Name: induction and testing of hypoxia in cell culture
Uploaded: 2011-08-12T13:00:00
Duration: 7 min 1 s
Description: Watch this Scientific Journal Video about Induction and Testing of Hypoxia in Cell Culture at JoVE.com

1. La hipoxia inducida por CoCl 2 solución Cobalto (II) hexahidratado Cloruro (CoCl 2 • 6H 2 O, PM = 237,9) es un inductor químico del factor inducible por hipoxia 1,3 8. Este producto es soluble en agua (100 mg / ml), dando una solución clara, de color rojo. <ol><li> Prepare una solución madre 25 mM en agua estéril dd, (prepararse inmediatamente antes de su uso) </li><li> Use CoCl 2 en la concentración final de 100μM en sus medios regulares de cultivo de células para inducir la hipoxia. </li><li> Agregue el CoCl 2 medios que contienen a las células y se incuban los cultivos durante 24 horas en una incubadora convencional (37 ° C, 5% C0 2). </li></ol> A la concentración de obras para las líneas celulares que hemos probado, pero cada línea celular debe ser probado en diferentes concentraciones (para establecer una curva dosis-dependiente), así como en diversos tiempos de incubación con el fin de limitar la toxicidad relacionada con las drogas y optimizar el ensayo. 2. Hipoxia inducida en Modular cámara de la incubadora <ol><li> Preparar al menos dos cultivos celulares idénticos (la cultura twin). Los cultivos de células pueden ser en cofres, los platos o la cultura. Para abrir la cámara, abrir por primera vez las dos abrazaderas de plástico blanco se encuentra en los tubos conectados a la cámara (tubos utilizados para la inyección / purgar el gas hipóxico) y abra suavemente el anillo de sujeción de acero. </li><li> Suelte la abrazadera. La tapa y bandejas de ahora se puede quitar. </li><li> Coloque el cultivo de células en la cámara de hipoxia. También colocar una placa de Petri que contiene agua estéril en la cámara para proporcionar humidificación adecuada de los cultivos. </li><li> Coloque el &quot;gemelo&quot; de cultivo de células en normoxia como control. </li><li> Asegúrese de que las bandejas están garantizados y no se mueve, y luego cerrar la cámara con su tapa. Correctamente la posición del anillo de sujeción de acero para garantizar el cierre hermético de la cámara y ciérrelo. </li><li> Para crear la hipoxia, conectar el tubo a un &quot;tanque de hipoxia&quot;, que contiene un 1% O 2 mezcla de gases. Si usted tiene un medidor de flujo conectado al tanque de su cámara se puede conectar directamente a él (el tanque de gas de flujo metros de la cámara). Nosotros utilizamos un medidor de flujo incorporado en nuestro organismo regulador. </li><li> Es importante para la mayoría, si no eliminar todos los presentes oxígeno en la cámara y en sus medios de comunicación, para hacerlo al ras de la cámara mediante la apertura del tanque de gas con un caudal de 20 litros por minuto durante 7-10 minutos, y luego rápidamente apagar el flujo de gas y se cierra completamente la cámara mediante el cierre de dos pinzas blancas. </li><li> Devolver la cámara a una incubadora convencional para el período de tiempo deseado. Si utiliza grandes culturas, que el material de las culturas de gas de 1 a 2 horas y luego repetir color. Los pasos anteriores se basan en las recomendaciones constructor &#39;(Billups-Rothenberg, Inc). Asegúrese de que el tanque de gas se fijan correctamente en todo momento. </li></ol> Notas: <ul><li> Con el fin de eliminar el O 2 contenido en los medios de comunicación, se aconseja volver a la cámara de gas una vez después de 3 horas 1. </li><li> Para el tiempo de incubación más largo que 48 horas también se recomienda a la cámara de re-gas. </li><li> Los sensores de oxígeno (electrodos / manómetro) que permiten la medición de la O 2 contenido en células / cámara proporcionará una medición más precisa de la O intracelular / cámara de 2 contienen). </li><li> El cultivo de células en diferentes longitudes de tiempo para hacer una curva de tiempo-podría ayudar a determinar el momento óptimo de expresión de HIF-1α en células específicas. </li></ul> 3. Evaluación de la hipoxia <ol><li> HIF-1 α detección por Western blot. <ol><li> Volver a abrir su cámara como se describe en la sección 2.2 e inmediatamente colocar sus cultivos en el hielo. </li><li> Lisis de la hipoxia células tratadas y no tratadas con 5% de solución de SDS en las placas luego transferir el lyzate celular a los tubos, ultrasonidos, spindown los restos celulares y recoger el sobrenadante. </li><li> Mediciones de concentración de proteínas utilizando BCA kit, preparar la muestra mediante la adición de tampón de carga y la calefacción a 95 º C durante 5 min. </li><li> Electroforesis de proteínas en un 8% SDS-gel de poliacrilamida y la transferencia a membranas de nitrocelulosa. </li><li> Bloquear la membrana en PBS que contenía 5% sin grasa leche en polvo y 0,1% Tween 20 a temperatura ambiente durante 1 hora o menos 4 ° C durante la noche en la coctelera. </li><li> Durante la noche con anti-HIF-1α anticuerpo monoclonal primario (1 / 600) en el mismo buffer inmunoblot a 4 ° C. </li><li> Lavar 3 veces en PBS que contenía 0,1% de Tween 20 a temperatura ambiente, 5 minutos cada vez y se incuban con HRP-secundaria de anticuerpos (1 / 2000) en PBS + 0,1% Tween 20 durante 45 minutos. </li><li> Lavar 3 veces en PBS que contenía 0,1% de Tween 20 a temperatura ambiente, 5 minutos / hora. </li><li> Use Pierce ECL kit y películas de rayos X para mostrar la banda de la proteína </li></ol></li><li> Elemento Sensible hipoxia (HRE). HIF-1α regula numerosos genes (es decir, VEGF, OEP), mediante la unión a una secuencia denominada Elemento de Regulación de hipoxia (HRE). Educación en derechos humanos asociados al uso de un gen marcador it es posible detectar la actividad de HIF 9. Para utilizar este método, primero que hay que transfectar las células con un plásmido de luciferasa HRE mediante un método de transfección adaptado a las células. Por ejemplo, podemos utilizar Lipofectamine 2000. Las células necesitan para ser transfectadas por lo menos 4 horas antes de ser incubados en hipoxia y normoxia. Este tiempo permite que el ADN de entrar en las células de modo que este primer paso no se ve afectada por la hipoxia. La señal de luciferasa se detectó mediante un Kit de ensayo de luciferasa. <ol><li> Incubar la educación en derechos humanos, las células transfectadas en la hipoxia en la cámara modular o el uso de EDH-transfectadas las células se incubaron con CoCl 2, incluir un control de normoxia. </li><li> Después del período de incubación se desea, eliminar el medio de cultivo de las células. </li><li> Enjuague las células en PBS, retire la PBS. </li><li> Dispensar 400μl del reactivo de lisis 1X en placa de cultivo y raspar las células adheridas, y luego los transferirá a un tubo de micro. </li><li> Pellet los escombros por centrifugación breve. </li><li> 20μl mezcla de lisados ​​celulares sobrenadante con 100μl del reactivo de ensayo de luciferasa y medir la luminiscencia usando un luminómetro. </li></ol></li></ol> 4. Los resultados representativos: En las células K562 (línea de células humanas de leucemia) cultivadas en dos días con poco oxígeno, en comparación con normoxia, la hipoxia induce un aumento de HIF-1α proteína que se detectó por Western blot (Figura 1). Utilizando HRE-luciferasa modificados células 293 (línea de células embrionarias de riñón) se cultivan en la hipoxia, un significativo aumento de HIF-1α se detectó actividad en las células hipóxicas (Figura 2). <img src="/files/ftp_upload/2899/2899fig1.jpg" alt="Figura 1"/> Figura 1: Aumento de HIF-1α en células hipóxicas. K562 células fueron la cultura en normoxia e hipoxia (cámara de hipoxia) durante 48 horas y se analizaron por Western blot utilizando un anticuerpo específico para HIF-1α. Un anticuerpo específico para la actina se utilizó para el control de carga. <img src="/files/ftp_upload/2899/2899fig2.jpg" alt="Figura 2"/> Figura 2: HIF-1α actividad. Educación en derechos humanos-luciferasa modificados 293 células fueron cultivadas en hipoxia y normoxia durante 48 horas y luego se lisaron para detectar la señal de luciferasa utilizando un kit de ensayo de luciferasa y un luminómetro. Los resultados se expresan como la unidad de luminiscencia relativa (RLU).

<ol>
	<li>Swinson, D. E., O'Byrne, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+hypoxia+and+epidermal+growth+factor+receptor+in+non-small-cell+lung+cancer.">Interactions between hypoxia and epidermal growth factor receptor in non-small-cell lung cancer.</a> Clin Lung Cancer. 7, 250-256 (2006).</li><li>van Laarhoven, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+in+relation+to+vasculature+and+proliferation+in+liver+metastases+in+patients+with+colorectal+cancer.">Hypoxia in relation to vasculature and proliferation in liver metastases in patients with colorectal cancer.</a> Int J Radiat Oncol Biol Phys. 64, 473-482 (2006).</li><li>Hockel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+pO2+predicts+survival+in+advanced+cancer+of+the+uterine+cervix.">Intratumoral pO2 predicts survival in advanced cancer of the uterine cervix.</a> Radiother Oncol. 26, 45-50 (1993).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1+and+human+disease:+one+highly+involved+factor.">HIF-1 and human disease: one highly involved factor.</a> Genes Dev. 14, 1983-1991 (2000).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor+1:+master+regulator+of+O2+homeostasis.">Hypoxia-inducible factor 1: master regulator of O2 homeostasis.</a> Curr Opin Genet Dev. 8, 588-594 (1998).</li><li>Tatum, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia:+importance+in+tumor+biology,+noninvasive+measurement+by+imaging,+and+value+of+its+measurement+in+the+management+of+cancer+therapy.">Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy.</a> Int J Radiat Biol. 82, 699-757 (2006).</li><li>Brahimi-Horn, M. C., Pouyssegur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF+at+a+glance.">HIF at a glance.</a> J Cell Sci. 122, 1055-1057 (2009).</li><li>Piret, J. P., Mottet, D., Raes, M., Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CoCl2,+a+chemical+inducer+of+hypoxia-inducible+factor-1,+and+hypoxia+reduce+apoptotic+cell+death+in+hepatoma+cell+line+HepG2.">CoCl2, a chemical inducer of hypoxia-inducible factor-1, and hypoxia reduce apoptotic cell death in hepatoma cell line HepG2.</a> Ann N Y Acad Sci. 973, 443-447 (2002).</li><li>Emerling, B. M., Weinberg, F., Liu, J. L., Mak, T. W., Chandel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEN+regulates+p300-dependent+hypoxia-inducible+factor+1+transcriptional+activity+through+Forkhead+transcription+factor+3a+(FOXO3a).">PTEN regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through Forkhead transcription factor 3a (FOXO3a).</a> Proc Natl Acad Sci U S A. 105, 2622-2627 (2008).</li></ol>

No hay conflictos de interés declarado.

La proliferación celular y la viabilidad en condiciones de hipoxia varían mucho en función de los tipos de células. Por lo tanto, usted debe ajustar el número de células o el número de placas de cultivo se comienza con asegurarse de que tendrá suficientes células / proteínas para sus experimentos. El método de cloruro de cobalto tiene la ventaja de ser barato y rápido. Este producto reproduce mediante la inducción de hipoxia HIF-1/3α pero también pueden regular otros genes, asegúrese de que este producto se adapta a su proyecto mediante la comprobación de lo que otros efectos que podría tener sobre la función y el fenotipo de las células en particular, independientemente de la &quot;imitar- hipóxica &quot;efecto. Otro medicamento también se utiliza para imitar la hipoxia es el mesilato de deferoxamina (DFO, que se utiliza en 100 mM concentración final). El uso de estos fármacos permite al experimentador para abrir la placa de cultivo / plato / frasco muchas veces sin afectar a la &quot;condición de hipoxia&quot;. El nivel de oxígeno contenido en la mezcla de gas puede variar en función de sus experimentos y tipos de células como los valores de la hipoxia varían dependiendo de los tejidos y tipos celulares. De hecho, algunas células hipóxicas en el 5% O 2 necesidad de otras menos del 1% de O 2 a la hipoxia. 5% de CO 2 se añade a la mezcla de gases para estabilizar el pH de los cultivos, el resto del gas es generalmente nitrógeno. Cámaras hipóxicas tiene la ventaja de no utilizar medicamentos que pueden alternar el comportamiento celular, independientemente de la tensión de oxígeno. Sin embargo, no todos los tipos de experimentación se puede hacer como el oxígeno vuelve a entrar en la cámara en cada abertura y por lo tanto disminuye la hipoxia. Debe tener en cuenta este factor en sus experimentos, la hipoxia / re-oxigenación es una condición específica que puede afectar a algunos tipos de células. Una alternativa es el uso de estaciones de trabajo de la hipoxia (conectados a los tanques de gas premezclado o los sistemas de mezcla de gases) o mayores hipoxia incubadora (cámara de tratamiento con la hipoxia de la guantera), que permite la experimentación de cambiar los medios de comunicación y de manipular las células en el ambiente hipóxico continua. Varias cámaras de hipoxia se han comercializado durante la última década y se debe elegir de acuerdo a su laboratorio utilizados, los proyectos, el espacio, tamaño y presupuesto. Asegúrese siempre de que el buen uso y condición de su cámara para garantizar las condiciones correctas de la cultura. En general cuando se utiliza una cámara, el nivel de hipoxia puede ser modulada por la selección de las mezclas de gases diferentes (es decir, un 1%, 5% al ​​10% de oxígeno). En cuanto a la detección de HIF-1α, es importante saber que algunas líneas celulares cancerosas expresan HIF-1α en normoxia. Por tanto, es fundamental utilizar un normoxia-control para determinar el nivel basal de HIF en estas líneas celulares. HIF-1α también pueden estar presentes en normoxia en las células no malignas después de la estimulación de células o el estrés. Esto también podría ocurrir si estas células están hambrientas, asegúrese de que los cultivos celulares se alimentan adecuadamente y se mantiene.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> Anti-HIF-1α </td><td> Invitrogen </td><td> 458400 </td></tr><tr><td> Cobalto (II) Cloruro </td><td> Sigma </td><td> C8661-25G </td></tr><tr><td> Incubadora de la Cámara </td><td> Billups-Rothenberg </td><td> MIC-101 </td></tr><tr><td> Educación en derechos humanos-Luciferase plásmido </td><td> Adgene </td><td> Plásmido 26731 </td></tr></tbody></table>

induction and testing of hypoxia in cell culture

La hipoxia se define como la reducción o falta de oxígeno en órganos, tejidos o células. Esta disminución de la tensión de oxígeno puede ser debido a una reducción del suministro de oxígeno (causas incluyen la red insuficiente de los vasos sanguíneos, los vasos sanguíneos defectuosos, y anemia) o de un mayor consumo de oxígeno en relación con la oferta (causado por una tasa más alta proliferación de las células) . La hipoxia puede ser fisiológico o patológico como en tumores sólidos, etc 1-3, aterosclerosis artritis reumatoide, ... Cada uno de los tejidos y las células tienen una capacidad diferente para adaptarse a esta nueva condición. Durante la hipoxia, la hipoxia inducible factor alfa (HIF) se estabiliza y regula varios genes como los que participan en la angiogénesis o el transporte de oxígeno 4. La estabilización de esta proteína es una característica de la hipoxia, por lo tanto, la detección de HIF se utiliza habitualmente para la detección de la hipoxia 5-7. En este artículo, se proponen dos métodos sencillos para inducir la hipoxia en cultivos celulares de mamíferos y pruebas sencillas para evaluar el estado de hipoxia de estas células.

Watch this Scientific Journal Video about Induction and Testing of Hypoxia in Cell Culture at JoVE.com

Induction and Testing of Hypoxia in Cell Culture

Aquí proponemos métodos sencillos para inducir la hipoxia en cultivos de células y pruebas sencillas para evaluar el estado de hipoxia de los cultivos.

Inducción y prueba de hipoxia en el cultivo celular

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

induction-and-testing-of-hypoxia-in-cell-culture

Research

JoVE Journal

Biology

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Cultura de ratón con células madre neurales Precursores

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Investigación

Biología

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

Inducción y Seguimiento de Adopción de hipersensibilidad retardada en ratas

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivación eficiente de los Derechos Humanos precursores cardiacos y cardiomiocitos de pluripotentes células madre embrionarias humanas mediante la inducción de moléculas pequeñas

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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%.

Aislamiento y cultivo primario de células hepáticas de ratas

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

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

El método de cultivo de la rebanada por Siguiendo desarrollo de gérmenes de dientes en Explante Cultura

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Aislamiento, cultivo y trasplante de células musculares satélite

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Análisis de RNA Global Síntesis en el nivel de células individuales siguiente Hipoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

La proteína c-Fos detección inmunohistoquímico: una herramienta útil como marcador de vías centrales implicados en las respuestas fisiológicas específicas<em&gt; En Vivo</em&gt; y<em&gt; Ex Vivo</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

La detección de las proteínas de unión a ARN por<em&gt; In Vitro</em&gt; ARN desplegable de adipocitos Cultura

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.