Gostaríamos de agradecer a Dan Carlin para ajudar na tomada de fluoresceína enjaulado. Além disso, gostaríamos de agradecer as fontes de financiamento seguintes: Vanderbilt University Medical School Programa de Grant Developmental Biology Formação de JAC, e NIH HD054534to JTG.

1. Síntese do Caged Fluorescein Dextran <ol><li> Meça 3,5-4 mg de aminodextran e adicione dentro do tubo Invitrogen fornecidos matizado contendo 1 mg de fluoresceína CMNB gaiolas SE. Em nossas mãos esta relação dá uma carga média de ~ 2,5 moléculas de corante por dextran. </li><li> Adicionar 500 mL de 0,1 M 2 B Na 4 tampão O 7 (borato de sódio) para o tubo. </li><li> Cap e vortex por 30 segundos para dissolver o aminodextran e fluoresceína enjaulado. </li><li> Vamos reagir durante a noite em um misturador de vórtice. </li><li> Retire a parte inferior de fechamento em uma coluna de spin Zeba e soltar a tampa. Marcar um ponto na coluna com uma caneta de feltro. Coloque em uma coluna de tubo de 15 mL cônico. </li><li> Centrifugar a 1000 xg por 2 min com o ponto de frente para o centro do rotor. Use coluna imediatamente após a compactação. </li><li> Colocar a coluna compactada em um tubo de 15 mL nova cônico. </li><li> Mistura piscina reação e transferência para o centro da cama compactado coluna de resina. Recoloque a tampa. </li><li> Centrifugar a 1000 xg por 2 min com o ponto de frente para o centro do rotor. Após a rodada, o eluente eo topo da coluna será mais ou menos o mesmo tom de cor amarela. </li><li> Transferir o eluente amarelo (~ mL 350) a um tubo de microcentrífuga de 1,5 mL. </li><li> Lyophilize à secura em um speedvac. Cobrir o speedvac com papel alumínio. </li><li> Re-suspender a ~ 2-3 mg resultantes de espuma amarelada na água para uma concentração final de 1% w / v. </li><li> Conservar a solução a -20 ° C em folhas cobertas de tubos. É uma boa idéia para a solução de dextran alíquota para evitar congelamento e descongelamento repetitivos. </li></ol> 2. A injeção de fluoresceína Caged Dextran em embriões Zebrafish <ol><li> Diluir 1% de ações w / v de fluoresceína enjaulado dextran 1:05 ou 1:10 em KCl 0,2 M. </li><li> Injetar 0,5 - 1.0nL de solução de fluoresceína enjaulado na gema de uma célula de embriões palco usando uma pressão injector. Uma vez injetadas, embreagens de embriões devem ser mantidos no escuro a 28,5 ° C para reduzir a não-específica uncaging. </li><li> Remover manualmente córions a partir de embriões. </li><li> Anestesiar embriões em tricaina 4%. </li><li> Embriões serão montados em agarose 0,8% em 35mmx10mm placa de Petri, em seguida, cobertos com água de ovo. </li><li> Equilibrar agarose derretida em 50 bloco de aquecimento ° C por pelo menos 15 minutos. </li><li> Usando uma pipeta de vidro, elaborar um embrião em uma quantidade mínima de água e colocá-la no Nota agarose:. É importante para resfriar o tubo de agarose em sua mão por aproximadamente 30 segundos antes de colocar o embrião para dentro do tubo para evitar matar o embrião. </li><li> Pipetar o embrião do tubo, juntamente com cerca de 50μL de agarose e ejetar o conteúdo para o centro da placa de Petri. </li><li> Orientar rapidamente o embrião usando um fio de plástico com as células a serem uncaged voltada para cima. Nota: A agarose com frio rapidamente para ser apressada com a orientação do embrião. Embriões podem ser danificadas se manipulada após a agarose torna-se rígida. </li><li> Permitir que a agarose para solidificar completamente (este processo leva alguns minutos) e encher o prato dois terços cheio com água contendo ovos tricaina 4% e 0,003% PTU. </li></ol> 3. Laser Uncaging de fluoresceína Dextran <ol><li> O microscópio utilizado para uncaging deve ter uma objetiva de imersão 40X água. </li><li> Nós usamos um Photonics Instruments laser com a célula de 365 nm corante e os 70% / 30% dividido espelho dicróico. </li><li> Antes de uncaging, o laser deve ser feita com o objetivo parfocal e atenuada com a potência adequada. </li><li> Para focar o laser, coloque uma lâmina de vidro espelhado com o objetivo e foco o objetivo até arranhões no slide são visíveis. </li><li> Ajustar a laser atenuador até que o laser pode produzir um risco claramente visível no espelho com um único pulso. </li><li> Ajuste o foco objetivo cima e para baixo. Se o laser pode produzir um risco quando o objetivo é fora de foco, ajustar o foco do laser ¼ de volta para cima ou para baixo. Repita até que o laser pode riscar apenas quando o objetivo é focada. </li><li> Lugar montado embrião no âmbito do objectivo e focar a área a ser uncaged. </li><li> Para soltar da jaula, o foco em uma célula de interesse e de pulso que 10-20 vezes menos 2-3 Hz. </li><li> Depois uncaging, embrião do livre por raspagem agarose afastado com uma pinça e colocá-los em água contendo ovos PTU 0,003%. Deixe os embriões se desenvolvem em uma caixa à prova de luz até que sejam corrigidos. </li></ol> 4. Rotulagem de anticorpos e Detecção de Fluoresceína Uncaged Dextran <ol><li> Fix embriões em Ab fixadora (paraformaldeído 4%, 0,3 CaCl 2, 8% de sacarose, fosfato de 1X tampão pH, solução salina 7,3) por 2-4 horas à temperatura ambiente (RT) ou durante a noite (O / N) a 4 ° C. Manter os embriões em uma caixa de luz apertado ou embrulhado em papel alumínio do tubo para todas as etapas rotulagem. </li><li> Remover fixador e substituir com MeOH 100%, deixe por 10 minutos a RT, remova e substitua MeOHce com MeOH 100% fresco. </li><li> Loja em MeOH a -20 ° C durante pelo menos 30 minutos. Nota: Você pode armazenar em MeOH por até duas semanas antes de começar a degradar epitopos. </li><li> Reidratar embriões com 5 lavagens minuto a RT de 75% de fosfato MeOH/1X solução salina tamponada com 0,1% Tween 20 (1X PBSTw), então 1XPBTw 50% MeOH/50%, então 25% MeOH/75% 1X PBSTw. </li><li> Lavar três vezes por 5 minutos em 1XPBSTw. </li><li> Se os embriões são&gt; 24 horas de idade, permiabilize embriões com 10 mcg / mL proteinase K em 1XPBSTw. Permiabilization tempo é dependente no palco. Cinco minutos em proteinase K é suficiente se os embriões são entre 24 e 48 horas pós-fertilização (hpf). Mais tempo pode ser necessária se para mais larvas. Re-fix em Ab fixadora por 20 minutos a RT </li><li> Lavar cinco vezes por 5 minutos em 1XPBSTw na RT. </li><li> Incubar em solução de bloco (1XPBS, Triton x100 0,1%, 10% de soro de ovelha, 10% de soro bovino albumina, 1% Dimetil sulfóxido) por 1-2 horas em temperatura ambiente. </li><li> Diluir anticorpos primários (anti-fluoresceína mais anticorpos contra células do tipo marcadores específicos) em solução de bloqueio. </li><li> Embriões incubar S / N, 4 ° C em solução contendo anticorpos primários. </li><li> Lavar quatro vezes por 20 minutos em 1XPhosphate Buffered Saline com Triton x100 0,1% (PBSTx) em temperatura ambiente. </li><li> Diluir fluorescente marcado com anticorpos secundários (geralmente 1:300) em solução de bloqueio. </li><li> Incubar S / N, 4 ° C em solução contendo anticorpos secundários. </li><li> Lavar quatro vezes por 20 minutos em 1XPBSTx na RT. </li><li> Embriões claro em 50% 1XPBS glycerol/50% para pelo menos duas horas em temperatura ambiente, em seguida, remover o glicerol / PBS mistura e adicione glicerol 100% e deixe descansar O / N a 4 ° C. </li></ol> 5. Resultados representativos: Depois de imunomarcação, deve haver uma área enriquecida de coloração nas células que foram uncaged (Figura.1). Não é incomum ver um sinal relativamente alta-ruído em&gt; zebrafish 48 horas de idade, devido a uncaging espontânea da fluoresceína. <img src="/files/ftp_upload/2672/2672fig1.jpg" alt="Figura 1" /> Figura 1. Lineage rotulagem de zebrafish complexo pineal. Aos 24 hpf, uma parte do primórdio anterior pineal, indicado por foxd3:. Expressão GFP, foi uncaged utilizando pulsos de laser UV A) Ao 48hpf, um cluster do lado esquerdo de células chamado de parapineal (círculo vermelho) surgiu a partir da uncaged domínio (círculo branco). B) sinal de fluoresceína Uncaged é enriquecido no círculo (parapineal vermelho) e do órgão pineal (círculo branco).

<ol>
	<li>Hatada, Y., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fate+map+of+the+epiblast+of+the+early+chick+embryo.">A fate map of the epiblast of the early chick embryo.</a> Development. 120, 2879-2889 (1994).</li><li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+the+blastomeres+of+the+32-cell-stage+Xenopus+embryo.">Fates of the blastomeres of the 32-cell-stage Xenopus embryo.</a> Dev Biol. 122, 300-319 (1987).</li><li>Wetts, R., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+Precursors+Can+Give+Rise+to+All+Major+Cell-Types+of+the+Frog+Retina.">Multipotent Precursors Can Give Rise to All Major Cell-Types of the Frog Retina.</a> Science. 239, 1142-1145 (1988).</li><li>Wetts, R., Fraser, S. E. <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+fluorescent+tracers+to+study+neural+cell+lineages.">Microinjection of fluorescent tracers to study neural cell lineages.</a> Development. , 1-8 (1991).</li><li>Woo, K., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Order+and+coherence+in+the+fate+map+of+the+zebrafish+nervous+system.">Order and coherence in the fate map of the zebrafish nervous system.</a> Development. 121, 2595-2609 (1995).</li><li>Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc Natl Acad Sci U S A. 99, 12651-12656 (2002).</li><li>Sato, T., Takahoko, M., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HuC:Kaede,+a+useful+tool+to+label+neural+morphologies+in+networks+in+vivo.">HuC:Kaede, a useful tool to label neural morphologies in networks in vivo.</a> Genesis. 44, 136-142 (2006).</li><li>Wiedenmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EosFP,+a+fluorescent+marker+protein+with+UV-inducible+green-to-red+fluorescence+conversion.">EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.</a> Proc Natl Acad Sci U S A. 101, 15905-15910 (2004).</li><li>Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Fluorescent Probes and Research Chemicals. , 707-711 (2002).</li><li>Kozlowski, D. J., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+cell+movement+and+tissue+patterning+in+the+zebrafish+embryo+revealed+by+fate+mapping+with+caged+fluorescein.">Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein.</a> Biochem Cell Biol. 75, 551-562 (1997).</li><li>Kozlowski, D. J., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+(caged)+fluorescein+as+a+cell+tracer+for+fate+mapping+in+the+zebrafish+embryo.">Photoactivatable (caged) fluorescein as a cell tracer for fate mapping in the zebrafish embryo.</a> Methods Mol Biol. 135, 349-355 (2000).</li><li>Russek-Blum, N., Nabel-Rosen, H., Levkowitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+fate+map+of+the+zebrafish+diencephalon.">High resolution fate map of the zebrafish diencephalon.</a> Dev Dyn. 238, 1827-1835 (2009).</li><li>Summers, R. G., Piston, D. W., Harris, K. M., Morrill, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+orientation+of+first+cleavage+in+the+sea+urchin+embryo,+Lytechinus+variegatus,+does+not+specify+the+axes+of+bilateral+symmetry.">The orientation of first cleavage in the sea urchin embryo, Lytechinus variegatus, does not specify the axes of bilateral symmetry.</a> Dev Biol. 175, 177-183 (1996).</li></ol>

Não há conflitos de interesse declarados.

Como descrito, este protocolo fornece um método de rotulagem relativamente rápido linhagem em zebrafish que é construída sobre as técnicas comumente usadas de microinjeção, microscopia e de imunofluorescência. Nós descobrimos que o laser uncaging ser a forma mais eficiente e de custo eficaz para libertar da gaiola de fluoresceína de uma forma localizada. Este método poderia ser usado para rotular linhagem com pontos de extremidade experimental tão tarde como 4 dpf. No entanto, como as células se dividem, a ...

A correction was made to <a href="http://www.jove.com/details.php?id=2672">Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran</a>. There was an error in the authors, Ilya A. Shestopalov and James K. Chen, names. The author's names have been corrected to: Ilya A. Shestopalov and James K. Chen instead of: Ilya Shestopalov and James Chen

A correction to the article Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran was made.

Erratum: Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<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>CMNB-caged fluorescein SE</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C20050</td>
<td>&nbsp;</td>
<tr>
<td>Aminodextran, 10kDa</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D1860</td>
<td>&nbsp;</td>
<tr>
<td>Zeba desalt spin columns</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>89889</td>
<td>&nbsp;</td>
<tr>
<td>goat anti-fluorescein antibody</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A11095</td>
<td>&nbsp;</td>
<tr>
<td>Alexa Fluor 633 Donkey anti-goat antibody</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A21082</td>
<td>&nbsp;</td>
<tr>
<td>35mmx10mm petri dishes</td>
<td>&nbsp;</td>
<td>Becton-Dickinson</td>
<td>351008</td>
<td>&nbsp;</td>
<tr>
<td>Upright compound microscope with 40x water immersion objective</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>DM6000B</td>
<td>&nbsp;</td>
<tr>
<td>MicroPoint Manual Laser Illumination System</td>
<td>&nbsp;</td>
<td>Photonic Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phenylthiourea (PTU)</td>
<td>&nbsp;</td>
<td>Alfa Aesar</td>
<td>L06690</td>
<td>&nbsp;</td>
<tr>
<td>Tricaine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E10521</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>03115836991</td>
<td>&nbsp;</td>
<tr>
<td>Plastic thread</td>
<td>&nbsp;</td>
<td>Any sewing supply store</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Agarose</td>
<td>&nbsp;</td>
<td>Research Products International</td>
<td>A20090</td>
<td>&nbsp;</td>
<tr>
<td>SpeedVac</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>Savant SPD131DDA</td>
<td>&nbsp;</td>
<tr>
<td>Vortexing mixer</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>Vortex Genie 2</td>
<td>&nbsp;</td>
<tr>
<td>Pressure injector</td>
<td>&nbsp;</td>
<td>Applied Scientific Instrumentation</td>
<td>MPPI-3</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

lineage labeling of zebrafish cells with laser uncagable fluorescein dextran

Um problema central na biologia do desenvolvimento é deduzir a origem da miríade de tipos de células presentes nos vertebrados que possam surgir a partir de precursores indiferenciados. Pesquisadores têm utilizado diversos métodos de rotulagem linhagem, como DII rotulagem 1 e injeção de pressão de enzimas rastreáveis ​​2 para verificar o destino de células em fases posteriores de desenvolvimento em sistemas-modelo. Os mapas destino primeiro peixe-zebra (Danio rerio) foram montados por meio de injeção de iontoforese de corantes fluorescentes, tais como a rodamina dextran, em células isoladas em regiões discretas do embrião e rastreamento destino da célula marcada ao longo do tempo 3-5. Apesar de eficaz, estes métodos são tecnicamente exigentes e que requerem equipamento especializado não comumente encontrados em laboratórios de zebrafish. Recentemente, photoconvertable proteínas fluorescentes, como o Eos e Kaede, que irreversivelmente mudar de verde para vermelho quando fluorescência expostos à luz ultravioleta, estamos vendo maior utilização em zebrafish 6-8. A claridade óptica do embrião zebrafish ea relativa facilidade de transgênese têm feito essas ferramentas particularmente atraente para a rotulagem de linhagem e de observar a migração das células in vivo 7. Apesar de sua utilidade, essas proteínas têm algumas desvantagens em comparação com corante mediada métodos de rotulagem linhagem. Crucial a maioria é a dificuldade que temos encontrado na obtenção de resolução em 3-D de alta durante fotoconversão destas proteínas. A esta luz, talvez a melhor combinação de resolução e facilidade de uso para a rotulagem linhagem em zebrafish faz uso de gaiolas de fluoresceína dextran, um corante fluorescente que é vinculado a um grupo de têmpera que mascara seus 9 de fluorescência. O corante pode ser &quot;uncaged&quot; (lançado a partir do grupo quenching) dentro de uma célula específica usando a luz UV de uma lâmpada a laser ou mercúrio, permitindo a visualização de sua fluorescência ou imunodetecção. Ao contrário dos métodos de iontoforese, fluoresceína enjaulado pode ser injetado com aparelhos de injeção padrão e uncaged com um microscópio de epifluorescência equipado com uma pinhole 10. Além disso, anticorpos contra fluoresceína detectar apenas a forma uncaged, eo epítopo sobrevive bem fixação 11. Finalmente, fluoresceína enjaulado pode ser ativada com muito alta resolução 3-D, especialmente se a microscopia de dois fótons é empregada 12,13. Este protocolo descreve um método de rotulagem linhagem de fluoresceína enjaulado e uncaging laser. Subsequenctly, uncaged fluoresceína é detectado simultaneamente com outros epitopos, como GFP pela marcação com anticorpos.

Watch this Scientific Journal Video about Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran at JoVE.com

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

Este protocolo delineia uma forma de rótulo e traçar o destino de pequenos grupos de células de embriões zebrafish utilizando UV-uncaging de fluoresceína enjaulado, seguido por montar todo imunomarcação para amplificar o sinal da fluoresceína uncaged.

Rotulagem de linhagem de células Zebrafish com Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

lineage-labeling-zebrafish-cells-with-laser-uncagable-fluorescein

Research

JoVE Journal

Biology

13.8K visualizações.  Vanderbilt University. Este protocolo delineia uma forma de rótulo e traçar o destino de pequenos grupos de células de embriões zebrafish utilizando UV-uncaging de fluoresceína enjaulado, seguido por montar todo imunomarcação para amplificar o sinal da fluoresceína uncaged.

Vídeo: Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

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

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

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

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 ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Ablação a laser da Pronephros Zebrafish para estudo da regeneração epitelial renal

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3.
Yet their existence is still a matter of controversy4,5. Indeed, lipid raft size has been estimated to be around 20 nm6, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells.
Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11.
FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12.

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.

Determinação da Raft Lipid Particionamento de Sondas marcadas com fluorescência em células vivas por Espectroscopia de Correlação de Fluorescência (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Análise fenotípica e Isolamento de Células-Tronco Hematopoéticas Murino e Lineage comprometidos Progenitores

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Laser-infligido Lesão de Zebrafish muscular esquelético embrionário

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotoxina microinjeção no peixe-zebra ao modelo de lesão renal aguda

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser radiation is strongly absorbed by water, and the cells at the bottom of the culture dish are heated through thermal diffusion. A laser fiber with a diameter of 365 &#181;m is set about 12 cm above the culture dish, without any optics, such that the laser beam diameter is almost equivalent to the inner diameter of the culture dish (30 mm). By keeping a consistent amount of culture medium in each experiment, it is possible to irradiate the cells with a highly reproducible temperature increase.
To calibrate the temperature increase and its distribution in one cell culture dish for each power setting, the temperature was measured during 10 s of irradiation at different positions and at the cellular level. The temperature distribution was represented using a mathematical graphics software program, and its pattern across the culture dish was in Gaussian form. After laser irradiation, different biological experiments could be performed to assess temperature-dependent cell responses. In this manuscript, viability staining (i.e., distinguishing live, apoptotic, and dead cells) is introduced to help determine the threshold temperatures for cell apoptosis and death after different points in time.
The advantages of this method are the preciseness of the temperature and the time of heating, as well as its high efficiency in heating cells in a whole cell culture dish. Furthermore, it allows for study with a wide variety of temperatures and time durations, which can be well-controlled by a computerized operating system.

An original experimental setup for heating cells in a culture dish using 1.94 &#181;m continuous-wave laser radiation is introduced here. Using this method, the biological responses of retinal pigment epithelial (RPE) cells after different thermal exposures can be investigated.

Laser de Thulium de onda contínua para aquecimento de células cultivadas para investigar efeitos térmicos celulares

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

Visualização e quantificação de células mesenquimais Adipogenic diferenciação potencial com um marcador específico da linhagem

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

Aplicação do movimento passivo da cabeça para gerar acelerações definidas nas cabeças dos roedores

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...