Preparação para a cirurgia Parte I. <ol><li> O dia antes da cirurgia de ratos, a certeza de que todas as soluções são feitas, mas não adicione nenhum enzimas ainda. Buffers A, B e do tampão de lavagem deve ser filtrado para todas as esterilidade antes do armazenamento. Os buffers devem ser todos armazenados a 4 ° C. </li><li> 6-bem placas de cultura de tecido deve ser revestida com 10-20 mcg / mL laminina em 2 mL 1XSFM no dia anterior, bem como, e deve ser armazenado a 37 ° C em uma incubadora de CO 2. </li><li> No dia da cirurgia, preparar os instrumentos cirúrgicos com a esterilização do grampo, tesouras e pinças com etanol 70% e deixe-os secar em papel toalha. </li><li> Uma seringa de 1 mL preenchidos com 0,5 mL da solução de heparina (90 U / ml) é preparado. </li><li> Agora é uma boa prática de lavar o aparelho de perfusão executando através de etanol 70%, usando a bomba peristáltica em sentido inverso. Uma vez terminado lavagem com etanol, enxaguar o aparelho com água destilada duas vezes e, finalmente, enxaguar com tampão A. O fluxo é coletado através de um copo colocado em banho-maria a 37 ° C. </li><li> Para preparar o aparelho de perfusão limpas, encher o tubo da seringa direita com 40 mL de tampão A e deixou o tubo da seringa com 40 mL de tampão B. </li><li> Primeiro o tubo de perfusão para a ponta do cateter, permitindo tampão A para fluir através do aparelho. Refil para o buffer de uma seringa ao nível de 40 ml. Certifique-se que não há bolhas de ar aprisionadas na tubulação após esta etapa. </li><li> Agora, feche o buffer Uma válvula torneira seringa e repita o processo com tampão B tal que o tubo de perfusão está agora preparado com tampão B. </li><li> Finalmente, o aparelho de perfusão fica com a torneira B buffer válvula aberta, mas com o fluxo parou de usar o regulador na linha IV. </li><li> Descartar o buffer de fluxo, embora no copo de coleta. </li><li> A última coisa a fazer antes da cirurgia é adicionar as enzimas necessárias à sua solução estoque, e, em seguida, filtrar a solução enzimática, assim como as outras soluções foram filtradas no dia anterior. Uma vez filtrado, esta solução pode ser deixado à temperatura ambiente. </li></ol> Parte II. A dissecção do coração de rato <ol><li> Protocolo padrão seguinte euthanize o rato com isoflurano em uma câmara de gás. </li><li> Esterilizar a área da incisão no tórax com etanol 70%. </li><li> Peito aberto com uma tesoura e expor o coração. </li><li> Injetar ventrículo esquerdo com 0,5 mL de solução de heparina (90 U / mL). </li><li> Remover coração com grande parcela de aorta lugar, intacto no gelo frio tampão B. </li></ol> Parte III. Isolamento miócitos <ol><li> Um coração de rato típico deve render uma grande fracção de miócitos em forma de bastonete (ao contrário de células mortas arredondado) se o procedimento a seguir é devidamente respeitado. </li><li> Para começar, clamp aorta ao cateter. A ponta do cateter não deve ser levada muito longe dentro do coração para garantir boa perfusão do coração pela artéria coronária. </li><li> Uma vez preso, gravata da aorta para o cateter com uma sutura. </li><li> Em seguida, abra o regulador da linha IV para permitir uma taxa de gotejamento rápido (~ 60 gotas / min) e permitirá B Buffer para lavar o coração. </li><li> Durante tampão de lavagem B, use a tesoura pequena e uma pinça para remover os átrios e qualquer tecido adiposo ou pulmão apego ao coração. </li><li> Depois de tampão B terminar, mude o fluxo de tampão A. </li><li> Enquanto tampão A é permitido fluir por 5 minutos, várias pequenas tarefas devem ser realizadas rapidamente. </li><li> Primeiro, aquecer a solução enzimática em um banho de água 37 ° C. </li><li> Finalmente, quando um buffer é esgotado da seringa (mas ainda presente no tubo), carga de 50 mL da solução enzimática em seringa do aparelho de perfusão </li><li> Agora é necessário descartar todas as fluxo através do copo coleção em banho-maria (pipetando), até que o coração Enzyme perfunde Solution. </li><li> Assim que a solução de enzima perfunde o coração, ative a bomba peristáltica que é configurado para transferir a solução enzimática do copo coleta para reabastecer seringa A. </li><li> Permitir a solução de enzima a fluir através do coração por 10 min. Como o coração digere, ele começa a olhar inchado. </li><li> Adicionar 37,5 mL de 0,1 M CaCl 2 para a solução de enzima em seringa A para dar uma concentração efetiva de 0,1 mM Ca 2 +. </li><li> Após 10 minutos aumentar a concentração de cálcio para 0,2 mM adicionando um adicional de 50 mL de 0,1 M CaCl 2 a seringa A e deixe a perfusão continuar por mais 10 minutos. </li><li> Cortar os ventrículos e transferir para um copo pequeno estéril contendo 20 mL da solução enzimática. </li><li> No copo aumentar a concentração de cálcio para 0,4 mM, adicionando mais 40 mL de 0,1 M CaCl 2. </li><li> Suavemente mince o coração em 10 ou mais peças com um par de pequenas tesouras esterilizadas. </li><li> Incubar o copo por 5 minutos a 37 ° C com balanço delicado. </li><li> Adicionar 40 mL de 0,1 M CaCl 2 adar concentração efetiva de 0,6 mM Ca 2 + e gentilmente triturar os pedaços do coração com uma pipeta de transferência de plástico 3-5 vezes. </li><li> Após incubação por mais 5 minutos a 37 ° C, adicionar 40 mL de 0,1 M CaCl 2 e gentilmente triturar como antes. </li><li> Separe digerida miócitos única de não-tecido conjuntivo digerido utilizando filtração através de uma malha mM estéril 500. </li><li> Permitir que as células se estabelecer em um tubo de 50 ml por 10 minutos em temperatura ambiente. </li><li> Elimine o sobrenadante com uma pipeta de transferência. </li><li> Delicadamente ressuspender as células em tampão de lavagem # 1 e permitir que as células de se contentar com 10-20 min em temperatura ambiente. </li><li> Enquanto as células estão se instalando, tomar uma alíquota pequena, e usar este tempo como uma oportunidade para avaliar a qualidade ea viabilidade das células em suspensão em um microscópio invertido. Uma boa preparação terá uma alta proporção (&gt; 80%) da haste, como as células com estrias nítidas. </li><li> Quando as células se instalaram, descarte as células sobrenadante e gentilmente ressuspender em tampão de lavagem 2. Vamos resolver as células à temperatura ambiente, que devem voltar a tirar 10-20 minutos, e desprezar o sobrenadante. </li><li> Repita o processo de lavagem mais uma vez com tampão de lavagem # 3, e as células estarão prontos para a cultura. </li></ol> Parte IV. Cultura de células miócitos <ol><li> Delicadamente ressuspender as células em 5% de soro de mídia. Antes de transferir as células a placas de cultura de tecidos, lavar os pratos com 1x SFM. Transferência das células com uma densidade desejada e incubar em um CO 2 de cultura de tecidos incubadora de 3-5 horas. </li><li> Mudar a mídia para 1x SFM. </li><li> As células podem ser cultivadas por até quatro dias e usado como necessária para os experimentos. </li></ol> Parte V. Representante Resultados / Resultados Deve haver mais de 70% miócitos cardíacos vivem sob microscópio invertido quando o protocolo é feito corretamente. <img src="/files/ftp_upload/1308/fig1.jpg" alt="figura 1" /> Tabela 1: Receita Solução para 1 L 10X Ca 2 + livre de buffer KH: <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> massa </td><td> Concentração Final </td></tr><tr><td> NaCl </td><td> 68,96 g </td><td> 1180 mM </td></tr><tr><td> KCl </td><td> 3,58 g </td><td> 48 mM </td></tr><tr><td> HEPES </td><td> 59,58 g </td><td> 250 mM </td></tr><tr><td> K 2 HPO 4 </td><td> 2,85 g </td><td> 12,5 mM </td></tr><tr><td> MgSO 4 </td><td> 3,08 g </td><td> 12,5 mM </td></tr></tbody></table> Tabela 2: Receita solução para um buffer L A: Adicionar 1,98 g de glicose a 100 mL de tampão KH 10X e levar a um volume final de 1 L. Ajuste osmolaridade à condição de fisiologia próximo (~ 300) com a glicose. Trazer para pH 7,4 com NaOH. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> - </td><td> Massa </td><td> Concentração Final </td></tr><tr><td> Tampão KH 10X </td><td> 100 mL </td><td> 100 ml </td></tr><tr><td> Glicose </td><td> 1,98 g </td><td> 11 mM </td></tr><tr><td> Trazer para 1 L </td><td></td><td></td></tr><tr><td> Trazer para pH 7,4 com NaOH </td><td></td><td></td></tr><tr><td colspan="3"> Ajuste osmolaridade à condição fisiológica próximo (~ 300) com a glicose. </td></tr></tbody></table> Tabela 3: Receita Solução para Buffers Enzyme <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> massa </td><td> Concentração Final </td></tr><tr><td> Colagenase tipo II </td><td> 25 mg </td><td> 0,05% (w / v) </td></tr><tr><td> Protease XIV </td><td> 10 mg </td><td> 0,02% (w / v) </td></tr><tr><td> BDM </td><td> 0,025 g </td><td> 5 mM </td></tr><tr><td> Carnitina </td><td> 0,020 g </td><td> 2 mM </td></tr><tr><td> Taurina </td><td> 0,031 g </td><td> 5 mM </td></tr><tr><td> Ácido glutâmico </td><td> 0,020 g </td><td> 2 mM </td></tr><tr><td> 0,1 M CaCl 2 </td><td> 12,5 uL </td><td> 25 uM </td></tr><tr><td> Um buffer </td><td></td><td> Preencha a 50 ml </td></tr></tbody></table> Tabela 4: Receita Solução para BDM 25X / Taurina / BSA (B / T / B) <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> massa </td><td> Concentração Final </td></tr><tr><td> BDM </td><td> 0,076 g </td><td> 125 mM </td></tr><tr><td> Taurina </td><td> 0,094 g </td><td> 125 mM </td></tr><tr><td> BSA </td><td></td><td> 0,1% (w / v) </td></tr><tr><td> Um buffer </td><td></td><td> Preencha a 6 mL </td></tr></tbody></table> Tabela 5: Receita Solução B Buffer e Buffers Wash <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Buffer: </td><td> B </td><td> # 1 </td><td> # 2 </td><td> # 3 </td></tr><tr><td> Adicionar: </td><td></td><td></td><td></td><td></td></tr><tr><td> 0,5 M CaCl 2 </td><td> 0,4 mL </td><td> 0,1 mL </td><td> 0,125 mL </td><td> 0,15 mL </td></tr><tr><td> Concentração final de CaCl 2 </td><td> 1 mM </td><td> 1 mM </td><td> 1,25 mM </td><td> 1,5 mM </td></tr><tr><td> 25X B / T / B </td><td> --- </td><td> 2 mL </td><td> 2 mL </td><td> 2 mL </td></tr><tr><td> Um buffer de volume para a final </td><td> 200 mL </td><td> 50 mL </td><td> 50 mL </td><td> 50 mL </td></tr></tbody></table> Tabela 6: Receita Solução para meios de cultura <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 2X sem soro Media (SFM) </td><td></td><td></td></tr><tr><td rowspan="2"> </td><td rowspan="2"></td><td> Concentração Final </td></tr><tr><td> (Após a diluição) </td></tr><tr><td> Carnitina </td><td> 0,099 g </td><td> 10 mM (5 mM) </td></tr><tr><td> Taurina </td><td> 0,063 g </td><td> 10 mM (5 mM) </td></tr><tr><td> Creatina </td><td> 0,075 g </td><td> 10 mM (5 mM) </td></tr><tr><td> Antibiótico / antimicótico </td><td> 0,5 ml </td><td> 1% (0,5%) (v / v) </td></tr><tr><td> Mídia 199 </td><td> 45 mL </td><td> Preencha a 50 mL </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 1X SFM </td><td></td><td></td></tr><tr><td></td><td></td><td> Concentração Final </td></tr><tr><td> 2X SFM </td><td> 25 mL </td><td> 1X </td></tr><tr><td> Mídia 199 </td><td></td><td> Preencha a 50 mL </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 1X de 5% de soro de mídia </td><td></td><td></td></tr><tr><td></td><td></td><td> Concentração Final </td></tr><tr><td> 2X SFM </td><td> 25 mL </td><td> 1X </td></tr><tr><td> FBS </td><td> 2,5 mL </td><td> 5% (v / v) </td></tr><tr><td> Mídia 199 </td><td></td><td> Preencha a 50 mL </td></tr></tbody></table>

<ol>
	<li>Miriyala, J., Nguyen, T., Yue, D. T., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CaVbeta+subunits,+and+lack+of+functional+reserve,+in+protein+kinase+A+modulation+of+cardiac+CaV1.2+channels.">Role of CaVbeta subunits, and lack of functional reserve, in protein kinase A modulation of cardiac CaV1.2 channels.</a> Circ Res. 102 (7), e54-e64 (2008).</li><li>SX, T. a. k. a. h. a. s. h. i., Mittman, S., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+modulatory+effects+of+five+human+auxiliary+beta2+subunit+splice+variants+on+L-type+calcium+channel+gating.">Distinctive modulatory effects of five human auxiliary beta2 subunit splice variants on L-type calcium channel gating.</a> Biophys. , 84-845 (2003).</li></ol>

Um passo crítico é a velocidade com que o coração isolado é preso no sistema de perfusão. A duração do período de digestão enzimática pode ser um pouco diferente para cada rato. O ajuste depende de como o coração se torna macia após o período regular de digestão. A recuperação lenta de Ca 2 + após digestão enzimática é essencial para a obtenção de Ca 2 + tolerante células saudáveis. Para cobaia, o protocolo é o mesmo, exceto hialuronidase é u...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S6191</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9541</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4034</td>
<td>&nbsp;</td>
<tr>
<td>K2HPO4</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9666</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>63068</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7021</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7902</td>
<td>&nbsp;</td>
<tr>
<td>Heparin Sodium Salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4784</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase Type II</td>
<td>&nbsp;</td>
<td>Worthington Biochemical</td>
<td>LS004176</td>
<td>&nbsp;</td>
<tr>
<td>Protease XIV</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5147</td>
<td>&nbsp;</td>
<tr>
<td>2,3-Butanedione Monoxime (BDM)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B0753</td>
<td>&nbsp;</td>
<tr>
<td>Carnitine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C9500</td>
<td>&nbsp;</td>
<tr>
<td>Taurine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T0625</td>
<td>&nbsp;</td>
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<td>Glutamic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1501</td>
<td>&nbsp;</td>
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<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3294</td>
<td>&nbsp;</td>
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<td>Creatine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C3630</td>
<td>&nbsp;</td>
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<td>Antibiotic</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-009-CI</td>
<td>&nbsp;</td>
<tr>
<td>Media 199</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11150</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>354232</td>
<td>&nbsp;</td>
<tr>
<td>water bath</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-462-5</td>
<td>&nbsp;</td>
<tr>
<td>variable flow chemical pump</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-077-67</td>
<td>&nbsp;</td>
<tr>
<td>6-well culture plate</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3516</td>
<td>&nbsp;</td>		</tbody>
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primary culture of adult rat heart myocytes

Cultura primária adulto células do coração de roedores são um sistema modelo importante para a pesquisa cardiovascular. No entanto, o estabelecimento de robusta, viável miócitos adulto culto pode ser um desafio técnico, passo limitante para muitos pesquisadores. Aqui nós descrevemos um protocolo para obter um alto rendimento de miócitos de ratos adultos coração que permanecem viáveis ​​em cultura por vários dias. O coração é isolado e perfundido com colagenase e protease em Ca baixo<sup&gt; 2 +</sup&gt; Condições para recuperar miócitos único. Ca<sup&gt; 2 +</sup&gt; Tolerante células são obtidas por um aumento gradual no Ca extracelular<sup&gt; 2 +</sup&gt; Concentração em três etapas de lavagem subseqüente. Células são filtrados, ressuspenso em meio de cultura, e banhado em deslizamentos laminina revestido. Miócitos cultivadas obtidos utilizando este protocolo são viáveis ​​por até quatro dias e são adequados para a maioria dos experimentos, incluindo eletrofisiologia, bioquímica, biologia molecular e de imagem.

Watch this Scientific Journal Video about Primary Culture of Adult Rat Heart Myocytes at JoVE.com

Primary Culture of Adult Rat Heart Myocytes

Neste artigo, descrevemos uma forma típica para isolar e cultura miócitos adulto coração de rato. Colagenase e protease são usadas para digerir e isolar miócitos único. Miócitos cultivadas seguir este protocolo de responder às necessidades mais experiência.

Cultura primária de Miócitos Coração Adulto Rat

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

primary-culture-of-adult-rat-heart-myocytes

Research

JoVE Journal

Biology

20.1K visualizações.  Columbia University. Neste artigo, descrevemos uma forma típica para isolar e cultura miócitos adulto coração de rato. Colagenase e protease são usadas para digerir e isolar miócitos único. Miócitos cultivadas seguir este protocolo de responder às necessidades mais experiência.

Vídeo: Primary Culture of Adult Rat Heart Myocytes

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

Cultura organotípicas de Retina Coelho Adulto

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

Biologia

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Implantação de engenharia de tecidos no coração de ratos

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

Isolamento e manipulação genética de adultos miócitos cardíacos for Imaging Confocal

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

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 ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

Preparação de coração de rato altamente acoplado mitocôndrias

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Isolamento e cultura primária de células hepáticas de ratos

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Métodos para o isolamento, Cultura e caracterização funcional de SINOATRIAL Nó miócitos de ratos adultos

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

Isolamento Simultâneo de Cardiomiócitos de Alta Qualidade, Células Endoteliais e Fibroblastos de um Coração de Rato Adulto

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolamento e Cultura de Queratinócitos de Rato Primário de Pele de Rato Neonatal e Adulto

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

Cultura primária de células Adrenocortical rato e ensaios das funções reguladora

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...