Este trabalho foi parcialmente financiado pelo National Institutes of Health (GM093282 para PW; 3U54AI057158-06S1 a RDS) e fundos de inicialização do Albert Einstein College of Medicine.

1. Coleta de ovos e Dechorionation <ol><li> Coletar e transferir os ovos zebrafish a 35mm placa de Petri, remova tanta água quanto possível e então adicione 1 E Pronease mg / ml em meio embrião E3 (5 mM NaCl, 0,17 mM KCl, 0,33 mM CaCl 2 · 2H 2 O, 0,33 mM MgSO 4, pH = 7,4) para digerir o córion. </li><li> Depois de 3-5 minutos, fundir o prato em um copo cheio de água de peixe (60 mg &quot;Ocean instantânea&quot; por litro destilada H 2 O), e gentilmente transferir os ovos para o copo e deixe os ovos para &quot;cair&quot; na água. </li><li> Lave os ovos com peixes de água três vezes. A maioria dos ovos serão liberados de suas córion. </li><li> Usando um fogo-de vidro polido Pasteur pipeta, transferir os ovos para dechorionated pratos agarose revestidas de petri preenchida com meio embrião E3. </li></ol> 2. Microinjeção com o PIB-FucAl <ol><li> Prepare os pratos injeção seguindo o protocolo relatados 7. </li><li> Transferência de ovos dechorionated em pratos cheios de injeção médio embrião E3. </li><li> Preparar a agulha, e carregar com 2 mL de solução de injeção. Esta solução contém 20 mM PIB FucAl sintetizado chemoenzymatically 8 e tanto Alexa Fluor 594-dextran (5% w / v) como um marcador ou corante vermelho de fenol de carga (0,1% w / v) em 0,2 M KCl. Como controle negativo, substitua-FucAl PIB com o PIB-fucose em solução de injeção. </li><li> Quebrar a agulha e ajustar a pressão de injeção e duração para produzir um 1 gota nL 9. </li><li> Injetar os ovos com 1 nL de uma solução. </li><li> Transferir os ovos em pratos agarose revestidas de petri preenchida com meio embrião E3. </li><li> Incubar os ovos a 28 ° C e remover os ovos não fertilizados dentro de três a quatro horas após a fertilização. </li></ol> 3. BTTES-Cu (I)-Reacção catalisada Clique Química <ol><li> Quando os embriões atingem desejado estágios de desenvolvimento (estádio atrasado, por exemplo, a segmentação dos tecidos e larva precoce), revestimento a base de uma placa de 96 poços com agarose. </li><li> Adicionar 92 mL de médio embrião E3 a cada poço, seguido por adição de 4 Alexa Fluor-488 mL de azida (a partir de um estoque de 2,5 mM em H 2 O), 2 mL BTTES-CuSO 4 06:01 complexo, e agitar suavemente para misturar. </li><li> Transferência de embriões no poço contendo o reagente química do clique utilizando um fogo-de vidro polido Pasteur pipeta. Cada poço deve conter menos de cinco embriões. </li><li> Adicionar 2,5 mL de ascorbato de sódio preparada na hora (de um estoque de 100 mm em H 2 O) para iniciar a reação clique em 3. Concentração final de cada reagente: Alexa Fluor azida-488: 100 mm; CuSO 4: 50 mM; BTTES: 300 mM; ascorbato de sódio: 2,5 mM. </li><li> Após 3 min, adicionar 2 mL bathocuproine sulfonato (50 mM de ações em H 2 O), um quelante de cobre biocompatível, para saciar a reação imediatamente, diluir com 100 mL de médio E3 embrião. </li><li> Transferência dos embriões para uma placa de Petri de vidro e lave os embriões tratados duas vezes com 15 mL de médio embrião E3. </li></ol> 4. Imagem <ol><li> Coloque uma gota de ultralow ponto de fusão agarose (1,2% (w / v) em meio embrião E3) em um copo prato fundo Mattek micropoços, e colocar um embrião para a queda de agarose. </li><li> Posição dos embriões dorsalmente ou lateralmente e colocar o prato no gelo por 5 min para solidificar a queda de agarose. Adicionar médio embrião E3 suavemente para o prato até cobrir a queda de agarose. </li><li> Imagens de fluorescência de campo e brilhantes são adquiridas seqüencialmente usando um microscópio confocal. Todas as imagens de embriões são adquiridos utilizando um intervalo de passo 5 mm. Figuras compostas são preparadas usando software ImageJ. </li></ol> 5. Resultados representante A Figura 1 mostra o fluxo de trabalho da nossa estratégia de rotulagem em duas etapas. A Figura 2 mostra a rotulagem de embriões zebrafish via BTTES mediada CuAAC em 9,5 horas pós-fertilização (hpf). BTTES é um tris (triazolylmethyl) ligand amina-based. Ele acelera drasticamente quando CuAAC coordenação com o in situ Cu gerado (I), e promove a reação de cicloadição rapidamente em sistemas vivos sem toxicidade aparente. Imediatamente após uma reação clique em 3-min, que são capazes de detectar etiquetagem robusta dos embriões PIB FucAl tratadas (Figura 2, os painéis à esquerda). Fluorescência de fundo só é detectado para controle de embriões microinjeções PIB-fucose (Figura 2, os painéis à direita). <img src="/files/ftp_upload/2686/2686fig1.jpg" alt="Figura 1" /> Figura 1. A estratégia de rotulagem glicanos fucosylated na camada envolvente de embriões zebrafish. <img src="/files/ftp_upload/2686/2686fig2.jpg" alt="Figura 2" /> Figura 2. Na geração de imagens in vivo de glicanos fucosylated durante a embriogênese zebrafish via BTTES-Cu (I)-catalisada química do clique. Uma célula-embriões zebrafish estágio são microinjeção com uma dose única do PIB-FucAl e permitiu a develop para 9,5 hpf. Os embriões são, então, reagiu com Alexa Fluor 488 azida-catalisada pela BTTES-Cu (I). Embriões reagiu são gravadas através de microscopia confocal. Máxima intensidade z projeção de imagens de Alexa Fluor 488 fluorescência (painel superior); campo Bright (painel inferior). Barra de escala: 100 mm. Solução de problemas <table border="1"><tbody><tr><td> Problema </td><td> Causa </td><td> Remédio </td></tr><tr><td> Os embriões olhar insalubres </td><td> A solução microinjeção contém contaminantes </td><td> A pureza dos açúcares nucleotídeos deverá ser superior a 85%. Verifique a sua fonte. </td></tr><tr><td> Não há rotulagem após a reação </td><td> Concentração de cobre é inferior a 30 mM </td><td> Tenha cuidado para não diluir a solução reacção adicionando médio de embriões em excesso E3 ao adicionar os embriões, como a velocidade da reação diminui significativamente quando a concentração de cobre é abaixo de 30 mM. </td></tr><tr><td> Os embriões morrem após a reação </td><td> Manuseio inadequado dos embriões </td><td> Verifique se a pipeta é fogo-polido eo vaso de reação é revestido com uma camada muito fina de agarose 0,5%. </td></tr><tr><td> As imagens parecem irregular </td><td> Os reagentes não são devidamente misturados antes de acrescentar os embriões </td><td> Seguir a ordem recomendada de adição para os reagentes, e certifique-se que os reagentes química do clique foram devidamente misturados antes de acrescentar os embriões. </td></tr><tr><td> As imagens mostram os embriões sejam danificados </td><td> Agitação vigorosa danos os embriões durante a reação </td><td> Agite suavemente para menos de 10 segundos, uma vez que os embriões são a solução. </td></tr><tr><td></td><td></td><td></td></tr></tbody></table>

<ol>
	<li>Laughlin, S. T., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+glycome.">Imaging the glycome.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 12-17 (2009).</li><li>Baskin, J. M., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+click+chemistry:+Covalent+labeling+in+living+systems.">Bioorthogonal click chemistry: Covalent labeling in living systems.</a> Qsar Comb. Sci. 26, 1211-1219 (2007).</li><li>Soriano del Amo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatible+copper(I)+catalysts+for+in+vivo+imaging+of+glycans.">Biocompatible copper(I) catalysts for in vivo imaging of glycans.</a> J. Am. Chem. Soc. 132, 16893-16899 (2010).</li><li>Chang, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+labeling+of+sialic+acids+in+living+animals+with+alkynyl+sugars.">Metabolic labeling of sialic acids in living animals with alkynyl sugars.</a> Angew. Chem. Int. Ed. 48, 4030-4033 (2009).</li><li>Laughlin, S. T., Baskin, J. M., Amacher, S. L., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+membrane-associated+glycans+in+developing+zebrafish.">In vivo imaging of membrane-associated glycans in developing zebrafish.</a> Science. 320, 664-667 (2008).</li><li>Baskin, J. M., Dehnert, K. W., Laughlin, S. T., Amacher, S. L., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+enveloping+layer+glycans+during+zebrafish+early+embryogenesis.">Visualizing enveloping layer glycans during zebrafish early embryogenesis.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 10360-10365 (2010).</li><li>Kemp, H. A., Carmany-Rampey, A., Moens, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+chimeric+zebrafish+embryos+by+transplantation.">Generating chimeric zebrafish embryos by transplantation.</a> J. Vis. Exp. , (2009).</li><li>Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoenzymatic+synthesis+of+GDP-L-fucose+and+the+Lewis+X+glycan+derivatives.">Chemoenzymatic synthesis of GDP-L-fucose and the Lewis X glycan derivatives.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 16096-16101 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J. Vis. Exp. , (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> THE ZEBRAFISH BOOK, A guide for the laboratory use of zebrafish (Danio rerio). , (2007).</li><li>Kimmel, C. B., Warga, R. M., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+organization+of+the+zebrafish+fate+map.">Origin and organization of the zebrafish fate map.</a> Development. 108, 581-594 (1990).</li></ol>

Não há conflitos de interesse declarados.

<p class="jove_content"&gt; Biomoléculas de imagem<em&gt; In vivo</em&gt; Fornece uma visão crítica de suas atividades biológicas em seus ambientes nativos. Neste vídeo, demonstramos como a rotulagem dos glicanos fucosylated na camada envolvente de embriões zebrafish é realizado por microinjecting embriões de uma célula palco com PIB FucAl e uma conjugação fluoróforo segunda etapa via BTTES mediada CuAAC biocompatível<sup&gt; 3</sup&gt;. Etiquetagem robusta pode ser conseguido dentro de 2-3 minutos, e os glicanos rotulados são det...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Cobre pentahidratado sulfato (II) </td><td> Sigma-Aldrich </td><td> 203165 </td></tr><tr><td> Alexa Fluor 488 azida </td><td> Invitrogen </td><td> A10266 </td></tr><tr><td> dextran, Alexa Fluor 594 </td><td> Invitrogen </td><td> D-22913 </td></tr><tr><td> (+) De sódio-L-ascorbato </td><td> Sigma-Aldrich </td><td> A7631 </td></tr><tr><td> Bathocuproinedisulfonic ácido </td><td> Acros Organics </td><td> 164060010 </td></tr><tr><td> Prato fundo de vidro micropoços </td><td> Mattek </td><td> P35G-1.5-14-C </td></tr></tbody></table>

imaging glycans in zebrafish embryos by metabolic labeling and bioorthogonal click chemistry

Glicanos de imagem in vivo foi recentemente ativado usando uma estratégia repórter bioorthogonal química por tratar células ou organismos com azida ou alcino marcadas monossacarídeos 1, 2. Os monossacarídeos modificado, processados ​​pela máquina biossintética glicano, são incorporados glicoconjugados de superfície celular. As tags bioorthogonal azida ou alcino então permitir a conjugação covalente com sondas fluorescentes para a visualização, ou afinidade com sondas para o enriquecimento e análise glycoproteomic. Este protocolo descreve os procedimentos normalmente utilizados para imagem não invasivos de glicanos fucosylated em embriões de peixe-zebra, incluindo: 1) microinjeção de um celular em embriões em estágio com o PIB-5-alkynylfucose (PIB FucAl), 2) rotulagem glicanos fucosylated na camada envolvente de embriões zebrafish com azida conjugada fluoróforos via Cu biocompatível (I)-catalisada cicloadição azida-alcino (CuAAC), e 3) de imagens por microscopia confocal 3. O método descrito aqui pode ser facilmente estendido para visualizar as outras classes de glicanos, glicanos por exemplo, contendo ácido siálico 4 e N-acetilgalactosamina 5, 6, em zebrafish em desenvolvimento e em outros organismos vivos.

Watch this Scientific Journal Video about Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry at JoVE.com

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Um método baseado em clique química que permite a rotulagem, rápida não invasiva, e robusta de alcino com tags glicanos em embriões de peixe-zebra é descrito. Glicanos Fucosylated na camada envolvente de embriões zebrafish na fase final de gastrulação foram fotografadas neste estudo.

Glicanos imagem em embriões Zebrafish por Labeling Metabólica e Química Clique Bioorthogonal

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

imaging-glycans-zebrafish-embryos-metabolic-labeling-bioorthogonal

Research

JoVE Journal

Biology

13.6K visualizações.  Albert Einstein College of Medicine, Yeshiva University. Um método baseado em clique química que permite a rotulagem, rápida não invasiva, e robusta de alcino com tags glicanos em embriões de peixe-zebra é descrito. Glicanos Fucosylated na camada envolvente de embriões zebrafish na fase final de gastrulação foram fotografadas neste estudo.

Vídeo: Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

Microinjeção de mRNA e Oligonucleotídeos Anti-Senso Morpholino em embriões Zebrafish.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Biologia

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Dois fótons axotomia e lapso de tempo de imagem confocal em embriões de peixe-zebra ao vivo

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

A criação de embriões quiméricos Zebrafish por Transplante

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

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 ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Imagem Alterar Forma Celular em Viver Drosophila Os embriões

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

Medindo a progressão do ciclo celular Cinética com marcação metabólica e Citometria de Fluxo

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

Análise do Perfil Metabólico de embriões Zebrafish

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Metabólica Rotulagem de Leucina rico Repita Kinases 1 e 2 com Radioactive Fosfato

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

Análise do estresse oxidativo em embriões Zebrafish

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Imagem semi-automatizada de Fluorescência de tecidos específicos em embriões Zebrafish

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...