<ul><li> Kai Johnsson </li><li> Ivan Correa </li><li> Andreas Brecht </li><li> Katrin Müentener </li><li> Chris Noren </li><li> Luo so </li><li> Ming Xu </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 Comb </li></ul>

Plating CHO-K1-Zellen: <ol><li> Add 50-100 &amp;mgr; von CHO-K1-Zellen, die nach Standardprotokollen auf 6ml komplette F-12K wurden trypsiniert. </li><li> Mix Zellsuspension durch Auf-und Abpipettieren mehrmals, dann aliquoten 300 pl trypsiniert Zellsuspension in jede Vertiefung einer sterilen 8-Kammer-Lab-Tek II Chambered Deckglas mit # 1.5 Borosilikat Deckglas Kammern (Nalgene Nunc Int;. Product # 155409; Lot: 100808-8-0). </li><li> Inkubieren Proben bei 37 ° C, 5% CO 2 über Nacht. </li></ol> Transiente Transfektion von CHO-K1-Zellen mit pSNAP-ADRB2 und pSNAP-H2B: <ol><li> Überprüfen Sie die Zellkulturkammern am Vortag ausgesät, um für die Zelldichte und Gesundheit suchen. </li><li> Beschriften Sie die entsprechende Anzahl von Mikrozentrifugenröhrchen. </li><li> In einer Sterilbank, bis Transfektion komplexe Gemische nach dem folgenden Beispiel-Tabelle setzen, entweder mit 0,3 ug pSNAP-ADBR2 Kontroll-Plasmid-DNA oder 0,3 pg pSNAP-H2B Kontroll-Plasmid-DNA, 1 ul Transpass D2 und 1 ul Transpass V pro Reaktion. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Plasmid </td><td> Anzahl der Reaktionen </td><td> ul Serum-freiem Medium </td><td> ul Transpass D2 </td><td> ul Transpass V </td><td> ul von Plasmid-DNA </td><td> ul Transpass D2/reaction </td><td> ul Transpass V / Reaktion </td><td> ng des DNA / Reaktion </td><td> DNA konz. (Ng / ul) </td><td> ul von Plasmid-DNA / Reaktion </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> Verspotten </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> Transfizierte </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> Zunächst mischen Sie die Serum-freien F-12K mit dem entsprechenden Plasmid-DNA, fügen Sie dann die Transpass D2 Transfektionsreagenz, fügen Sie dann die Transpass V Transfektionsreagenz. Mischen Sie die Komponenten gut durch Auf-und Abpipettieren vorsichtig mehrmals nach der Zugabe der einzelnen Reagenzien. </li><li> Inkubieren Sie die Reaktion bei Raumtemperatur für 30 Minuten. </li><li> Während der Inkubation waschen Sie die Zellen einmal mit 600 ul von kompletten DMEM und fügen 450 ul DMEM komplett zu jeder Probe. </li><li> Add 50 ul der Transfektion komplexen Mischung zu jeder Probe, langsam und in einem Drop-weise Mode. </li><li> Inkubieren Sie die Proben über Nacht bei 37 ° C, 5% CO 2. </li></ol> SNAP-Cell TMR Star-Kennzeichnung von CHO-K1 Zellen transient mit pSNAP-ADRB2 und pSNAP-H2B Transfizierte: <ol><li> Entfernen Sie vorsichtig die Transfektion komplexen Medien von jeder Probe mittels Vakuum abgesaugt. Wash Zellen zweimal mit 600 ul der kompletten F-12K und ersetzen Medien in jede Vertiefung mit 600 ul der kompletten F-12K. </li><li> Mix Farbstoffmarkierung Medien, indem 995 ul der kompletten F-12K und 51 ul 0,6 mM SNAP-Cell TMR-Star Substrat. Die Endkonzentration von SNAP-Cell TMR-Star sollte 3mm. Pipette nach oben und unten mehrere Male, um nach der Zugabe von Substrat-Mix. </li><li> Entfernen Sie vorsichtig das Wachstum Medien von Proben und 200 ul der Farbstoff Kennzeichnung Medien in jede Vertiefung. Inkubieren Proben bei 37 ° C, 5% CO 2 für 30 Minuten. </li><li> Während Proben inkubiert werden, bereiten Hoechst 33342-Lösung für die Kernfärbung der Proben durch Mischen von 1 ul der Hoechst 33342, Tri-, Trihydrat (10 mg / ml Lösung in Wasser, 16,2 mM Lösung; Invitrogen Molecular Probes) in 10 ml komplettem F-12K . </li><li> Entfernen Sie die Medien aus aller Proben und fügen 600 ul der verdünnten Hoechst-Lösung zu jeder Probe. Inkubieren Proben bei 37 ° C, 5% CO 2 für 5 Minuten. </li><li> Wash Proben dreimal mit 600 ul der kompletten F-12K. </li><li> Inkubieren Sie die Proben bei 37 ° C, 5% CO 2 für zusätzliche 30 Minuten, damit Personengesellschaften Fluorophor zu diffundieren aus den Zellen heraus. </li><li> Nach 30 Minuten wechseln die Medien über die SNAP-Cell-Proben ein letztes Mal, und fahren Sie Bildgebung. </li><li> Bild der Zellen unter Verwendung eines Zeiss Axiovert 200M Apotome Fluoreszenzmikroskop. </li></ol> Repräsentative Ergebnisse <img src="/files/ftp_upload/1876/1876fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Hier sindeinige repräsentative Ergebnisse der Fluoreszenz-Imaging mit Standard-Fluoreszenz-Mikroskopie von lebenden COS-7 Zellen, die zum Ausdruck SNAP-tag Fusionen werden. Das erste Bild zeigt Live-COS-7-Zellen, die pSNAP-H2B (nuclear Histon) mit SNAP-Cell TMR-Star bezeichnet. Die pSNAP-H2B-Konstrukt wurde mit Hilfe des pSNAP-tag (m)-Vektor. Die Zellen wurden mit SNAP-Cell TMR-Star für 30 Minuten beschriftet und mit Hoechst 33342 (blau) für Kerne. Die rosa Fluoreszenz zeigt die Überlagerung der roten Fluoreszenz, wo Histon-Protein vorhanden ist zusätzlich zu den blaue Fluoreszenz zeigt den Kern. Der Ausdruck der H2B Protein ist klar und leicht zu identifizieren. <img src="/files/ftp_upload/1876/1876fig2.jpg" alt="Abbildung 2" /> <img src="/files/ftp_upload/1876/1876fig3.jpg" alt="Abbildung 3" /> Abbildungen 2 und 3. Die nächsten beiden Bilder zeigen Live-COS-7 Zellen transient mit pSNAP-ADRβ2 transfiziert. Die Zellen wurden mit SNAP-Cell TMR-Star (rot) markiert und mit Hoechst 33342 (blau). Die pSNAP-ADRβ2 Konstrukt wurde mit Hilfe des pSNAP-tag (m)-Vektor. Die Zellen wurden mit SNAP-Cell TMR-Star für 30 Minuten beschriftet und mit Hoechst 33342 (blau) für Kerne. Die rote Fluoreszenz zeigt die Anwesenheit von Zelloberflächen-Rezeptor-Protein ADRB2 während blaue Fluoreszenz identifiziert den Kern.

<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>

Die Autoren werden von New England Biolabs, dass die Reagenzien und Instrumente in diesem Artikel verwendet produziert beschäftigt.

Es gibt mehrere Variablen, die die beobachteten Ergebnisse wie Fokus, Laserintensität, Geräte-Funktionen, verwendeten Objektiv, etc. Die Photostabilität des Fluorophors bestimmt, wie schnell die Fluoreszenz müssen, bevor sie verblassen erfasst werden. Diese Methode der Protein-Kennzeichnung ist vielseitig und für viele Anwendungen geeignet. SNAP-tag und CLIP-tag-Technologien für die spezifische Markierung von Fusionsproteinen mit synthetischen Sonden ermöglichen die verschiedenen Aspekte der Funktion von Proteinen, einschließlich einer Vielzahl von dynamischen Prozessen in lebenden Zellen und in Zelllysaten. SNAP-, CLIP-, MCP-und AKP-tag-Technologien bieten Einfachheit und außergewöhnliche Vielseitigkeit für die Bildgebung von Proteinen in lebenden und fixierten Zellen und die Untersuchung von Proteinen in vitro. Die Schaffung eines einheitlichen Genkonstrukt ergibt eine getaggt Fusionsprotein in der Lage, eine kovalente Verknüpfung mit einer Vielzahl von funktionellen Gruppen, einschließlich der Fluorophore, Biotin oder Perlen. Das Protein von Interesse sind kloniert und nur einmal und dann die Fusion kann mit einer Vielzahl von fluoreszierenden Substrate für zahlreiche Downstream-Anwendungen wie zB die gleichzeitige Proteinmarkierung in lebenden Zellen, Protein-Lokalisation und Translokation, Puls-Chase-Experimente, Rezeptor-Internalisierung Studien verwendet werden , selektive Zelloberfläche Kennzeichnung, Protein-Pull-Down-Assays, Protein-Nachweis in SDS-PAGE, Durchflusszytometrie, High-Throughput-Bindungs-Assays in Mikrotiterplatten, Biosensor-Interaktion Experimente und FRET-basierten Bindungsassays.

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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-tag und CLIP-tag Proteinmarkierung Systeme ermöglichen die spezifische, kovalente Bindung von Molekülen, einschließlich Fluoreszenzfarbstoffe, um ein Protein von Interesse in lebenden Zellen. Diese Systeme bieten eine breite Auswahl an fluoreszierende Substrate für eine Reihe von Imaging-Instrumenten optimiert. Einmal kloniert und exprimiert, kann die markierte Protein mit einer Vielzahl von Substraten für zahlreiche nachgelagerte Anwendungen, ohne sich erneut Klon verwendet werden. Es gibt zwei Schritte, um mit diesem System: Klonierung und Expression des Proteins von Interesse als SNAP-tag-Fusion, und die Kennzeichnung der Fusion mit der SNAP-tag Substrat der Wahl. Die SNAP-tag ist ein kleines Protein auf die menschliche O Basis<sup&gt; 6</sup&gt;-Alkylguanin-DNA-Alkyltransferase (hAGT), ein DNA-Reparatur-Protein. SNAP-tag-Etiketten sind Farbstoffe konjugiert Guanin oder Chlorpyrimidin Abgangsgruppen über eine Benzyl-Linker. Bei der Kennzeichnung Reaktion wird das substituierte Benzylgruppe des Substrates kovalent an die SNAP-tag befestigt. CLIP-tag ist eine modifizierte Version des SNAP-tag, entwickelt, um mit benzylcytosine anstatt Benzylguanin Derivaten reagieren. In Verbindung mit SNAP-tag verwendet, ermöglicht CLIP-tag der orthogonalen und komplementären Kennzeichnung von zwei Proteine ​​gleichzeitig in den gleichen Zellen.

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 und CLIP-tag Proteinmarkierung Systeme ermöglichen die spezifische, kovalente Bindung von Molekülen, einschließlich Fluoreszenzfarbstoffe, um ein Protein von Interesse in lebenden Zellen. Einmal kloniert und exprimiert, kann die markierte Protein mit einer Vielzahl von Substraten für zahlreiche nachgelagerte Anwendungen, ohne sich erneut Klon verwendet werden.

Fluoreszenzmarkierung von COS-7 mit dem Ausdruck SNAP-tag-Fusionsproteinen für das Live Cell Imaging

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 Aufrufe.  New England Biolabs. SNAP-tag und CLIP-tag Proteinmarkierung Systeme ermöglichen die spezifische, kovalente Bindung von Molekülen, einschließlich Fluoreszenzfarbstoffe, um ein Protein von Interesse in lebenden Zellen. Einmal kloniert und exprimiert, kann die markierte Protein mit einer Vielzahl von Substraten für zahlreiche nachgelagerte Anwendungen, ohne sich erneut Klon verwendet werden.

Serie: 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.

Labeling Stammzellen mit fluoreszierenden Farbstoffen zur nicht-invasiven Detektion mit 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 ...

Forschung

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).

Live Cell Imaging von F-Aktin Dynamics über 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 ...

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.

Fluoreszenzmarkierung Drosophila Herz Structures

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.

Die Transplantation von GFP-exprimierenden Blastomeren für Live Imaging von Retinal und Entwicklung des Gehirns in Chimäre Zebrafischembryonen

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.

Photokonversion von gereinigtem fluoreszierende Proteine ​​und Dual-Sonde Optische Hervorhebung in lebenden Zellen

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.

Zwei-und dreidimensionale Live Cell Imaging der DNA-Reparatur-Proteine

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.

Live-Imaging von GFP-markierten Proteine ​​in<em&gt; Drosophila</em&gt; Oozyten

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.

Live-Imaging von apoptotischen Ausverkauf während<em&gt; Drosophila</em&gt; Embryogenese

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.

Intravitalmikroskopie zur Imaging subzellulärer Strukturen in lebenden Mäusen Ausdruck Fluoreszierende Proteine

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.

Ausdruck von fluoreszierenden Fusionsproteinen in murinen Knochenmark gewonnenen dendritischen Zellen und Makrophagen

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