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

Chapeamento de células CHO-K1: <ol><li> Adicionar 50-100μl de células CHO-K1 que foram tripsinizados de acordo com protocolos padrão de 6ml de completo F-12K. </li><li> Mix suspensão celular por pipetagem cima e para baixo várias vezes, então alíquota 300 ml de suspensão de células tripsinizados a cada poço de uma câmara estéril 8-Lab-Tek II Chambered lamínula com # 1.5 borosilicato lamela câmaras (Nalgene Nunc Int;. Produto # 155409; Lote: 100808-8-0). </li><li> Incubar as amostras a 37 ° C CO, 5% 2 durante a noite. </li></ol> Transfecção transiente de células CHO-K1 com pSNAP-ADRB2 e pSNAP-H2B: <ol><li> Verifique as câmaras de cultura de células semeadas no dia anterior para procurar densidade celular e da saúde. </li><li> Rótulo o número apropriado de tubos de microcentrífuga. </li><li> Em uma capela de fluxo laminar, configurar misturas complexas transfecção acordo com a tabela de exemplo a seguir, usando 0,3 mg pSNAP-ADBR2 DNA Controle Plasmid ou 0,3 mg de pSNAP-H2B DNA Controle Plasmid, 1 ml de D2 TransPass e 1 ml de TransPass V por reação. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> plasmídeo </td><td> número de reações </td><td> l de soro livre de médio </td><td> mL de D2 TransPass </td><td> mL de TransPass V </td><td> mL de DNA plasmídeo </td><td> mL de D2/reaction TransPass </td><td> mL de TransPass V / reação </td><td> ng de DNA / reação </td><td> DNA conc. (Ng / mL) </td><td> mL de DNA plasmídeo / reação </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> Simulado </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> Primeiro, misture o soro livre F-12K com o DNA adequado plasmídeo, em seguida, adicione o reagente TransPass Transfection D2, em seguida, adicionar o reagente TransPass Transfection V. Misture bem os componentes pipetando cima e para baixo suavemente várias vezes após a adição de cada reagente. </li><li> Incubar a reação em temperatura ambiente por 30 minutos. </li><li> Durante a incubação, lavar as células uma vez com 600 mL de meio DMEM completo e adicionar 450 mL de meio DMEM completo para cada amostra. </li><li> Adicionar 50 ul da mistura de transfecção complexo para cada amostra, lentamente e de forma gota a gota. </li><li> Incubar as amostras durante a noite a 37 ° C, 5% CO 2. </li></ol> SNAP-Cell Labeling Estrela TMR de células CHO-K1 transitoriamente transfectadas com pSNAP-ADRB2 e pSNAP-H2B: <ol><li> Remova cuidadosamente os meios de comunicação transfecção complexas a partir de cada amostra por sucção a vácuo. Lave as células duas vezes com 600 mL de completo F-12K e substituir a mídia em cada poço com 600 mL de completo F-12K. </li><li> Mix de mídia rotulagem dye, adicionando 995 mL de completo F-12K e 51 mL 0,6 mM SNAP-Cell substrato TMR-Star. A concentração final da SNAP-Cell TMR-Estrela deve ser de 3mm. Pipeta cima e para baixo várias vezes para misturar após a adição de substrato. </li><li> Remova cuidadosamente meios de crescimento a partir de amostras e adicionar 200 mL de mídia rotulagem corante a cada poço. Incubar as amostras a 37 ° C, 5% CO 2 por 30 minutos. </li><li> Enquanto as amostras são incubação, preparar solução Hoechst 33342 para a coloração nuclear das amostras através da mistura de 1 ml de Hoechst 33342, trihydrochloride, tri-hidratado (10 mg / ml solução em água, 16,2 mM solução; Sondas Invitrogen Molecular) em 10 ml de completo F-12K . </li><li> Remova a mídia de todas as amostras e adicionar 600 mL da solução de Hoechst diluir para cada amostra. Incubar as amostras a 37 ° C, 5% CO 2 por 5 minutos. </li><li> Lavar três vezes com amostras de 600 mL de completo F-12K. </li><li> Incubar as amostras a 37 ° C, 5% CO 2 por mais 30 minutos para permitir que fluoróforo unincorporated difundir para fora das células. </li><li> Após 30 minutos, altere os meios de comunicação nas amostras SNAP-Cell, uma última vez e vá para a imagem. </li><li> Imagem as células usando um microscópio Zeiss ApoTome Axiovert 200M fluorescente. </li></ol> Resultados representante <img src="/files/ftp_upload/1876/1876fig1.jpg" alt="Figura 1" /> Figura 1. Aqui estãoalguns resultados representativos das imagens fluorescentes por microscopia de fluorescência padrão de viver COS-7 células que estão expressando tag SNAP-fusões. Esta primeira imagem mostra ao vivo COS-7 células que expressam pSNAP-H2B (nuclear histona) marcada com SNAP-Cell TMR-Star. A construção pSNAP-H2B foi gerada usando o tag pSNAP-vetor (m). As células foram marcadas com SNAP-Cell TMR-Estrela por 30 minutos e contrastado com Hoechst 33342 (azul) para núcleos. A fluorescência rosa demonstra a sobreposição da fluorescência vermelha onde a proteína histona está presente, além da fluorescência azul indicando o núcleo. A expressão da proteína H2B é clara e facilmente identificados. <img src="/files/ftp_upload/1876/1876fig2.jpg" alt="Figura 2" /> <img src="/files/ftp_upload/1876/1876fig3.jpg" alt="Figura 3" /> Figuras 2 e 3. Essas duas imagens seguintes mostram ao vivo COS-7 transfectadas transitoriamente com células-pSNAP ADRβ2. As células foram marcadas com SNAP-Cell TMR-Star (vermelho) e contrastado com Hoechst 33342 (azul). A construção pSNAP-ADRβ2 foi gerada usando o tag pSNAP-vetor (m). As células foram marcadas com SNAP-Cell TMR-Estrela por 30 minutos e contrastado com Hoechst 33342 (azul) para núcleos. A fluorescência vermelha indica a presença de superfície celular ADRB2 proteína receptora de fluorescência azul, enquanto identifica o núcleo.

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

Os autores são empregados por New England Biolabs, que produz os reagentes e instrumentos utilizados neste artigo.

Há diversas variáveis ​​que afetam os resultados observados, tais como foco intensidade do laser, a capacidade de equipamentos, lentes usadas, etc A fotoestabilidade do fluoróforo determina a rapidez com que a fluorescência deve ser capturado antes de desaparecer. Este método de rotulagem proteína é versátil e adequada para muitas aplicações. SNAP-tag e CLIP tag tecnologias para a rotulagem específica de proteínas de fusão com sondas sintéticas permitem vários aspectos da função da proteína, incluindo uma variedade de processos dinâmicos em células vivas e em lisados ​​celulares. SNAP, CLIP, MCP e ACP-tag tecnologias oferecem versatilidade e simplicidade extraordinária para a imagem de proteínas em células vivas e fixos e para o estudo das proteínas in vitro. A criação de uma construção de um único gene produz uma proteína de fusão com a tag capaz de formar uma ligação covalente a uma variedade de grupos funcionais, incluindo fluoróforos, biotina, ou grânulos. A proteína de interesse devem ser clonados e expressos apenas uma vez e, em seguida, a fusão pode ser usado com uma variedade de substratos fluorescentes para diversas aplicações a jusante como a rotulagem de proteína simultânea no interior das células vivas, localização de proteínas e translocação, pulse-chase experimentos, estudos de internalização do receptor , rotulagem superfície seletiva de células, proteínas puxar para baixo os ensaios, a detecção de proteínas em SDS-PAGE, citometria de fluxo, de alto rendimento ensaios de ligação em placas de microtitulação, as experiências de interação biosensor, e FRET baseado em ensaios de ligação.

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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 e CLIP tag-sistemas de rotulagem proteínas permitem o anexo, covalentes de moléculas específicas, incluindo corantes fluorescentes, a uma proteína de interesse em células vivas. Estes sistemas oferecem uma ampla seleção de substratos fluorescentes otimizado para uma faixa de instrumentação de imagem. Uma vez clonado e expresso, a proteína com a tag pode ser usada com uma variedade de substratos para inúmeras aplicações a jusante sem ter de clonar novamente. Há duas etapas para usar este sistema: a clonagem e expressão da proteína de interesse como uma fusão SNAP-tag, e rotulagem da fusão com o substrato SNAP-tag de escolha. O SNAP-tag é uma pequena proteína humana com base em O<sup&gt; 6</sup&gt;-Alkylguanine-DNA-alkyltransferase (hAGT), uma proteína de reparo do DNA. SNAP tag rótulos são corantes conjugado com guanina ou chloropyrimidine grupos deixando através de um linker benzílico. Na reação de rotulagem, o grupo benzila substituído do substrato é covalentemente ligado ao SNAP-tag. CLIP-tag é uma versão modificada do SNAP-tag, projetado para reagir com benzylcytosine ao invés de derivados benzylguanine. Quando usado em conjunto com SNAP-tag, tag-CLIP permite a rotulagem ortogonais e complementares de duas proteínas simultaneamente nas mesmas celas.

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 e CLIP tag-sistemas de rotulagem proteínas permitem o anexo, covalentes de moléculas específicas, incluindo corantes fluorescentes, a uma proteína de interesse em células vivas. Uma vez clonado e expresso, a proteína com a tag pode ser usada com uma variedade de substratos para inúmeras aplicações a jusante sem ter de clonar novamente.

Labeling fluorescentes de COS-7 expressando as proteínas SNAP tag-Fusion para imagens de células vivas

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

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

Research

JoVE Journal

Biology

22.9K visualizações.  New England Biolabs. SNAP-tag e CLIP tag-sistemas de rotulagem proteínas permitem o anexo, covalentes de moléculas específicas, incluindo corantes fluorescentes, a uma proteína de interesse em células vivas. Uma vez clonado e expresso, a proteína com a tag pode ser usada com uma variedade de substratos para inúmeras aplicações a jusante sem ter de clonar novamente.

Vídeo: 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.

Rotulagem células-tronco com corantes fluorescentes para detecção não invasiva com imagens ópticas

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

Biologia

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).

Imagens de células vivas de F-actina Dynamics via Microscopia fluorescente Speckle (FSM)

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

Fluorescent Labeling of Drosophila Heart Structures

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

Fluorescent Labeling of Drosophila Heart Structures

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

Labeling fluorescentes de Drosophila Estruturas Coração

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.

Transplante de GFP-expressando Blastômeros for Imaging ao vivo de Desenvolvimento da retina e do cérebro em embriões Zebrafish Quimérico

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.

Fotoconversão de proteínas purificadas fluorescentes e Dual-sonda Destacando Optical em células vivas

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

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

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

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

Duas e três dimensões imagens de células vivas de Proteínas de DNA resposta a danos

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.

Imagens ao vivo de Proteínas GFP-rotulados em<em&gt; Drosophila</em&gt; Oócitos

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.

Imagens ao vivo de Apuramento celular por apoptose durante<em&gt; Drosophila</em&gt; Embriogênese

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

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

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

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

Microscopia intravital para Estruturas de imagem Subcelulares em ratos vivos Expressando fluorescentes Proteínas

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.

Expressão de proteínas da fusão fluorescente em murino derivadas da medula óssea as células dendríticas e macrófagos

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