O financiamento foi fornecido por &quot;Progetto um concorso Fondazione IRCCS Ospedale Maggiore Policlinico 2009-2010&quot; para MD, e Associazione Bambino Nefropatico ABN Onlus, Milano

Todos os experimentos com animais foram realizados de acordo com a regulamentação em vigor da Europa em matéria de protecção dos animais utilizados para fins científicos e foram analisados ​​e aprovados pelo comitê de ética da Universidade de Milão e Nacional. 1. Isolamento de osteoblastos primários O método, com modificações menores, segue o procedimento descrito por Dodig et al 14. <ol><li> Prepare digerindo médio, por adição de 0,1% de colagenase P e 0,05% de tripsina (a partir de 2,5% de tripsina 10x, sem vermelho de fenol) com meio HBSS (Solução Salina Equilibrada de Hanks, sem cálcio, magnésio, ou vermelho de fenol). </li><li> Use 3-4 dias os ratos velhos (o processo pode ser aplicado para tão poucos como um ratinho). </li><li> Sacrifício por decapitação. Pulveriza-se a cabeça com etanol a 70% e manter em gelo. Remover as camadas da pele e dos músculos por pinças e dissecação / tesoura cirúrgica. </li><li> Remova cuidadosamente os ossos parietais esepará-las após a sutura sagital, em duas metades. Certifique-se que os ossos são livres de suturas e qualquer tecido aderente e coloque pedaços de ossos individuais em bem-placas contendo meio HBSS até que o processo seja concluído. </li><li> Descartar HBSS e adicionar a cada poço meio de digerir durante 15 minutos a 37 ° C. </li><li> Desprezar o sobrenadante. </li><li> Adicionar mais uma vez o meio para digerir durante 15 minutos a 37 ° C. </li><li> Este tempo de guardar o sobrenadante e colocá-lo em alfa-MEM (Meio Essencial Mínimo) suplementado com 10% de FBS (Soro Fetal Bovino) e 1% de estreptomicina / penicilina. </li><li> Repita os passos 1.7 e 1.8 2x. </li><li> Reúnem-se os três fracções recolhidas, as células de rotação por centrifugação a 425 g durante 5 minutos, descarta-se o sobrenadante, adicionar 3 ml de alfa-MEM suplementado para ressuspender o sedimento de células, e transferir para um frasco de 25 cm 2. </li><li> Mude o meio após 24 horas para remover detritos e células mortas. </li><li> Adicionar 50 ug / ml de ácido ascórbico (AA) e 3 mM de gliCerol 2-fosfato de hidrato de sal de dissódio (GP) para o meio alfa-MEM. </li><li> Depois disso, mudar o meio (alfa-MEM mais AA / PG) a cada dois dias. </li></ol> 2. Protocolo ATRA <ol><li> Realizar todos os experimentos com ATRA (ácido trans-retinóico), em uma sala escura para evitar a inativação. </li><li> Prepara-se uma solução de tripsina 1X em meio HBSS e manter a 37 ° C até à sua utilização. </li><li> Remova o meio de células confluentes e cuidadosamente lave-os com HBSS. </li><li> Adicionar 1 ml de tripsina, rodar suavemente o frasco para permitir que todas as células a serem cobertas pela tripsina e incubar durante 3 min a 37 ° C. </li><li> Verifique se o desprendimento real de células sob um microscópio invertido. </li><li> Adicionar 3 ml de meio alfa-MEM para inactivar a tripsina. </li><li> Recuperar as células e centrifugar durante 15 min a 425 g. </li><li> Ressuspender o sedimento de células em meio fresco. </li><li> Coloque uma gota de uma câmara de contagem do hemocitômetro, deixar repousar por alguns minutos e contar o cenúmero ll. </li><li> Colocar as células em frascos de 25 centímetros 2 a uma densidade de 10.000 células / cm 2 com alfa-MEM mais AA / GP. Esteja ciente de que a densidade celular é relevante para o melhor desenvolvimento de processos celulares e contatos intercelulares. Uma densidade de partida maior do que 10.000 células / cm 2 prejudica o bom desenvolvimento de processos celulares, mas os números de células mais baixos resultam em morte celular prematura, devido à ausência de contactos intercelulares. </li><li> Adicionar 5 mM ATRA ao meio cada 24 horas. </li><li> Monitorar as células diariamente para avaliar a morfologia: alterações morfológicas são geralmente observadas após 48 horas. Se, após a primeira 24-48 horas, as células estão ainda em proliferação, replate na densidade acima. Uma população homogénea de osteócitos madura está presente após 4-5 dias. Dependendo das necessidades, utilizar as células, em qualquer ponto de tempo, até 12-15 dias. </li><li> Quando as células são colocadas em outros suportes (como lamelas ou bem-placas), certifique-se que a densidade acima é mantido econtinuar a adicionar ATRA cada 24 horas até à sua utilização. Deixar as células a aderir ao novo suporte para, pelo menos, 24 horas antes da experiência. </li></ol> 3. Imunocoloração <ol><li> Para imunocoloração, placa das células sobre lamínulas e seguir metodologias estabelecidas (ver, por exemplo Burry 15). </li><li> Por exemplo, por imunofluorescência indirecta, fixar em 4% de paraformaldeído ou acetona fria, sequencialmente incubar com o anticorpo primário, à TA, durante 1 hora numa câmara húmida, em seguida, com o anticorpo secundário marcado com fluorescência, no escuro, à TA, durante 30 min, numa câmara húmida. </li><li> Use DAPI ou análogos para contracoloração nuclear, e montar em meio anti-desbotamento. </li></ol> 4. Alizarin coloração vermelha e Extração <ol><li> Placa as células em lamelas. </li><li> Fixar as células imergindo as lamelas com etanol a 70% durante 10 min. </li><li> Cobrir as células com algumas gotas de solução de coloração de vermelho de alizarina (1 mg / ml, pH 5,5)durante 30 min. </li><li> Lave as lamelas com água da torneira. Monte com uma gota de glicerol em lâminas de vidro e tirar fotos usando um microscópio vertical em 10x e 20x de ampliação. </li><li> Após o procedimento acima, recuperar as lamelas por imersão das lâminas em água da torneira. </li><li> Coloque cada lamela em um prato bem. </li><li> Adicionar 60% de ácido perclórico (a quantidade deve ser suficiente para cobrir completamente as células) e deixar de O / N a 4 ° C com agitação suave. </li><li> Leia a densidade óptica do sobrenadante por espectrofotometria a 450 nm de comprimento de onda. </li><li> Para normalizar os valores para o número de células, adicionar Janus verde (solução a 0,2% em PBS) para cobrir a preparação de células durante 10 min. </li><li> Lave cuidadosamente com água ultrapura. </li><li> Adicionar 0,5 N HCl (a quantidade deve ser suficiente para cobrir todas as células) e agitar suavemente com a mão por alguns segundos. </li><li> Leia a absorvância a 615 nm por espectrofotometria. </li></ol>

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Nos últimos anos, o osteócito emergiu como a célula mais central do osso. Avanços da pesquisa são progressivamente revelando um número de propriedades osteócitos anteriormente insuspeitas ou não comprovadas, que são de enorme valor para a concepção de novos e melhores tratamentos para uma variedade de doenças ósseas. No entanto, a investigação de biologia osteócito tem vindo a sofrer de disponibilidade limitada de modelos in vitro de tal forma que os osteoblastos têm sido utilizados como substi...

Osteócitos, o tipo de célula mais abundante do osso, são terminalmente diferenciados, células altamente ramificados profundamente situadas dentro do esqueleto. O corpo celular de osteócitos maduros estão contidas em lacunas de osso e têm diversas formas; osteócitos com corpos celulares alongados são encontrados no osso cortical, enquanto osteocytes arredondadas são mais freqüentes no osso trabecular 1. Dendritos ramificados estender a partir do corpo celular e residem em pequenos canais chamados canalículos, formando uma intrincada rede que faz com que vários contatos não só com outros osteócitos, mas também com outros tipos de células do osso, medula óssea, vasos sanguíneos e pericitos associados. Através do fluido intersticial contido nas lacunas e canalículos, osteócitos estão também em última análise, ligado ao sistema de circulação, pelo que pode influenciar não só local, mas também efeitos sistémicos, e vice-versa, o seu comportamento pode ser regulada por ambas as alterações locais e sistémicas 2. Oestudo de osteócitos recentemente ganhou impulso, graças a uma série de avanços técnicos, tais como a geração de-tecidos e animais transgénicos específicos de células, a utilização de técnicas de microscopia potentes e de rastreio molecular de alto rendimento 3,4. No entanto, o conhecimento destas células ainda é incompleta, principalmente devido à escassez relativa de adequada em modelos in vitro. Na verdade, osteócitos sempre foram difíceis de obter e manter na cultura devido à localização profunda, e ao baixo nível de proliferação que caracteriza esse tipo de célula diferenciada. Ao longo dos anos, um número de métodos tem sido desenvolvida para isolar osteócitos primário 5-7, embora eles geralmente produzem rendimentos celulares baixas e comporta o risco de que mesmo poucos fibroblastos contaminantes irá cobrir rapidamente osteócitos 8. Por esta razão, a maior parte do trabalho experimental in vitro tem sido realizada até agora para a célula osteócito bem estabelecida line MLO-Y4 9. Metodologias in vitro adicionais podem aumentar a possibilidade de estudar essas células e pode melhorar a análise da biologia osteócito e fisiopatologia. Para ser amplamente adotada, tais métodos devem ser fáceis de reproduzir, e não exigiria instrumentos especiais ou tempos muito longos para alcançar a maturação celular. Mais importante, se for o caso de células primárias, elas tornam possível tirar proveito de animais transgénicos. Descrevemos recentemente que o tratamento de células da linha osteoblástica MC3T3-E1 e em osteoblastos primários com ácido retinóico induz uma mudança de fenótipo rápida que leva ao desenvolvimento de uma população de células homogéneas ramificadas rolamento características morfológicas e moleculares de osteócitos 10. All-trans ácido retinóico (ATRA) é um produto metabólico ativa da vitamina A, que regula a transcrição de genes pelos receptores de ácido retinóico nucleares (RAR) vinculativo. RARse ligam ao DNA, como heterodímeros com os receptores de retinóide X (RXRs), em última análise conduzindo à modulação de ácido retinóico (RA)-responsivo genes alvo 11. ATRA foi mostrado para modular a diferenciação e maturação de vários tipos de células, entre as quais as outras células ramificadas, tais como as células neuronais e podócitos 12 13. O método aqui descrito baseia-se na adição de ATRA para osteoblastos primários. Para ser eficaz, a ATRA tem de ser adicionado a uma fase de maturação precisas sobre células plaqueadas a uma densidade definida; em nossa experiência essas condições são fundamentais para obter a chave de fenótipo de osteoblastos para osteócitos.

<table border="0" cellpadding="0" cellspacing="0" style="width:923px;" width="923">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:283px;">Name</td>
			<td style="width:239px;">Company</td>
			<td style="width:219px;">Cat #</td>
			<td style="width:183px;">comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase P</td>
			<td>Roche Applied Science</td>
			<td>11213857001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Gibco, Life Technologies</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco, Life Technologies</td>
			<td>14175129</td>
			<td>Pre-warm at 37&#176;C before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alpha-MEM</td>
			<td>Invitrogen, Life Technologies</td>
			<td>22571038</td>
			<td>Pre-warm at 37&#176;C before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptomycin/Penicillin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4403</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glycerol 2-phosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ATRA</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td>Protect ATRA from light</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Dissolve in PBS and filter before use. Work always under a chemical hood.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>32670</td>
			<td>Can be added to the secondary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alizarin red</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Janus Green</td>
			<td>Sigma-Aldrich</td>
			<td>201677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Perchloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>176745</td>
			<td>Use with caution (skin and eye protection are recommended)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>320331</td>
			<td>Use with caution (skin and eye protection are recommended)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fluorsave</td>
			<td>Calbiochem - Merck</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Economy Tweezers #7, 0.40 x 0.5mm tips</td>
			<td>World Precision Instruments</td>
			<td>501981</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Economy Tweezers #4, 0.40 x 0.45mm tips</td>
			<td>World Precision Instruments</td>
			<td>501978</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Scissors, straight,10cm curved</td>
			<td>World Precision Instruments</td>
			<td>14394</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Scissors, 14 cm, straight, S/S</td>
			<td>World Precision Instruments</td>
			<td>501218</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Culture flasks</td>
			<td>Corning</td>
			<td>430168</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Well-plates</td>
			<td>Corning</td>
			<td>3335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermanox coverslips</td>
			<td>Thermo Scientific Nunc</td>
			<td>12-565-27</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microscope</td>
			<td>Zeiss Apotome</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">spectrometer</td>
			<td>Safas Xenius</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

application of retinoic acid to obtain osteocytes cultures from primary mouse osteoblasts

<html> A necessidade de culturas osteócitos é bem conhecido para a comunidade de pesquisadores de osso; isolamento de osteócitos primário é difícil e produz números de celular de baixa. Portanto, o sistema celular mais extensamente utilizado é a linha de células MLO-Y4-osteócito semelhantes. O método aqui descrito refere-se à utilização de ácido retinóico para gerar uma população de células homogéneas ramificadas com características morfológicas e osteócitos moleculares. Após o isolamento de osteoblastos de calvárias de ratinho, o ácido all-trans retinóico (ATRA) é adicionado ao meio celular, e monitorização celular é conduzida por dia, sob um microscópio invertido. Primeiros alterações morfológicas são detectáveis ​​após 2 dias de tratamento e diferenciação está geralmente completa em 5 dias, com o desenvolvimento progressivo de dendrites, a perda da capacidade para produzir matriz extracelular, a infra-regulação de marcadores de osteoblastos e a sobre-regulação de moléculas específicas de osteócitos. conteúdo &quot;&gt; monitoramento de células diária é necessária devido à variabilidade inerente de pilhas, eo protocolo pode ser adaptado com variação mínima de células obtidas a partir de diferentes linhagens de camundongos e aplicadas a modelos transgênicos. O método é fácil de realizar e não requerem instrumentação especial, que é altamente reprodutível, e rapidamente gera uma população osteócito madura em completa ausência de matriz extracelular, que permite a utilização destas células para aplicações biológicas ilimitadas.

Os resultados foram obtidos 5-10 experiências independentes. Morfologia celular AA / GP tratado pilhas têm características principalmente de paralelepípedos-like, característica dos osteoblastos maduros. Células ramificadas intercaladas (conforme indicado pelas setas vermelhas na Figura 1A) pode ser encontrado, o que provavelmente representa cerca de osteócitos. Com o tratamento com ATRA, as células começam rapidam...

Watch this Scientific Journal Video about Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts at JoVE.com

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

Tratamento de osteoblastos primários de rato com ácido retinóico produz uma população homogênea de células ramificadas que carregam características morfológicas e moleculares de osteócitos. O método supera a dificuldade de obtenção e manutenção de osteócitos primárias em cultura, e pode ser vantajosa para o estudo de células derivadas de modelos transgénicos.

A aplicação de ácido retinóico para obter culturas de osteócitos principal do mouse osteoblastos

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

application-retinoic-acid-to-obtain-osteocytes-cultures-from-primary

Research

JoVE Journal

Biology

13.3K visualizações.  Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico.  Tratamento de osteoblastos primários de rato com ácido retinóico produz uma população homogênea de células ramificadas que carregam características morfológicas e moleculares de osteócitos. O método supera a dificuldade de obtenção e manutenção de osteócitos primárias em cultura, e pode ser vantajosa para o estudo de células derivadas de modelos transgénicos. 

Vídeo: Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

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

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 ...

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 ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

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 ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

Isolar células de melanócito-like primárias do coração mouse

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

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&amp;#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 ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

Isolamento de primárias Miofibroblastos de Mouse e tecido do cólon humano

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Preparação de Primário Miogênica de Células Precursoras / Culturas de mioblastos de Vertebrados basais Linhagens

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.