una variedad de grupos funcionales, incluyendo fluoróforos, biotina, o de granos. La proteína de interés debe ser clonado y expresado una sola vez y después de la fusión se puede utilizar con una variedad de sustratos fluorescentes para numerosas aplicaciones posteriores, como el etiquetado de proteínas simultáneamente dentro de células vivas, localización de la proteína y el traslado, el pulso-caza experimentos, estudios de internalización del receptor , el etiquetado selectivo de la superficie celular, proteínas desplegable ensayos, la detección de proteínas en SDS-PAGE, citometría de flujo, de alto rendimiento de los ensayos de unión en placas de microtitulación, experimentos biosensor interacción, y FRET basado en ensayos de unión.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CHO-K1 Cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>CCL-61</td>
<td>&nbsp;</td>
<tr>
<td>Serum free F-12K</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Lab-Tek II Chambered Coverglass with #1.5 borosilicate coverglass chambers</td>
<td>&nbsp;</td>
<td>Nalgene Nunc Int.</td>
<td>155409</td>
<td>Lot: 100808-8-0</td>
</tr>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hoechst 33342, trihydrochloride, trihydrate (10 mg/ml solution in water, 16.2 mM solution)</td>
<td>&nbsp;</td>
<td>Invitrogen Molecular Probes</td>
<td>H3570</td>
<td>470519</td>
</tr>
<tr>
<td>Zeiss Apotome Axiovert 200M fluorescent microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Transpass V</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>M2561S</td>
<td>0030709</td>
</tr>
<tr>
<td>Transpass D2</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>M2554S</td>
<td>0060802</td>
</tr>
<tr>
<td>SNAP-Cell TMR Star</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>S9105S</td>
<td>0010811</td>
</tr>
<tr>
<td>pSNAP-ADBR2 Control Plasmid</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>N9178S</td>
<td>0010811</td>
</tr>
<tr>
<td>pSNAP-H2B Control Plasmid </td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>N9179S</td>
<td>0010811</td>
</tr>		</tbody>
		</table>
		</div>

fluorescent labeling of cos-7 expressing snap-tag fusion proteins for live cell imaging

SNAP-etiqueta y una etiqueta CLIP-los sistemas de etiquetado de proteínas permiten la conexión específico, covalente de moléculas, incluyendo tintes fluorescentes, con una proteína de interés en las células vivas. Estos sistemas ofrecen una amplia gama de sustratos fluorescentes optimizado para una amplia gama de instrumentos de imagen. Una vez clonado y expresado, la proteína marcada se puede utilizar con una variedad de sustratos para numerosas aplicaciones posteriores sin tener que clonar otra vez. Hay dos pasos para usar este sistema: la clonación y expresión de la proteína de interés como una fusión SNAP-etiqueta, y el etiquetado de la fusión con el sustrato SNAP-etiqueta de la elección. El SNAP-tag es una pequeña proteína humana sobre la base de O<sup&gt; 6</sup&gt;-Alkylguanine-DNA-alquiltransferasa (hAGT), una proteína de reparación del ADN. Etiqueta de SNAP-etiquetas son tintes conjugados con guanina o cloropirimidina grupos salientes a través de un conector de bencilo. En la reacción de marcaje, el grupo bencilo sustituido del sustrato se une covalentemente a la SNAP-etiqueta. CLIP-etiqueta es una versión modificada de la SNAP-tag, diseñado para reaccionar con benzylcytosine en lugar de derivados bencilguanina. Cuando se utiliza junto con SNAP-tag, CLIP-etiqueta permite el etiquetado ortogonales y complementaria de dos proteínas simultáneamente en las mismas celdas.

Watch this Scientific Journal Video about Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging at JoVE.com

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-etiqueta y una etiqueta CLIP-los sistemas de etiquetado de proteínas permiten la conexión específico, covalente de moléculas, incluyendo tintes fluorescentes, con una proteína de interés en las células vivas. Una vez clonado y expresado, la proteína marcada se puede utilizar con una variedad de sustratos para numerosas aplicaciones posteriores sin tener que clonar otra vez.

Marcaje fluorescente de COS-7 Expresando etiqueta SNAP-proteínas de fusión de imágenes de células vivas

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

fluorescent-labeling-cos-7-expressing-snap-tag-fusion-proteins-for

Research

JoVE Journal

Biology

22.9K vistas.  New England Biolabs. SNAP-etiqueta y una etiqueta CLIP-los sistemas de etiquetado de proteínas permiten la conexión específico, covalente de moléculas, incluyendo tintes fluorescentes, con una proteína de interés en las células vivas. Una vez clonado y expresado, la proteína marcada se puede utilizar con una variedad de sustratos para numerosas aplicaciones posteriores sin tener que clonar otra vez.

Video: Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Etiquetado de las células madre con tintes fluorescentes para la detección no invasiva con imágenes ópticas

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Investigación

Biología

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

Imágenes de células vivas de la F-actina Dynamics a través de Microscopía Fluorescente Speckle (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

Marcaje fluorescente de Drosophila Estructuras del Corazón

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

El trasplante de las buenas prácticas agrarias-que expresa Blastómeros de imágenes en vivo del desarrollo de la retina y el cerebro en embriones de pez cebra quiméricos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles.
Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion.
This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.

Fotoconversión purificada de proteínas fluorescentes y de doble sonda óptica destacar en células vivas

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

De dos y tres dimensiones imágenes de células vivas de las proteínas de respuesta al daño del ADN

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

Imágenes en directo de las proteínas GFP-etiquetados en<em&gt; Drosophila</em&gt; Los ovocitos

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Imágenes de Apoptotic Liquidación de células vivas durante<em&gt; Drosophila</em&gt; Embriogénesis

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Microscopia intravital para formar imágenes de estructuras subcelulares en ratones vivos que expresan proteínas fluorescentes

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Expresión de proteínas fluorescentes de fusión en murinas médula ósea células dendríticas y macrófagos

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...