<ul><li> Kai Johnsson </li><li> Ivan Correa </li><li> Andreas Brecht </li><li> Katrin Müentener </li><li> Chris Noren </li><li> Luo dim. </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> Nicolas Labarthe </li><li> James MacFarland </li><li> John Buswell </li><li> Brenda Desmond </li><li> James Ellard </li><li> Peigne Don </li></ul>

Placage cellules CHO-K1: <ol><li> Ajouter 50 100 pi de cellules CHO-K1 qui ont été traitées à la trypsine selon des protocoles standard pour 6ml de complet F-12K. </li><li> Mélanger la suspension cellulaire par aspiration et refoulement plusieurs fois, puis aliquote de 300 ul de suspension cellulaire trypsinisées à chaque puits d&#39;une chambre de stériles 8-Lab-Tek II lamelle couvre-objet Chambré avec # 1.5 borosilicate lamelle chambres (Nalgene Nunc Int;. Produit # 155409; Lot: 100808-8-0). </li><li> Incuber les échantillons à 37 ° C, 5% CO 2 pendant la nuit. </li></ol> Transfection transitoire de cellules CHO-K1 avec pSNAP-ADRB2 et pSNAP-H2B: <ol><li> Vérifiez les chambres de culture de cellules ensemencées sur les jours précédents à chercher densité cellulaire et la santé. </li><li> Étiquetez le nombre approprié de tubes de microcentrifugeuse. </li><li> Dans une hotte à flux laminaire, mis en place des mélanges complexes de transfection selon le tableau de l&#39;échantillon suivant, en utilisant soit 0,3 ug pSNAP-ADBR2 l&#39;ADN plasmidique de contrôle ou 0,3 pg d&#39;ADN de contrôle pSNAP-H2B plasmidique, 1 pl de D2 Transpass et 1 ul de Transpass V par réaction. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> plasmide </td><td> nombre de réactions </td><td> pl d&#39;un milieu sans sérum </td><td> ul de D2 Transpass </td><td> pl d&#39;Transpass V </td><td> ul d&#39;ADN plasmidique </td><td> pl d&#39;D2/reaction Transpass </td><td> pl d&#39;Transpass V / réaction </td><td> ng d&#39;ADN / réaction </td><td> L&#39;ADN conc. (Ng / l) </td><td> ul d&#39;ADN plasmidique / réaction </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> Moquer </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> Non transfectées </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> Tout d&#39;abord, mélanger les sans sérum F-12K avec l&#39;ADN plasmidique appropriée, puis ajouter le réactif de transfection Transpass D2, puis ajouter le Réactif de transfection Transpass V. Mélanger les composants bien par aspiration et refoulement en douceur à plusieurs reprises après l&#39;addition de chaque réactif. </li><li> Incuber la réaction à température ambiante pendant 30 minutes. </li><li> Durant l&#39;incubation, laver les cellules une fois avec 600 pi de DMEM complet et ajouter 450 ul de DMEM complet à chaque échantillon. </li><li> Ajouter 50 ul du mélange de transfection complexe pour chaque échantillon, lentement et de manière goutte à goutte. </li><li> Incuber les échantillons nuit à 37 ° C, 5% de CO 2. </li></ol> SNAP-Cell Étiquetage Étoile TMR des cellules CHO-K1 transfectées transitoirement avec pSNAP-ADRB2 et pSNAP-H2B: <ol><li> Retirer délicatement le support de transfection complexes à partir de chaque échantillon par aspiration sous vide. Laver les cellules deux fois avec 600 pi de terminer F-12K et de remplacer les médias dans chaque puits avec 600 pi de terminer F-12K. </li><li> Mix média étiquetage colorant en ajoutant 995 ul de complet F-12K et 51 ul 0,6 mM SNAP-Cell TMR-Star substrat. La concentration finale de SNAP-Cell TMR-Star doit être de 3mm. Pipette de haut en bas plusieurs fois pour mélanger après l&#39;ajout de substrat. </li><li> Retirez délicatement les milieux de croissance à partir d&#39;échantillons et ajouter 200 ul de médias étiquetage colorant pour chaque puits. Incuber les échantillons à 37 ° C, 5% de CO 2 pendant 30 minutes. </li><li> Alors que les échantillons sont en incubation, préparer Hoechst 33342 solution pour la coloration nucléaire d&#39;échantillons en mélangeant 1 pl de Hoechst 33342, trichlorhydrate, trihydraté (10 mg / ml solution dans l&#39;eau, 16,2 mM solution; sondes moléculaires Invitrogen) dans 10 ml d&#39;achever F-12K . </li><li> Retirez le support de tous les échantillons et ajouter 600 ul de la solution diluée Hoechst pour chaque échantillon. Incuber les échantillons à 37 ° C, 5% de CO 2 pendant 5 minutes. </li><li> Lavez échantillons trois fois avec 600 pi de terminer F-12K. </li><li> Incuber les échantillons à 37 ° C, 5% de CO 2 pour 30 minutes supplémentaires pour permettre fluorophore non constituée en société à diffuser à l&#39;extérieur des cellules. </li><li> Après 30 minutes, le changement des médias sur les échantillons SNAP-Cell une dernière fois et procéder à l&#39;imagerie. </li><li> Image des cellules en utilisant un Zeiss Axiovert 200M Apotome microscope à fluorescence. </li></ol> Les résultats représentatifs <img src="/files/ftp_upload/1876/1876fig1.jpg" alt="Figure 1" /> Figure 1. Voiciquelques résultats représentatifs de l&#39;imagerie de fluorescence en utilisant la norme de la microscopie à fluorescence direct cellules COS-7 qui sont l&#39;expression SNAP-tag fusions. Cette première image montre direct cellules COS-7 exprimant pSNAP-H2B (nucléaire histones) marqué avec SNAP-Cell TMR-Star. La construction pSNAP-H2B a été généré en utilisant l&#39;pSNAP-tag (m) du vecteur. Les cellules ont été marquées avec SNAP-Cell TMR-Star pour 30 minutes et de contraste avec Hoechst 33342 (bleu) pour les noyaux. La fluorescence rose montre la superposition de la fluorescence rouge où protéine histone est présent dans plus de la fluorescence bleue indiquant le noyau. L&#39;expression de la protéine H2B est clairement et facilement identifiables. <img src="/files/ftp_upload/1876/1876fig2.jpg" alt="Figure 2" /> <img src="/files/ftp_upload/1876/1876fig3.jpg" alt="Figure 3" /> Les figures 2 et 3. Ces deux images suivantes montrent direct cellules COS-7 transfectées transitoirement avec pSNAP-ADRβ2. Les cellules ont été marquées avec SNAP-Cell TMR-Star (rouge) et de contraste avec Hoechst 33342 (bleu). La construction pSNAP-ADRβ2 a été généré en utilisant l&#39;pSNAP-tag (m) du vecteur. Les cellules ont été marquées avec SNAP-Cell TMR-Star pour 30 minutes et de contraste avec Hoechst 33342 (bleu) pour les noyaux. La fluorescence rouge indique la présence de protéines de surface cellulaire ADRB2 récepteurs tout en fluorescence bleue identifie le noyau.

<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>

Les auteurs sont employés par New England Biolabs qui produit les réactifs et les instruments utilisés dans cet article.

Il ya plusieurs variables qui affectent les résultats observés tels que l&#39;intensité du laser se concentrer, capacités de l&#39;équipement, l&#39;objectif utilisé, etc photostabilité du fluorophore détermine la vitesse de la fluorescence doit être capturé avant de s&#39;estomper. Cette méthode de marquage des protéines est polyvalent et convient pour de nombreuses applications. Technologies de SNAP-tag et CLIP-tag pour le marquage spécifique de protéines de fusion avec des sondes synthétiques permettent divers aspects de la fonction des protéines, y compris une variété de processus dynamiques dans les cellules vivantes et dans les lysats cellulaires. SNAP-CLIP-MCP-et ACP-tag technologies offrent une polyvalence extraordinaire simplicité et à l&#39;imagerie de protéines dans les cellules vivantes et fixes, et à l&#39;étude des protéines in vitro. La création d&#39;un seul gène de construire donne une protéine de fusion taggés capable de former une liaison covalente à une variété de groupes fonctionnels, y compris les fluorophores, biotine, ou des perles. La protéine d&#39;intérêt doit être cloné et exprimé qu&#39;une seule fois et ensuite la fusion peut être utilisé avec une variété de substrats fluorescents pour de nombreuses applications en aval tels que l&#39;étiquetage de protéines simultanément à l&#39;intérieur des cellules vivantes, la localisation des protéines et translocation, pulse-chase expériences, des études de l&#39;internalisation du récepteur , sélectives étiquetage surface de la cellule, les protéines déroulant tests, la détection de protéines en SDS-PAGE, la cytométrie en flux à haut débit dans les dosages de liaison des plaques de microtitration, des expériences d&#39;interaction biocapteur, et FRET basé dosages de liaison.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<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>
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fluorescent labeling of cos-7 expressing snap-tag fusion proteins for live cell imaging

SNAP-tag et CLIP-tag systèmes d&#39;étiquetage de protéines permettre au particulier, la fixation covalente de molécules, y compris les colorants fluorescents, à une protéine d&#39;intérêt dans les cellules vivantes. Ces systèmes offrent une large sélection de substrats fluorescents optimisé pour une gamme d&#39;instruments d&#39;imagerie. Une fois cloné et exprimé, la protéine marquée peut être utilisé avec une variété de substrats pour de nombreuses applications en aval sans avoir à cloner à nouveau. Il ya deux étapes à l&#39;aide de ce système: le clonage et l&#39;expression de la protéine d&#39;intérêt comme une fusion SNAP-tag, et l&#39;étiquetage de la fusion avec le substrat de SNAP-tag de choix. Le SNAP-tag est une petite protéine humaine basée sur O<sup&gt; 6</sup&gt;-Alkylguanine-ADN-alkyltransférase (hAGT), une protéine de réparation d&#39;ADN. SNAP-tag étiquettes sont des colorants conjugués à la guanine ou chloropyrimidine groupes partants via un linker benzylique. Dans la réaction de l&#39;étiquetage, le groupe benzyle substitué du substrat est fixée de manière covalente à l&#39;SNAP-tag. CLIP-tag est une version modifiée du SNAP-tag, conçu pour réagir avec benzylcytosine plutôt que dérivés benzylguanine. Lorsqu&#39;il est utilisé en conjonction avec SNAP-tag, CLIP-tag permet l&#39;étiquetage orthogonaux et complémentaires de deux protéines simultanément dans les mêmes cellules.

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-tag et CLIP-tag systèmes d&#39;étiquetage de protéines permettre au particulier, la fixation covalente de molécules, y compris les colorants fluorescents, à une protéine d&#39;intérêt dans les cellules vivantes. Une fois cloné et exprimé, la protéine marquée peut être utilisé avec une variété de substrats pour de nombreuses applications en aval sans avoir à cloner à nouveau.

Marquage fluorescent du COS-7 exprimant des protéines de fusion SNAP-tag pour imagerie de cellules vivantes

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

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Biology

22.9K vues.  New England Biolabs. SNAP-tag et CLIP-tag systèmes d'étiquetage de protéines permettre au particulier, la fixation covalente de molécules, y compris les colorants fluorescents, à une protéine d'intérêt dans les cellules vivantes. Une fois cloné et exprimé, la protéine marquée peut être utilisé avec une variété de substrats pour de nombreuses applications en aval sans avoir à cloner à nouveau.

Vidéo: 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.

Étiquetage des cellules souches avec des colorants fluorescents pour les non-invasive de détection avec l&#39;imagerie optique

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 ...

Recherche

Biologie

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).

Imagerie de cellules vivantes de la F-actine fluorescente Dynamics via chatoiement de microscopie (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.

Marquage fluorescent Drosophile</emOuvrages d&#39;art> Coeur

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.

La transplantation de blastomères exprimant la GFP pour l&#39;imagerie en direct sur le développement de la rétine et du cerveau dans les embryons de poissons zèbres chimériques

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.

Photoconversion de protéines purifiées fluorescentes et double-sonde optique dans Soulignant cellules vivantes

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.

Deux et trois dimensions imagerie de cellules vivantes de protéines endommagent l&#39;ADN de réponse

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.

Vivez l&#39;imagerie des protéines GFP-étiquetés<em&gt; Drosophila</em&gt; Les ovocytes

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.

Vivre imagerie de dédouanement cellulaire par apoptose au cours<em&gt; Drosophila</em&gt; Embryogenèse

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.

Microscopie intravitale pour l&#39;imagerie Subcellular ouvrages chez des souris vivantes exprimant des protéines 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.

Expression des protéines de Fusion Fluorescent en Murine dérivés de la moelle osseuse des cellules dendritiques et 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 ...