Kai Johnsson Ivan Correa Andreas Brecht Katrin Müentener Chris Noren Luo Sun Ming Xu Theodore Davis Salvatore Russello Aihua Zhang Inca Ghosh Elissa Maunus Nicholas Labarthe James MacFarland John Buswell Brenda Desmond James Ellard Don Tarak

CHO-K1 Hücreler Kaplama: <ol><li> F-12K tam 6ml standart protokollere göre tripsinize CHO-K1 hücreleri 50-100μl ekleyin. </li><li> Birkaç kez aşağı yukarı pipetleme hücre süspansiyonu karıştırın, 1.5 borosilikat coverglass odaları (tripsinize hücre süspansiyonu daha sonra steril bir 8-odacık Lab-Tek II odacıklı Coverglass her kuyuya kısım 300 ul Nalgene Nunc Int. Ürün # 155.409; Yeri: 100808-8-0). </li><li> Numuneleri 37 ° C,% 5 CO 2 gece boyunca inkübe edin. </li></ol> PSNAP ADRB2 ve pSNAP-H2B ile CHO-K1 Hücre Geçici Transfeksiyon <ol><li> Hücre yoğunluğu ve sağlık için önceki gün seribaşı hücre kültürü odaları kontrol edin. </li><li> Mikrofuge&#39;de tüplerin uygun sayıda etiketleyin. </li><li> Laminer akış kaputu, transfeksiyon kompleks karışımlar 0.3 mikrogram pSNAP ADBR2 Kontrol Plazmid DNA veya pSNAP-H2B Kontrol Plazmid DNA, TransPass D2 1 ul ve 1 TransPass V ul 0.3 mikrogram ile aşağıdaki örnek grafiğe göre ayarlamak her reaksiyon için. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> plazmid </td><td> reaksiyon sayısı </td><td> serum orta, ul </td><td> TransPass D2, ul </td><td> TransPass V, ul </td><td> plazmid DNA, ul </td><td> TransPass D2/reaction, ul </td><td> TransPass V / reaksiyon, ul </td><td> DNA / reaksiyon ng </td><td> DNA kons. (Ng / ul) </td><td> plazmid DNA / reaksiyon, ul </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> Sahte </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> İlk olarak, F-12K uygun plazmid DNA ile serum serbest, daha sonra sonra TransPass V Transfeksiyon Reaktif eklemek TransPass D2 Transfeksiyon Reaktif ekleyip karıştırın. Bileşenlerin her reaktifin ilavesinden sonra birkaç kez yavaşça yukarı ve aşağı pipetleme iyi karıştırın. </li><li> Reaksiyon oda sıcaklığında 30 dakika inkübe edin. </li><li> İnkübasyon sırasında, tam DMEM 600 ul kez hücreleri yıkayın ve her bir örnek için 450 tam DMEM ul ekleyin. </li><li> Yavaş yavaş ve akıllıca bir damla moda, her bir örnek 50 ul transfeksiyon karmaşık bir karışımı ekleyin. </li><li> 37 ° C,% 5 CO 2 gecede örnekleri inkübe edin. </li></ol> SNAP-geçici pSNAP ADRB2 ve pSNAP-H2B ile transfekte CHO-K1 Hücre Hücre TMR Yıldız Etiketleme: <ol><li> Dikkatle vakum emme her örnekten transfeksiyon karmaşık ortamı çıkarın. F-12K tam iki kez 600 ul hücreleri yıkayın ve F-12K tam 600 ul ile her bir kuyunun medya yerine. </li><li> 995 F-12K ve 51 ul 0.6 mM SNAP-Cell TMR-Star substrat ul ekleyerek boya etiketleme medya karıştırın. SNAP-Cell TMR-Star final konsantrasyon 3mm olmalıdır. Substrat eklenmesinden sonra karıştırmak için birkaç kez aşağı ve yukarı Pipet. </li><li> Dikkatle örneklerinden büyüme ortamı çıkarın ve her bir kuyunun 200 ul boya etiketleme medya ekleyin. 37 numune ° C&#39;de,% 5 CO 2 30 dakika. </li><li> Tam F-12K 10 ml; örnekleri kuluçka iken, Hoechst 33.342, trihydrochloride, trihidrat (Invitrogen Molecular Probes suda 10 mg / ml solüsyon, 16,2 mM çözüm) 1 ul karıştırılarak Hoechst 33.342 çözüm örnekleri nükleer boyanması için hazırlamak . </li><li> Tüm örneklerde medyayı çıkartın ve her numune için seyreltilmiş Hoechst çözüm 600 ul ekleyin. 37 numune ° C&#39;de,% 5 CO 2 5 dakika. </li><li> F-12K tam 600 ul örnekleri üç kez yıkayın. </li><li> Numuneleri 37 ° ° C&#39;de,% 5 CO 2 adi fluorofor hücrelerin diffüz izin vermek için ek bir 30 dakika . </li><li> 30 dakika sonra, SNAP-Cell örnekleri, son bir kez medya değiştirmek ve görüntüleme geçin. </li><li> Zeiss Apotome Axiovert 200M floresan mikroskop kullanılarak Görüntü hücreler. </li></ol> Temsilcisi Sonuçlar <img src="/files/ftp_upload/1876/1876fig1.jpg" alt="Şekil 1" /> Şekil 1.SNAP-tag füzyonlar ifade canlı COS-7 hücreleri standart floresan mikroskopi kullanılarak floresan görüntüleme bazı temsilcisi sonuçları. Bu ilk görüntü, SNAP-Cell TMR-Star etiketli pSNAP-H2B (histon nükleer) ifade COS-7 hücreleri canlı gösterir. PSNAP-H2B inşa pSNAP-tag (m) vektörü kullanılarak oluşturulmuştur. Hücreler 30 dakika boyunca SNAP-Cell TMR-Star etiketli ve çekirdekleri Hoechst 33.342 (mavi) ile zıt. Pembe floresan kırmızı floresan histon protein çekirdeği gösteren mavi floresan yanı sıra mevcut kaplamayı göstermektedir. H2B protein ifadesi, açık ve kolay bir şekilde tespit edilir. <img src="/files/ftp_upload/1876/1876fig2.jpg" alt="Şekil 2" /> <img src="/files/ftp_upload/1876/1876fig3.jpg" alt="Şekil 3" /> Şekil 2 ve 3 Bu önümüzdeki iki görüntü geçici pSNAP-ADRβ2 transfekte canlı COS-7 hücreleri göstermektedir. Hücreler SNAP-Cell TMR-Star (kırmızı) etiketli ve Hoechst 33.342 (mavi) ile zıt. PSNAP ADRβ2 inşa pSNAP-tag (m) vektörü kullanılarak oluşturulmuştur. Hücreler 30 dakika boyunca SNAP-Cell TMR-Star etiketli ve çekirdekleri Hoechst 33.342 (mavi) ile zıt. Mavi floresan çekirdeği tanımlar iken kırmızı floresan hücre yüzeyinde reseptör protein ADRB2 varlığını gösterir.

<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>

Bu makalede kullanılan reaktifler ve araçlar üreten New England Biolabs yazarları tarafından istihdam edilmektedir.

Fluorofor fotostabilite solma önce ele olmalıdır ne kadar hızlı floresan belirler odak, lazer yoğunluğu, ekipman özellikleri, lens kullanılır, vs gibi gözlenen sonuçlar etkileyen birçok değişken vardır. Protein etiketleme Bu yöntem, çok yönlü ve birçok uygulama için uygundur. Sentetik problar ile füzyon proteinleri belirli bir etiketleme için SNAP-etiket ve CLIP-etiket teknolojileri bir çok canlı hücreler ve hücre lizatları dinamik süreçleri de dahil olmak üzere protein fonksiyonu çeşitli yönleriyle sağlar. SNAP, CLIP, MCP-ve ACP-etiket teknolojileri, canlı ve sabit hücrelerin proteinlerin görüntüleme ve proteinlerin in vitro çalışmada, sadelik ve olağanüstü çok yönlülük sağlar. Tek bir gen yapının oluşturulması fluorophores, biotin veya boncuk gibi çeşitli fonksiyonel gruplar, bir kovalent bağ kurma yeteneğine sahip etiketli füzyon proteini verir. Ilgi protein klonlanmış ve ifade sadece bir kez ve daha sonra füzyon floresan substratlar canlı hücrelerin içinde aynı anda protein etiketleme, protein yerelleştirme ve translokasyon, darbe-kovalamaca deneyler, reseptör içselleştirilmesi çalışmaları gibi çok sayıda alt uygulamalar için çeşitli kullanılabilir olmalıdır , seçici hücre yüzeyinde etiketleme, protein testleri, SDS-PAGE, akış sitometri, microtiter plakalar, biyosensör etkileşim deneyleri yüksek verim bağlayıcı deneyleri protein algılama ve FRET-temelli bağlayıcı deneyleri aşağı çekme.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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-etiket ve CLIP-tag protein etiketleme sistemleri, canlı hücreler ilgi bir protein, floresan boyalar da dahil olmak üzere moleküller, kendine özgü, kovalent eklenti sağlar. Bu sistemler, görüntüleme enstrümantasyon bir dizi için optimize edilmiş floresan substratların geniş bir seçki sunuyor. Klonlanmış ve ifade sonra tekrar clone kalmadan, etiketli protein çok sayıda downstream uygulamalar için çeşitli substratlar kullanılmalıdır. Bu sistemi kullanarak iki adım vardır: SNAP-tag füzyon, füzyon seçim SNAP-tag substrat etiketlenmesi ve protein gibi ilgi klonlama ve ifade. SNAP-etiket insan O dayalı küçük bir protein<sup&gt; 6</sup(HAGT)&gt; alkylguanine-DNA alkyltransferase bir DNA tamir proteini. SNAP-tag etiketleri benzil ilintileyici üzerinden guanin konjuge boyalar veya chloropyrimidine bırakarak grupları. Etiketleme tepki olarak, yüzey yerine benzil grubu kovalent SNAP-etiketi eklenir. CLIP-etiket benzylcytosine ziyade benzylguanine türevleri ile reaksiyona için tasarlanmış SNAP-tag değiştirilmiş bir versiyonu. SNAP-tag ile birlikte kullanıldığında, CLIP-etiketi aynı hücrelerde aynı anda iki proteinin dik ve tamamlayıcı etiketleme sağlar.

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-etiket ve CLIP-tag protein etiketleme sistemleri, canlı hücreler ilgi bir protein, floresan boyalar da dahil olmak üzere moleküller, kendine özgü, kovalent eklenti sağlar. Klonlanmış ve ifade sonra tekrar clone kalmadan, etiketli protein çok sayıda downstream uygulamalar için çeşitli substratlar kullanılmalıdır.

Canlı Hücre Görüntüleme için SNAP-tag Fusion Proteinler ifade COS-7 Floresan Etiketleme

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 Görüntüleme.  New England Biolabs. SNAP-etiket ve CLIP-tag protein etiketleme sistemleri, canlı hücreler ilgi bir protein, floresan boyalar da dahil olmak üzere moleküller, kendine özgü, kovalent eklenti sağlar. Klonlanmış ve ifade sonra tekrar clone kalmadan, etiketli protein çok sayıda downstream uygulamalar için çeşitli substratlar kullanılmalıdır.

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.

Optik Görüntüleme ile non-invaziv Algılama için Floresan Boyalar Kök Hücreler Etiketleme

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 ...

Biyoloji

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).

Floresan üzerinden F-aktin Dynamics Canlı Hücre Görüntüleme speckle Mikroskopi (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.

VE Floresan Etiketleme Drosophila Kalp Yapıları

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.

Transplantasyon GFP of-Kimerik Zebra balığı Embriyolar Retina ve Beyin Gelişimi Canlı Görüntüleme blastomer ifade

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.

Canlı Hücrelerde Floresan Proteinleri ve Çift prob Optik Vurgulama Saf, Fotoçevrim

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.

DNA Hasarı Tepki Proteinler İki ve Üç Boyutlu Canlı Hücre Görüntüleme

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.

GFP etiketli Proteinlerin Görüntüleme Canlı<em&gt; Drosophila</em&gt; Oosit

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.

Apoptotik hücresi Temizliği görüntülenmesi sırasında Canlı<em&gt; Drosophila</em&gt; Embriyo

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.

Floresan Proteinler ifade Canlı Farelerde Görüntüleme hücrealtı Yapıların Intravital Mikroskopi

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.

Kemik iliği türevi fare dendritik hücreleri ve makrofajlar floresan Fusion proteinlerin ifade

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