JB foi apoiado pelo Damon Runyon Cancer Research Foundation Fellowship DRG-1716-1702. JZ foi apoiado pelo NIH / NIGMS Fellowship GM082174 F32-02. NP é um investigador do Instituto Médico Howard Hughes.

1. Preparação de gaiolas voar <ol><li> Crescer Drosophila (linhagens selvagens, como Oregon-R ou qualquer genótipo) em recipientes convenientes, tais como copos de 32 oz de insetos. </li><li> Transferência de moscas recém eclosed a grande colheita de embriões em gaiolas ou voar gaiolas população. </li><li> Mover as gaiolas em um quarto com uma temperatura de 25 ° C e umidade de 65% ~ em uma luz de 12 h, e um ciclo de 12 h escuro para facilitar a coleta de embriões síncrona. </li><li> Manter as moscas nas gaiolas, alimentando-os mortos colar fermento semeado em placas de melaço. Mudar as placas de melaço (com pasta de leveduras mortas) uma vez ao dia por 2-3 d. </li></ol> 2. Colheita de embriões síncrona <ol><li> No dia da primária de células, preparação alimentar as moscas wth placas revestidas com fermento fresco morto colar para um pré-lay h 1-2 período. </li><li> Pouco antes do ciclo de 12 h escuro, substitua as placas pré deitou com placas de melaço doce listradas com uma quantidade mínima de pasta de levedura. Descartar a coleção pré leigos, que contém assíncrona embriões mais velhos. </li><li> Coletar embriões por um período de 1-2 h. </li><li> Remova as placas contendo a população relativamente síncrona de embriões e armazenar a 25 ° C por 5-6 h. Embriões será no estádio de gástrula avançados neste momento. </li></ol> 3. Coleta e lavagem de embriões <ol><li> Solte os embriões das placas com um pincel molhado, e enxaguar abundantemente com água da torneira morna através de um conjunto de peneiras empilhadas, de modo que os embriões recolher na peneira de malha mais fina na parte inferior. Enxaguar por alguns minutos para remover o fermento e detritos voam muito possível. </li><li> Dica para as peneiras que os embriões se acumulam de um lado, e gentilmente lavá-las a partir da peneira para uma cesta dechorionation. </li><li> Mover para a área de cultura de tecidos capô, sem deixar que os embriões secar. </li></ol> 4. Homogeneização de embriões e revestimento de células <ol><li> Esterilizar e dechorionate os embriões por imersão em 50% (vol / vol) lixívia para 5-10 min. Enxágüe os embriões fora do muro da cesta com 70% (vol / vol) de etanol. Alternativamente, lave a lixívia off embriões com água destilada antes do tratamento de etanol. Deste ponto em diante os embriões devem ser tratados em condições estéreis e no capô cultura de tecidos. </li><li> Enxágüe os embriões completamente para remover a água sanitária e / ou etanol com água destilada autoclavada. </li><li> Durante a etapa de dechorionation, preencher um homogeneizador Dounce com o volume apropriado de meio ambiente M3 temperatura com base na quantidade de embriões. A relação entre o volume de embriões e de meios de comunicação não deve ser superior a 0,5 ml embriões: 40 ml de mídia (ou ~ 100-200 embriões / ml). </li><li> Coloque os embriões lavado num copo esterilizados com a quantidade de M3 media suficiente para submergir os embriões. Deixe o molho por 2-5 embriões min. </li><li> Desmonte da cesta dechorionation e blot a malha Nitex seco com toalhas de papel esterilizados. </li><li> Transferência dos embriões para o homogeneizador com um pincel esterilizados. </li><li> Homogeneizar suavemente com movimentos curtos usando um pilão solta sem atingir o fundo do tubo até que todos os embriões são suspensas. Em seguida, suavemente, mas firmemente, homogeneizar com oito golpes completo. Não torça o pilão como ele se move para cima e para baixo. </li><li> Transferir o homogeneizado em um tubo de centrifugação estéril de 50 ml cônico ou por derramamento ou pipetagem, ea tampa do tubo. </li><li> Centrifugar por 10 min a 40g, a temperatura ambiente até o pellet restos de tecido, pedaços grandes células e membranas vitelínico. Transferir o sobrenadante contendo as células e gema para um tubo limpo e repita os passos de centrifugação por 5 min. </li><li> Transferir cuidadosamente o sobrenadante por derramamento ou pipetando para um tubo limpo e gire por 10 minutos a 360g, temperatura ambiente, para sedimentar as células. Desprezar o sobrenadante. Ressuspender o pellet celular em 10 ml de meio de cultura (sem soro meio de experimentos RNAi). </li><li> Contagem de células viáveis ​​utilizando um hemocitômetro para estimar a densidade das células. Enquanto isso, repita o passo 4,10 para agregar as células. </li><li> Volte a suspender as células a uma concentração desejada. Para começar, tente um intervalo de 1 ~ 5 x10 6 células / ml e placa para fora em 1,7-2,5 x10 5 células / cm 2. </li></ol> 5. Primárias de células RNAi para células cultivadas em uma placa de 384 poços <ol><li> Dispense 5 mL de dsRNAs em cada poço a uma concentração final de ~ 250 ng por poço em uma placa de 384 poços. </li><li> Use uma pipeta multi-canal, dispensar 10 ml (1 ~ 4x106 células / ml) de pilhas por poço em meio M3 sem soro em uma placa de 384 poços contendo um dsRNA diferentes em cada poço. Alternativamente, as células podem ser cultivadas em 8 de bem-câmara de lâminas de vidro para imagens confocal. </li><li> Centrifugar as placas durante 1 min em 300g, temperatura ambiente. Cultura as células em meio livre de soro a 25 ° C por 22 h ou a 18 ° C por 1-2 d. </li><li> Adicionar 30 ml de soro contendo meio de cultura em cada poço. </li&gt; <li> Centrifugar as placas durante 1 min em 300g, temperatura ambiente. </li><li> Coloque as placas em câmara úmida e cultura as células primárias para um adicional de 5 ~ 7 d a 25 ° C. </li><li> Imagem das células ao vivo ou depois de imunofluorescência. </li></ol> 6. Resultados representativos: Em condições ideais, as células na cultura vai olhar saudável, com uma morfologia rodada. A cultura terá restos celulares pouco, e de 50 confluentes ~ 80% após o início do cultivo. Estas células sofrerão alterações morfológicas após ser cultivadas por um longo período de tempo. Ele normalmente leva cerca de 5-8 dias para as células na cultura a ser totalmente diferenciada, a 25 ° C. Culturas primárias de boa qualidade geralmente são julgadas por quão bem as células de interesse são diferenciados em cultura. Por exemplo, células musculares primárias muitas vezes têm uma morfologia ramo e tornar-se-like miotubos multinucleados com listra-like miofibrilas consistem de uma série de sarcômeros. Miotubos totalmente diferenciadas, normalmente de forma espontânea mover-se na cultura. Em contraste, diferenciada neurônios primários formam aglomerados com seus corpos celulares, e se conectar com outros clusters com cabos de seus axônios. <img src="/files/ftp_upload/2215/2215fig1.jpg" alt="Figura 1" /> Figura 1. Pilhas sofrer alterações morfológicas após ser banhado na cultura (A) A imagem de campo claro de pilhas direita depois de ser banhado em um poço de uma placa de 384 poços. A maioria das células são redondas e intacta e bem separados. (B) Uma imagem de campo claro da cultura de células primárias 20 horas após o plaqueamento. Pilhas já formam aglomerados (grande ponta de seta) e músculos com uma morfologia ramo-like (seta longa) pode ser reconhecida nesta fase. (C) Uma imagem de contraste de fase de cultura primária de cinco dias após o plaqueamento. A cultura parece muito mais avançada, com extensões neuronal (setas pequenas), clusters neuronal (setas grandes) e músculos (médias seta). <img src="/files/ftp_upload/2215/2215fig2.jpg" alt="Figura 2" /> Figura 2. Exemplos de pilhas tratadas com dsRNAs cultivadas em uma placa de 384 poços Pilhas foram isolados a partir da embriões portadores de Dmef2-Gal4, D42-Gal4, UAS-mito-GFP transgenes, em que Dmef2-Gal4 e D42-Gal4 expressão da unidade do mito-GFP nos músculos e neurônios motores, respectivamente. As células foram tratadas com dsRNAs segmentação tanto lacZ (AC) ou inflados (se) (DF). Ambos os músculos primários e de neurônios pode ser visto por GFP (A, C, D, F). As setas brancas apontam para as extensões neuronal. Faloidina coloração de actina (setas) revela uma estrutura muscular ramo, como na cultura tratada com lacZ dsRNAs (B e C), mas round-up morfologia muscular na cultura tratada com se dsRNAs (E e F). Nas imagens mescladas (C, F), os músculos estão manchadas amarelo, mas vermelho neurônios negativos mostra e suas extensões (setas brancas). <img src="/files/ftp_upload/2215/2215fig3.jpg" alt="Figura 3" /> Figura 3. Micrografias confocal de músculos primários cultivados em humanos vitronectina revestido slides tampa de vidro Músculos primários foram immunofluorescently corados com anti-Drosophila titina (KZ) (vermelho na mesclagem) e com phalloidin para actina (verde na mala direta). Os painéis de topo são o músculo controle do tipo selvagem e as de fundo são as cessões (CG31374) RNAi muscular com sarcômeros encurtado.

<ol>
	<li>Ashburner, M., Roote, J., Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory culture of Drosophila. , 588-599 (1999).</li><li>Bai, J., Hartwig, J. H., Perrimon, N. S. A. L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+WH2-domain-containing+protein,+promotes+sarcomeric+actin+filament+elongation+from+pointed+ends+during+Drosophila+muscle+growth.">a WH2-domain-containing protein, promotes sarcomeric actin filament elongation from pointed ends during Drosophila muscle growth.</a> Dev. Cell. 13, 828-842 (2007).</li><li>Bai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+screening+in+Drosophila+primary+cells+for+genes+involved+in+muscle+assembly+and+maintenance+.">RNA interference screening in Drosophila primary cells for genes involved in muscle assembly and maintenance .</a> Development. 135, 1439-1449 (2008).</li><li>Sepp, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neural+outgrowth+genes+using+genome-wide+RNAi.">Identification of neural outgrowth genes using genome-wide RNAi.</a> PloS. Genet. 4, e1000111-e1000111 (2008).</li><li>Donady, J. J., Fyrberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+culturing+of+Drosophila+embryonic+cells+in+vitro.">Mass culturing of Drosophila embryonic cells in vitro.</a> TCA Manual. 3, 685-687 (1977).</li><li>Seecof, R. L., Alleaume, N., Teplitz, R. L., Gerson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+neurons+and+myocytes+in+cell+cultures+made+from+Drosophila+gastrulae.">Differentiation of neurons and myocytes in cell cultures made from Drosophila gastrulae.</a> Exp. Cell Res. 69, 161-173 (1971).</li><li>Bai, J., Sepp, K. J., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+Drosophila+primary+cells+dissociated+from+gastrula+embryos+and+their+use+in+RNAi+screening.">Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening.</a> Nature Protocols. 4, 1502-1512 (2009).</li></ol>

Não há conflitos de interesse declarados.

O padrão de desenvolvimento de culturas primárias de células Drosophila pode variar muito, possivelmente devido a diversas variáveis ​​durante o processo primário de células de preparação. Primeiro de tudo, voa usado para postura de ovos devem ser jovens (dentro de uma semana de idade) e saudável (livre de infecção viral ou bacteriana). Embriões não fertilizados ou infectado devem ser evitados, já que são inúteis e células derivadas de embriões não os pode diferenciar e só vai sobreviver...

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		<th>Catalogue Number</th>
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<tr>
<td>Shields and Sang M3 medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3652</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>JRH Biosciences</td>
<td>12103-500M</td>
<td>&nbsp;</td>
<tr>
<td>Insulin from bovine pancreas</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I-6634</td>
<td>&nbsp;</td>
<tr>
<td>Large embryo collection cages</td>
<td>&nbsp;</td>
<td>Genesee Scientific</td>
<td>59-101</td>
<td>&nbsp;</td>
<tr>
<td>#25 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-454</td>
<td>&nbsp;</td>
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<td>#45 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-462</td>
<td>&nbsp;</td>
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<td>#120 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-474</td>
<td>&nbsp;</td>
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<td>Dounce tissue grinder, Wheaton 7 mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>62400-620</td>
<td>&nbsp;</td>
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<td>Dounce tissue grinder,Kontes 40 mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>KT885300-0040</td>
<td>&nbsp;</td>
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<td>Dounce tissue grinder,Kontes 40 mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>KT885300-0100</td>
<td>&nbsp;</td>
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<td>Costar, 384-well plate</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>29444-078</td>
<td>&nbsp;</td>
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<td>Lab-Tek II Chambered coverglass</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
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primary cell cultures from drosophila gastrula embryos

Aqui nós descrevemos um método para a preparação e a cultura de células primárias dissociada de embriões Drosophila gástrula. Em suma, uma grande quantidade de embriões de moscas encenado jovens e saudáveis ​​são recolhidos, esterilizados, e depois fisicamente dissociada em uma suspensão única célula usando um homogeneizador de vidro. Depois de ser semeadas em placas de cultura ou slides câmara em uma densidade adequada em meio de cultura, essas células podem diferenciar em vários tipos de células morfologicamente-distintas, que podem ser identificadas por seus marcadores celulares específicos. Além disso, apresentamos as condições para o tratamento destas células com fita dupla (ds) RNAs para provocar knockdown do gene. RNAi em células de Drosophila eficiente primário é realizado por simplesmente banhar as células do dsRNA contendo meio de cultura. A capacidade de realizar perturbação RNAi eficaz, juntamente com outros molecular, bioquímica, análises de imagens de células, permitirá uma série de perguntas a serem respondidas em células de Drosophila primária, especialmente aquelas relacionadas ao músculo diferenciadas e células neuronais.

Watch this Scientific Journal Video about Primary Cell Cultures from Drosophila Gastrula Embryos at JoVE.com

Primary Cell Cultures from Drosophila Gastrula Embryos

Nós fornecemos um protocolo detalhado para preparar células primárias dissociada<em&gt; Drosophil</em&gt; A embriões. A capacidade de realizar a perturbação RNAi eficaz, juntamente com outros métodos de imagem molecular, bioquímica e celular permitirá que uma variedade de questões a serem abordadas na<em&gt; Drosophila</em&gt; Pilhas.

Culturas de células primárias de Drosophila Os embriões Gástrula

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

primary-cell-cultures-from-drosophila-gastrula-embryos

Research

JoVE Journal

Biology

Primary Cell Cultures from Drosophila Gastrula Embryos

13.8K visualizações.  Harvard Medical School. Nós fornecemos um protocolo detalhado para preparar células primárias dissociada Drosophil A embriões. A capacidade de realizar a perturbação RNAi eficaz, juntamente com outros métodos de imagem molecular, bioquímica e celular permitirá que uma variedade de questões a serem abordadas na Drosophila Pilhas.

Vídeo: Primary Cell Cultures from Drosophila Gastrula Embryos

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

Culturas primárias neuronais do cérebro de Tarde Stage Drosophila pupas

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Biologia

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Preparação de culturas de neurônios a partir de embriões Midgastrula Stage Drosophila

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Principal Dissociated Midbrain dopamina culturas de células de recém-nascidos de roedores

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Tecido alvo quimeras embrionárias: Zebrafish celular Gástrula Transplante

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

Estabelecer culturas de fibroblastos adultos primária de roedores

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Imagem vertical de Drosophila Os embriões

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

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 ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Isolamento e Purificação de cinesina partir Drosophila Os embriões

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells isolamento de culturas primárias de fibroblastos humanos

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...