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. L&#39;hypoxie induite par CoCl 2 solutions Hexahydrate de chlorure de cobalt (II) (CoCl 2 • 6H 2 O, MO = 237,9) est un inducteur chimique de hypoxia-inducible factor-1.3 8. Ce produit est soluble dans l&#39;eau (100 mg / ml), ce qui donne une solution claire, rouge. <ol><li> Préparer une solution stock de 25 mM jj eau stérile, (préparer immédiatement avant utilisation) </li><li> Utilisez CoCl 2 à la concentration finale de 100 microns dans votre réguliers milieux de culture cellulaire pour induire une hypoxie. </li><li> Ajoutez les 2 CoCl support contenant vos cellules et incuber les cultures pour les 24 heures dans un incubateur conventionnelle (37 ° C, 5% C0 2). </li></ol> La concentration au-dessus fonctionne pour les lignées cellulaires, nous avons testé, mais chaque lignée cellulaire devrait être testé à différentes concentrations (d&#39;établir une courbe dose-dépendante) ainsi qu&#39;à différents temps d&#39;incubation afin de limiter la toxicité des médicaments liés et d&#39;optimiser le dosage. 2. L&#39;hypoxie induit dans Modulaire Incubateur Chambre <ol><li> Préparer au moins deux cultures cellulaires identiques (double culture). Les cultures cellulaires peuvent être dans des flacons, des plaques ou des boîtes de culture. Pour ouvrir la chambre, ouvrez tout d&#39;abord les deux pinces en plastique blanc situé sur les tubes attachés à la chambre (tubes utilisés pour l&#39;injection / la purge du gaz hypoxique) et ouvrir doucement la pince anneau en acier. </li><li> Communiqué de la pince. Le couvercle et les plateaux peuvent maintenant être supprimés. </li><li> Placez la culture de cellules dans la chambre hypoxique. Également placer une boîte de Petri contenant de l&#39;eau stérile dans la chambre pour procurer une humidification adéquate des cultures. </li><li> Placer la culture &quot;jumeau&quot; de cellules en normoxie comme contrôle. </li><li> Assurez-vous que les plateaux sont sécurisés et ne bouge pas, et puis fermer la chambre avec son couvercle. Positionner correctement le collier bague en acier pour assurer la fermeture hermétique de la chambre et le fermer. </li><li> Pour créer une hypoxie, fixez le tuyau d&#39;un «réservoir d&#39;hypoxie&quot; contenant un mélange de 1% O 2 du gaz. Si vous avez un compteur de débit connecté à votre réservoir de votre chambre sera directement relié à celui-ci (citerne de gaz à débit mètre de chambre). Nous utilisons un débitmètre incorporé dans notre régulateur. </li><li> Il est important pour la plupart, sinon tous retirer l&#39;oxygène présent dans la chambre et dans vos médias, de le faire rincer la chambre en ouvrant le réservoir de gaz à un débit de 20 litres par minutes pour les 7-10 minutes, puis éteindre rapidement la débit de gaz et de fermer complètement la chambre en fermant deux colliers blancs. </li><li> Retour dans la chambre à une installation classique incubateur pour la période de temps désirée. Si vous utilisez de grandes cultures, permettre aux médias dans les cultures à dégazer pour 1 - 2 heures, puis répétez rincer. Les étapes ci-dessus sont basées sur les recommandations du constructeur (Billups-Rothenberg, Inc). Assurez-vous du réservoir de gaz sont correctement sécurisés à tout moment. </li></ol> Notes: <ul><li> Afin d&#39;éliminer l&#39;O 2 contenu dans les médias, il est conseillé de re-gaz de la chambre une fois après 1-3 heures. </li><li> Pour le temps d&#39;incubation plus longue que 48 heures, il est également conseillé de re-gaz de la chambre. </li><li> Les capteurs d&#39;oxygène (électrodes / manomètre) qui permettent des mesures de la O 2 contenu dans les cellules / chambre va fournir une mesure plus précise de l&#39;O intracellulaire / chambre 2 contiennent). </li><li> La culture de cellules à des longueurs différentes de temps pour faire un temps courbe permettrait de déterminer l&#39;expression du temps optimal de HIF-1α dans vos cellules particulières. </li></ul> 3. Evaluation de l&#39;hypoxie <ol><li> HIF-1 α la détection par Western blot. <ol><li> Re-ouvrir votre chambre comme décrit dans la section 2.2 et placer immédiatement vos cultures sur la glace. </li><li> Lyse hypoxie cellules traitées et non traitées avec une solution de SDS à 5% dans les assiettes, puis transférer le lysat cellulaire à des tubes, soniquer, spindown les débris cellulaires et recueillir le surnageant. </li><li> Mesurer la concentration de protéines en utilisant kit BCA, préparer l&#39;échantillon par addition de tampon de chargement et de chauffage à 95 ° C pendant 5 min. </li><li> Electrophorèse des protéines sur un 8% gel SDS-polyacrylamide et le transfert des membranes de nitrocellulose. </li><li> Bloc de la membrane dans du PBS contenant 5% non gras du lait en poudre et 0,1% de Tween 20 à la température ambiante pendant 1 heure ou à 4 ° C pendant la nuit sur le shaker. </li><li> Nuit avec des anti-HIF-1α anticorps monoclonal primaire (1 / 600) dans le même tampon immunoblot à 4 ° C. </li><li> Laver 3 fois en PBS contenant 0,1% de Tween 20 à la température ambiante, 5min à chaque fois et incuber avec HRP-anticorps secondaire (1 / 2000) dans du PBS + 0,1% Tween 20 pendant 45 min. </li><li> Laver 3 fois en PBS contenant 0,1% de Tween 20 à la température ambiante, 5min / heure. </li><li> Utilisez Pierce ECL kit et X-ray du film pour montrer la bande protéique </li></ol></li><li> L&#39;hypoxie responsive element (HRE). HIF-1α régule de nombreux gènes (c.-VEGF, OEB) en se liant à une séquence appelée hypoxie élément régulateur (HRE). En utilisant l&#39;EDH associé à un gène marqueur it est possible de détecter l&#39;activité de HIF 9. Pour utiliser cette méthode, vous devez d&#39;abord vous transfecter des cellules avec un plasmide HRE-luciférase en utilisant une méthode de transfection adapté à vos cellules. Par exemple, nous avons utilisé Lipofectamine 2000. Les cellules transfectées ont besoin d&#39;être au moins 4 heures avant d&#39;être incubés dans l&#39;hypoxie et normoxie. Ce temps permet à l&#39;ADN de pénétrer dans les cellules de sorte que cette première étape n&#39;est pas affecté par l&#39;hypoxie. Le signal de la luciférase est détectée en utilisant un kit Luciferase Assay. <ol><li> Incuber votre EDH cellules transfectées en hypoxie dans la chambre modulaire ou l&#39;utilisation de cellules transfectées HRE-incubées avec CoCl 2, comprennent un contrôle normoxie. </li><li> Après la période d&#39;incubation désirée, retirez le milieu de croissance des cellules. </li><li> Rincer les cellules dans du PBS, enlever le CPE. </li><li> Distribuer 400μl de réactif de lyse 1X dans un plat à la culture et racler les cellules attachées, puis les transférer dans un tube de micro. </li><li> Des débris par centrifugation brève. </li><li> Mélange de 20 pi lysats cellulaires surnageant avec 100 pi de réactif de dosage luciférase et de mesurer la luminescence en utilisant un luminomètre. </li></ol></li></ol> 4. Les résultats représentatifs: Dans les cellules K562 (lignée cellulaire humaine de leucémie) cultivées dans les deux jours en oxygène est faible, par rapport à la normoxie, hypoxie induit une augmentation de HIF-1α protéine qui a été détectée par western blot (figure 1). Utiliser HRE-luciférase modifiée 293 cellules (embryonnaires lignée cellulaire de rein) cultivées en hypoxie, une augmentation significative de HIF-1α activité n&#39;a été détectée dans les cellules hypoxiques (Figure 2). <img src="/files/ftp_upload/2899/2899fig1.jpg" alt="Figure 1"/> Figure 1: Augmentation de HIF-1α dans les cellules hypoxiques. K562 ont été la culture en normoxie et hypoxie (chambre hypoxique) pendant 48 heures et analysés par western blot utilisant un anticorps spécifique de HIF-1α. Un anticorps spécifique de l&#39;actine a été utilisée pour le contrôle du chargement. <img src="/files/ftp_upload/2899/2899fig2.jpg" alt="Figure 2"/> Figure 2: HIF-1α activité. HRE-luciférase modifiée 293 cellules ont été cultivées en hypoxie et normoxie pendant 48 heures puis lysées pour détecter le signal luciférase en utilisant un kit de dosage de la luciférase et un luminomètre. Les résultats sont exprimés comme l&#39;unité de luminescence relatives (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>

Aucun conflit d&#39;intérêt déclaré.

La prolifération cellulaire et la viabilité dans des conditions hypoxiques varient beaucoup selon les types de cellules. Par conséquent, vous devriez ajuster le nombre de cellules ou le nombre de plaques cultures vous commencez avec de s&#39;assurer que vous aurez suffisamment de cellules / protéines pour vos expériences. La méthode au chlorure de cobalt a l&#39;avantage d&#39;être peu coûteux et rapide. Ce produit imite l&#39;hypoxie en induisant HIF-1/3α mais peut également réguler d&#39;autres gènes, assurez-vous que ce produit est adapté à votre projet en vérifiant quelles sont les autres effets qu&#39;elle pourrait avoir sur la fonction et le phénotype de vos cellules particulières indépendamment de la «synoptique hypoxique &quot;effet. Un autre médicament également utilisé pour imiter l&#39;hypoxie est mésylate de déféroxamine (DFO, utilisé à une concentration de 100 uM final). L&#39;utilisation de ces médicaments permet à l&#39;expérimentateur d&#39;ouvrir la plaque de culture / plat / flacon de nombreuses fois sans affecter la «condition hypoxique». Le niveau d&#39;oxygène contenue dans le mélange gazeux peut varier en fonction de vos expériences et de types de cellules comme des valeurs hypoxie varient selon les tissus et types cellulaires. En effet, certaines cellules sont hypoxiques dans 5% O 2 ont besoin d&#39;autres moins de 1% d&#39;O 2 à l&#39;hypoxie. 5% de CO 2 est ajoutée au mélange de gaz pour stabiliser le pH des cultures, le reste du gaz est habituellement de l&#39;azote. Chambres hypoxiques ont l&#39;avantage de ne pas utiliser des médicaments qui peuvent alterner le comportement des cellules, indépendamment de la tension d&#39;oxygène. Cependant, tous les types d&#39;expérimentation qui peut être fait que l&#39;oxygène rentre dans la chambre à chaque ouverture et diminue ainsi l&#39;hypoxie. Vous devriez considérer ce facteur dans vos expériences; hypoxie / réoxygénation est une condition spécifique qui peut affecter certains types cellulaires. Une alternative est d&#39;utiliser les postes de travail hypoxie (connecté à des réservoirs de gaz pré-mélangés ou à des systèmes de mélange de gaz) ou plus hypoxie incubateur (chambre de traitement hypoxie avec boîte à gants) qui permet l&#39;expérimentation de changer de support et de manipuler des cellules dans un environnement hypoxique continu. Diverses chambres hypoxiques ont été commercialisés au cours de la dernière décennie et devrait être choisi en fonction de votre laboratoire utilisés, les projets, l&#39;espace, la taille et du budget. Toujours s&#39;assurer du bon usage et l&#39;état de votre chambre afin de garantir des conditions de culture correcte. En général lors de l&#39;utilisation d&#39;une chambre, le niveau d&#39;hypoxie peut être modulée par la sélection des différents mélanges de gaz (soit 1%, 5% à 10% d&#39;oxygène). Concernant la détection de HIF-1α, il est important de savoir que certains lignée de cellules cancéreuses expriment HIF-1α en normoxie. Il est donc essentiel d&#39;utiliser une normoxie de contrôle pour déterminer le niveau basal de HIF dans ces lignées cellulaires. HIF-1α peut également être présent en normoxie de cellules non malignes après stimulation de cellules ou de stress. Cela pourrait également se produire si ces cellules sont affamés, assurez-vous des cultures de cellules sont nourrir correctement et entretenus.

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th></tr><tr><td> Anti-HIF-1α </td><td> Invitrogen </td><td> 458400 </td></tr><tr><td> Cobalt (II) chlorure </td><td> Sigma </td><td> C8661-25G </td></tr><tr><td> Incubateur Chambre </td><td> Billups-Rothenberg </td><td> MIC-101 </td></tr><tr><td> HRE-luciférase plasmidique </td><td> Adgene </td><td> Plasmidique 26731 </td></tr></tbody></table>

induction and testing of hypoxia in cell culture

L&#39;hypoxie est définie comme la réduction ou l&#39;absence d&#39;oxygène dans les organes, tissus ou cellules. Cette diminution de la tension d&#39;oxygène peut être due à une alimentation réduite en oxygène (causes incluent l&#39;insuffisance du réseau de vaisseaux sanguins, des vaisseaux sanguins défectueux, et l&#39;anémie) ou à une consommation accrue d&#39;oxygène par rapport à l&#39;offre (causée par un taux plus élevé soudaine prolifération cellulaire) . L&#39;hypoxie peut être physiologique ou pathologique, comme dans les cancers solides 1-3, etc polyarthrite rhumatoïde athérosclérose, ... Chaque tissus et les cellules ont une capacité différente pour s&#39;adapter à cette nouvelle condition. Pendant l&#39;hypoxie, l&#39;hypoxie inducible factor alpha (HIF) est stabilisée et réglemente divers gènes tels que ceux impliqués dans l&#39;angiogenèse ou le transport d&#39;oxygène 4. La stabilisation de cette protéine est une caractéristique de l&#39;hypoxie, donc la détection de HIF est couramment utilisé pour dépister 5-7 hypoxie. Dans cet article, nous proposons deux méthodes simples pour induire l&#39;hypoxie dans des cultures cellulaires de mammifères et des tests simples pour évaluer l&#39;état de ces cellules hypoxiques.

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

Ici, nous proposons des méthodes simples pour induire l&#39;hypoxie dans des cultures cellulaires et des tests simples pour évaluer l&#39;état des cultures hypoxiques.

Induction et les essais de l&#39;hypoxie en culture cellulaire

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.

Culture de la souris de neurones précurseurs de cellules souches

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

Recherche

Biologie

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.

L&#39;induction et la surveillance des parents adoptifs hypersensibilité de type retardé chez les rats

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.

Dérivation efficace des ressources humaines précurseurs cardiaques et les cardiomyocytes à partir pluripotentes cellules souches embryonnaires humaines à l&#39;induction de petites molécules

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%.

L&#39;isolement et la culture primaire de cellules de foie de rat

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.

La méthode la Culture Slice pour la suite du développement des germes de dents dans explantation Culture

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;

L&#39;isolement, la culture, et la transplantation de cellules satellites du muscle

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.

Analyse de Global ARN de synthèse à la Cellule Plain suivante hypoxie

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 protéine c-FOS détection immunohistologique: Un outil utile comme marqueur des voies centrales impliquées dans les réponses physiologiques spécifiques<em&gt; In Vivo</em&gt; et<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 détection des protéines liant l&#39;ARN par<em&gt; In vitro</em&gt; ARN Pull-down en adipocytes 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 ...

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.