El financiamiento fue proporcionado por el &quot;Progetto una concorso Fondazione IRCCS Ospedale Maggiore Policlinico 2009-2010&quot; de MD, y Associazione Bambino Nefropatico ABN Onlus, Milano

Todos los experimentos con animales se realizaron de acuerdo con la normativa vigente de Europa en cuanto a la protección de los animales utilizados para fines científicos y que fueron revisados ​​y aprobados por el comité de ética de la Universidad de Milán y el Nacional. 1. Aislamiento de osteoblastos primarios El método, con pequeñas modificaciones, sigue el procedimiento descrito por Dodig et al 14. <ol><li> Preparar la digestión de medio, mediante la adición de 0,1% de colagenasa P y 0,05% de tripsina (a partir de 2,5% de tripsina 10X, sin rojo de fenol) a medio HBSS (solución salina equilibrada de Hanks, sin calcio, magnesio, o rojo fenol). </li><li> Utilice 3-4 días de edad ratones (el procedimiento se puede aplicar a tan pocos como 1 ratón). </li><li> Sacrificio por decapitación. Rocíe la cabeza con etanol al 70% y mantener en hielo. Retire las capas de piel y músculo por pinzas de disección y / tijeras quirúrgicas. </li><li> Retire con cuidado los huesos parietales ysepararlos siguiendo la sutura sagital en dos mitades. Asegúrese de que los huesos están libres de las suturas y los tejidos adherentes y colocar piezas óseas individuales en placas de pocillos que contenían medio HBSS hasta que se complete el procedimiento. </li><li> Deseche HBSS y añadir a cada pocillo el medio digerir durante 15 minutos a 37 ° C. </li><li> Eliminar el sobrenadante. </li><li> Añadir de nuevo el medio de la digestión durante 15 minutos a 37 ° C. </li><li> Esta vez guardar el sobrenadante y colocarlo en alfa-MEM (medio esencial mínimo) suplementado con 10% de FBS (suero bovino fetal) y 1% de estreptomicina / penicilina. </li><li> Repita los pasos 1.7 y 1.8 2x. </li><li> Reunir las tres fracciones recogidas, las células de spin por centrifugación a 425 g durante 5 min, descartar el sobrenadante, añadir 3 ml de alfa-MEM suplementado para volver a suspender el sedimento de células, y la transferencia a un matraz de 25 cm 2. </li><li> Cambie el medio después de 24 horas para eliminar los desechos y células muertas. </li><li> Añadir 50 mg / ml de ácido ascórbico (AA) y 3 mM de GlyCerol 2-fosfato de hidrato de sal disódica (GP) al medio de alfa-MEM. </li><li> A partir de entonces, cambiar el medio (alfa-MEM más AA / GP) cada dos días. </li></ol> 2. Protocolo de ATRA <ol><li> Llevar a cabo todos los experimentos con ATRA (all-trans retinoico) en una habitación a oscuras para evitar la inactivación. </li><li> Preparar una solución de tripsina 1X en medio HBSS y mantener a 37 ° C hasta su uso. </li><li> Retire el medio de las células subconfluentes y lave cuidadosamente con HBSS. </li><li> Añadir 1 ml de tripsina, gire suavemente el frasco para permitir que todas las células que se cubrirán por la tripsina, y se incuba durante 3 min a 37 ° C. </li><li> Compruebe si el desprendimiento real de las células bajo un microscopio invertido. </li><li> Añadir 3 ml de medio alfa-MEM para inactivar la tripsina. </li><li> Recuperar las células y centrifugar durante 15 minutos a 425 g. </li><li> Resuspender el sedimento de células en medio fresco. </li><li> Coloque una gota en una cámara de recuento del hemocitómetro, dejar reposar durante unos minutos y cuente el cenúmero ll. </li><li> Coloque las células en matraces de 25 cm2 a una densidad de 10.000 células / cm 2 con alfa-MEM más AA / GP. Tenga en cuenta que la densidad celular es relevante para el desarrollo óptimo de los procesos de las células y los contactos intercelulares. Una densidad de partida superior a 10.000 células / cm 2 perjudica el desarrollo adecuado de los procesos celulares, pero menor número de células resulta en la muerte celular prematura, debido a la ausencia de los contactos intercelulares. </li><li> Añadir 5 M ATRA al medio cada 24 horas. </li><li> Monitorear las células diariamente para evaluar la morfología: cambios morfológicos se observan generalmente después de 48 hr. Si, después de las primeras 24-48 horas, las células se siguen proliferando, replate a la densidad anterior. Una población homogénea de osteocitos maduros está presente después de 4-5 días. Dependiendo de las necesidades, utilizar las células en cualquier punto del tiempo, hasta 12-15 días. </li><li> Cuando las células se colocan en otros soportes (como cubreobjetos o-y placas), asegúrese de que se mantiene la densidad de arriba ycontinuar agregando ATRA cada 24 horas hasta su uso. Deja células para adherirse al nuevo soporte para al menos 24 horas antes del experimento. </li></ol> 3. Inmunotinción <ol><li> Por inmunoticción, placa las células sobre cubreobjetos y seguir metodologías establecidas (véase, por ejemplo Burry 15). </li><li> Por ejemplo, para inmunofluorescencia indirecta, fijar en paraformaldehído al 4% o acetona fría, se incuban secuencialmente con el anticuerpo primario a temperatura ambiente durante 1 h en una cámara humidificada, a continuación, con el anticuerpo marcado fluorescente secundaria en la oscuridad, a temperatura ambiente durante 30 min, en una cámara humidificada. </li><li> Utilice DAPI o análogos para contratinción nuclear, y montar en un medio de lucha contra la decoloración. </li></ol> 4. Rojo de alizarina Coloración y Extracción <ol><li> Placa las células sobre cubreobjetos. </li><li> Fijar las células sumergiendo el cubreobjetos en etanol al 70% durante 10 min. </li><li> Cubrir las células con algunas gotas de solución de tinción con rojo de alizarina (1 mg / ml, pH 5,5)durante 30 min. </li><li> Lavar los portaobjetos con agua del grifo. Montar con una gota de glicerina en portaobjetos de vidrio y tomar las imágenes utilizando un microscopio vertical a 10X y 20X. </li><li> Después de que el procedimiento anterior, la recuperación de los cubreobjetos sumergiendo las muestras en agua del grifo. </li><li> Ponga cada cubreobjetos en una placa. </li><li> Añadir ácido perclórico al 60% (la cantidad debe ser suficiente para cubrir completamente las células) y dejar de O / N a 4 º C con agitación suave. </li><li> Leer la densidad óptica del sobrenadante mediante espectrofotometría a 450 nm de longitud de onda. </li><li> Para normalizar los valores para el número de células, añadir verde de Janus (solución 0,2% en PBS) para cubrir la preparación de células durante 10 min. </li><li> Lave cuidadosamente con agua ultrapura. </li><li> Añadir 0,5 N HCl (la cantidad debe ser suficiente para cubrir todas las células) y agitar suavemente con la mano durante unos segundos. </li><li> Lea la absorbancia a 615 nm por espectrofotometría. </li></ol>

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Los autores no tienen nada que revelar.

En los últimos años la osteocitos se ha convertido en la celda más central en la médula. Avances de investigación están revelando progresivamente una serie de propiedades de osteocitos antes insospechados o no probados, que son de gran valor para el diseño de nuevos y mejores tratamientos para una variedad de enfermedades óseas. Sin embargo, la investigación de la biología osteocitos ha estado sufriendo de la disponibilidad limitada de modelos in vitro hasta el punto de que los osteoblastos se han uti...

Los osteocitos, el tipo de célula ósea más abundantes, son terminales diferenciadas, células altamente ramificadas profundamente ubicadas dentro del esqueleto. El cuerpo de la célula de osteocitos maduros están contenidas en las lagunas de hueso y tener diversas formas; osteocitos con cuerpos celulares alargados se encuentran en el hueso cortical, mientras que los osteocitos redondeadas son más frecuentes en el hueso trabecular 1. Dendritas ramificadas se extienden desde el cuerpo celular y residen en pequeños canales llamados canalículos, formando una intrincada red que hace múltiples contactos no sólo con otros osteocitos, sino también con otros tipos de células de hueso, médula ósea, vasos sanguíneos y los pericitos asociados. A través del fluido intersticial contenida en lagunas y canalículos, osteocitos son también conectado en última instancia al sistema de circulación, por lo tanto, pueden influir no sólo local, sino también eventos sistémicos, y viceversa su comportamiento puede ser regulada por ambos cambios locales y sistémicos 2. Laestudio de osteocitos ha cobrado impulso recientemente, gracias a varios avances técnicos, tales como la generación de tejidos y animales transgénicos específicos de células, el uso de técnicas de microscopía de gran alcance y de alto rendimiento de cribado molecular de 3,4. Sin embargo, el conocimiento de estas células es todavía incompleta, debido principalmente a la escasez relativa de adecuada en modelos in vitro. En realidad, los osteocitos han sido siempre difíciles de obtener y mantener en cultivo debido a la ubicación de profundidad, y el bajo nivel de proliferación que caracteriza a un tipo de célula diferenciada tal. A lo largo de los años, un número de métodos se han desarrollado para aislar osteocitos primaria 5-7, a pesar de que generalmente producen bajos rendimientos celulares y llevan el riesgo de que incluso unos pocos fibroblastos contaminantes se crecer demasiado rápidamente la osteocitos 8. Por esta razón, la mayor parte experimental en el trabajo in vitro ha llevado a cabo hasta ahora en la bien establecida de células osteocitos Line MLO-Y4 9. Metodologías adicionales in vitro podría aumentar la posibilidad de estudiar estas células y podría mejorar el análisis de la biología osteocitos y fisiopatología. Para ser adoptado en gran parte, tales métodos deben ser fáciles de reproducir, y no requieren instrumentos especiales o tiempos muy largos para llegar a la maduración de células. Es importante destacar que, si es aplicable a células primarias, que harían posible tomar ventaja de los animales transgénicos. Recientemente hemos descrito que el tratamiento de la línea celular de osteoblastos MC3T3-E1 y de los osteoblastos primarios con ácido retinoico induce un cambio de fenotipo rápida que conduce al desarrollo de una población homogénea de células ramificadas que llevan características morfológicas y moleculares de los osteocitos 10. El ácido trans-retinoico (ATRA) es un producto metabólico activa de la vitamina A, que regula la transcripción de genes mediante la unión a los receptores del ácido retinoico nucleares (RAR). RARunirse al ADN como heterodímeros con los receptores de retinoides X (RXRs), en última instancia conduce a la modulación de ácido retinoico (RA)-sensible genes diana 11. ATRA se ha demostrado que modulan la diferenciación y la maduración de varios tipos de células, entre ellos otras células ramificadas tales como las células neuronales 12 y podocitos 13. El método aquí descrito se basa en la adición de ATRA a osteoblastos primarios. Para ser eficaz, el ATRA tiene que ser añadido en una etapa de maduración precisa sobre células cultivadas en placas a una densidad definida; en nuestra experiencia, estas condiciones son fundamentales para obtener el interruptor fenotipo de osteoblastos a osteocitos.

<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> La necesidad de las culturas osteocitos es bien conocido por la comunidad de investigadores de hueso; aislamiento de osteocitos primaria es difícil y produce números bajos de células. Por lo tanto, el sistema celular más utilizado es la línea celular MLO-Y4-osteocitos similares. El método aquí descrito se refiere al uso de ácido retinoico para generar una población homogénea de células ramificadas con características de osteocitos morfológicos y moleculares. Después del aislamiento de los osteoblastos de bóveda craneal de ratón, se añade ácido trans-retinoico (ATRA) a medio celular, y seguimiento de células se lleva a cabo todos los días en un microscopio invertido. Primeros cambios morfológicos son detectables después de 2 días de tratamiento y la diferenciación se completa generalmente en 5 días, con el desarrollo progresivo de las dendritas, la pérdida de la capacidad de producir la matriz extracelular, la regulación por disminución de los marcadores de los osteoblastos y la regulación de las moléculas-osteocitos específico. contenido &quot;&gt; monitoreo de celda diaria es necesaria debido a la variabilidad inherente de las células primarias, y el protocolo se puede adaptar con una mínima variación de las células obtenidas a partir de diferentes cepas de ratón y aplicados a los modelos transgénicos. El método es fácil de realizar y no requiere instrumentación especial, es altamente reproducible, y genera rápidamente una población de osteocitos maduros en ausencia completa de la matriz extracelular, permitiendo el uso de estas células para aplicaciones biológicas ilimitadas.

Resultados Se obtuvieron De 5 a 10 experimentos independientes. Morfología Celular AA / GP tratados células primarias tienen características en su mayoría empedradas similar, característico de los osteoblastos maduros. Células ramificadas intercalados (como indican las flechas rojas en la Figura 1A) se pueden encontrar, lo que probablemente representan algunos osteocitos. Con el tratamiento de ATRA, las células comie...

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

Tratamiento de los osteoblastos primarios de ratón con ácido retinoico produce una población homogénea de células ramificadas que llevan características morfológicas y moleculares de los osteocitos. El método supera la dificultad de obtención y mantenimiento de osteocitos primarios en cultivo, y puede ser ventajoso para estudiar las células derivadas de los modelos transgénicos.

La aplicación de ácido retinoico para obtener cultivos de osteocitos principal del ratón 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 vistas.  Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico.  Tratamiento de los osteoblastos primarios de ratón con ácido retinoico produce una población homogénea de células ramificadas que llevan características morfológicas y moleculares de los osteocitos. El método supera la dificultad de obtención y mantenimiento de osteocitos primarios en cultivo, y puede ser ventajoso para estudiar las células derivadas de los modelos transgénicos. 

Video: 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.

Cultivos neuronales primarios del cerebro de Drosophila pupas etapa tardía

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

Investigación

Biología

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.

Primaria disociada Mesencéfalo culturas dopamina celulares de roedores recién nacidos

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.

La preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

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.

El establecimiento de cultivos primarios de fibroblastos de adultos de los 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.

Cultivos de células primarias de Drosophila Gástrula embriones

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;

Aislar las células melanocitos como primarios del corazón alfombrillas

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 Aislamiento de Primaria cultivos 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.

Aislamiento de Miofibroblastos primarias de ratón y el tejido del colon 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.

Preparación de Primaria Myogenic precursor de célula / cultivos de mioblastos de basales Vertebrados Linajes

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.