Nous remercions Haruya Ohno, Kosaku Shinoda, Louis Sharp, Emi Tomoda, et Lauren Ruiz de discussion, une aide technique et aide à la rédaction du manuscrit. Ce travail a été soutenu par des subventions du NIH (DK087853), du Programme de recherche biomédicale et percée de Asubio Pharm Inc SKULA a été pris en charge par un ACTIONS bourses de doctorat de l&#39;Université de Copenhague et l&#39;UE projet FP7 Diabat (SANTÉ-F2 -2011 à 278373) à Lise Madsen et Karsten Kristiansen. Nous reconnaissons aussi le centre DERC subvention (NIH P30 DK063720).

1. Préparer milieu de digestion Faire 5 ml par 5 souris par du tissu (environ 1 ml / 1 g tissu adipeux). <ol><li> Peser des enzymes de digestion: - Collaginase D: 1,5 u / ml (1108874103, 1 g, Roche, 70334223) - Dispase II: 2,4 u / ml (04942078001, 0.980 mg / lyo, Roche, 11466200) </li><li> Ajouter 25 ml de PBS et bien mélanger pour dissoudre </li><li> Ajouter CaCl 2 juste avant la digestion du tissu à une concentration finale de 10 mM </li></ol> 2. Disséquer les tissus adipeux des souris <ol><li> La dissection doit être effectuée avec soin et immédiatement après les souris (6-8 semaines) sont sacrifiés. Travailler avec de l&#39;éthanol stérile pulvérisé sur la fourrure de la souris avant de l&#39;ouvrir. Placez le tissu directement dans PBS propre, distincte BAT interscapulaire et sous-cutanée inguinale WAT. </li><li> Pour BAT interscapulaire: avoir la souris placé à l&#39;arrière fait face, découpé le long du dos et tout le chemin vers le cou. Le brunle tissu adipeux se trouve juste sous la peau entre les épaules (interscapulaire). MTD peuvent être considérés comme les deux lobes en forme de papillon,. Une fine couche de graisse blanche couvrant la MTD devrait être soigneusement enlevés. </li><li> Pour sous-cutanée WAT (WAT inguinale): enlever la peau. Inguinale WAT se trouve directement sous la peau des deux côtés, en commençant presque sur le dos et aller à côté, à l&#39;intérieur des cuisses et le bas en direction du testicule. </li></ol> 3. Couper et digérer le tissu adipeux <ol><li> Supprimer rapidement toute contamination, comme les cheveux, les muscles squelettiques et du tissu conjonctif des coupes de tissus. </li><li> Séchez les tissus rapidement sur le papier pour ne pas diluer le milieu de dissection avec du PBS et le placer sur une plaque sèche. </li><li> Ajouter milieu de digestion (ajouté CaCl 2 avant la digestion) et émincer le tissu en petits morceaux. Mélangez bien à la pipette de haut en bas avec une pipette de 5 ml. Transférez les tissus hachés dans des tubes de 50 ml avec le reste de la digestion moyen, mélangez encore plus par pipetage de haut en bas. </li><li> Digestion à 37 ° C sous agitation constante à 150 tours par minute pendant 40-50 min. Vérifiez toutes les 10-15 minutes pour vous assurer que la digestion se passe bien et pour éviter une sur-digestion. </li></ol> Remarque: milieu de digestion correcte et le temps sont importants. Le tissu doit être bien digéré, mais la digestion excès peut endommager les cellules. 4. Suspension Cellulaire filtre <ol><li> Arrêtez la digestion en ajoutant 5 ml de milieu complet (DMEM/F12 contenant FPS de 10% et P / S). Les cellules doivent être presque complètement homogène. Bien mélanger par pipetage. </li><li> Centrifuger à 700 xg pendant 10 min. </li><li> SVF peut désormais être considérée comme une pastille brunâtre sur le fond du tube. Aspirer le gras des adipocytes matures couche sur le dessus et plus de la couche liquide - garder le culot et le dissoudre dans 10 ml de milieu complet. Mélangez bien. </li><li> Placez un tamis cellulaire (50-70 um de diamètre) sur une 50 nouveaux<html> Tube ml et filtrer la suspension de cellules. </li></ol> 5. Cellules de la plaque <ol><li> Transfert suspension de cellules dans un tube de 15 ml et centrifuger à 700 xg pendant 10 min. </li><li> Aspirer à moyen et à remettre en suspension le culot dans 10 ml de milieu complet. Pipette et bien mélanger. </li><li> Estimer combien de plaques sont nécessaires. Cela peut varier et dépend de la grosseur des granules. Une règle générale est de 2 des plaques de 10 cm de 5 souris pour inguinale WAT et une plaque de 10 cm pour les MTD. </li><li> Cellules de la plaque sur les plats revêtus de collagène (R &amp; D) dans un milieu complet </li><li> 1-2 heures après l&#39;étalement des cellules, moyen aspirer, laver avec du PBS deux fois et ajouter du milieu frais. Cette étape est importante, car cela peut éliminer les globules rouges, les cellules immunitaires et d&#39;autres contaminants. </li></ol> 6. Différencier les cellules <ol><li> Faire l&#39;entretien et les médiums à induction. </li></ol> Un milieu d&#39;entretien Milieu complet additionné de: ent &quot;&gt; L&#39;insuline, concentration finale. 5 pg / ml (5 mg / ml, *** 100 ul d&#39;acide acétique dans 10 ml H 2 O pour préparer pH de l&#39;eau acidifiée 2.5. actions magasin dissoudre l&#39;insuline dans l&#39;eau acidifiée. à -20 ° C) 3,3 &#39;,5-triiodo-L-thyronine (T3), concentration finale. 1 nM (10 uM actions, *** dissoudre T3 dans NaOH 1N et ajouter du milieu pour faire 10 en stock uM. Sigma cat # T-2877) Conserver à 4 ° C, bon pour une semaine Un milieu d&#39;induction Un milieu d&#39;entretien contenant des composés suivants: Indométhacine, concentration finale. 125 microns (0,125 stock M dans l&#39;éthanol, le chat Sigma # I-7378). Indomethacin doit être chauffé à 60 ° C doit être dissoute. Dexaméthasone, concentration finale. 2 pg / ml (2 mg / ml dans de l&#39;éthanol chaussette, Sigma cat # D-1756) 3-isobutyl-1-méthylxanthine (IBMX), concentration finale. 0,5 mM (0,25 stock M dans le DMSO, Sigma cat # I-5879) La rosiglitazone, concentration finale. 0,5 uM(10 actions mM dans du DMSO, Sigma cat # R-2408) Remarque: Assurez-induction du milieu frais à chaque fois. <ol start="2"><li> Cultivez adipocytes primaires jusqu&#39;à la confluence de 95-97% en milieu complet (confluente mais pas trop emballé) </li><li> Changez régulièrement milieu complet avec un milieu d&#39;induction (jour 0) </li><li> Après 48 heures (jour 2), modifier un média à l&#39;entretien avec la rosiglitazone (0,5 uM) </li><li> Après plus de 48 heures (jour 4), changer de milieu d&#39;entretien frais avec la rosiglitazone (1 uM) pour 2-3 jours supplémentaires. 6-7 jours après l&#39;ajout du milieu d&#39;induction, les cellules sont complètement différenciées de cellules adipeuses matures et sont remplis de gouttelettes d&#39;huile. Gouttelettes apparaissent autour de 3-4 jours après l&#39;induction. </li></ol> Remarque: moyen Changer tous les 2-3 jours jusqu&#39;à ce que les cellules sont complètement différenciées. Les cellules peuvent maintenant être récoltées et expression de l&#39;ARNm de Ucp1 et otheR Brown gènes spécifiques des adipocytes peut être mesurée par qRT-PCR. Western blot est utilisé pour détecter des protéines.

<ol>
	<li>Barness, L. A., Opitz, J. M., Gilbert-Barness, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity:+genetic,+molecular,+and+environmental+aspects.">Obesity: genetic, molecular, and environmental aspects.</a> American Journal of Medical Genetics. Part A. 143A, 3016-3034 (2007).</li><li>Rothwell, N. J., Stock, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+effects+of+cafeteria+and+tube-feeding+on+energy+balance+in+the+rat.">Combined effects of cafeteria and tube-feeding on energy balance in the rat.</a> The Proceedings of the Nutrition Society. 38, 5A (1979).</li><li>Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue:+function+and+physiological+significance.">Brown adipose tissue: function and physiological significance.</a> Physiological Reviews. 84, 277-359 (2004).</li><li>Cypess, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+importance+of+brown+adipose+tissue+in+adult+humans.">Identification and importance of brown adipose tissue in adult humans.</a> The New England Journal of Medicine. 360, 1509-1517 (2009).</li><li>van Marken Lichtenbelt, W. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold-activated+brown+adipose+tissue+in+healthy+men.">Cold-activated brown adipose tissue in healthy men.</a> The New England Journal of Medicine. 360, 1500-1508 (2009).</li><li>Virtanen, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+brown+adipose+tissue+in+healthy+adults.">Functional brown adipose tissue in healthy adults.</a> The New England Journal of Medicine. 360, 1518-1525 (2009).</li><li>Zingaretti, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+presence+of+UCP1+demonstrates+that+metabolically+active+adipose+tissue+in+the+neck+of+adult+humans+truly+represents+brown+adipose+tissue.">The presence of UCP1 demonstrates that metabolically active adipose tissue in the neck of adult humans truly represents brown adipose tissue.</a> FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology. 23, 3113-3120 (2009).</li><li>Saito, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+incidence+of+metabolically+active+brown+adipose+tissue+in+healthy+adult+humans:+effects+of+cold+exposure+and+adiposity.">High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity.</a> Diabetes. 58, 1526-1531 (2009).</li><li>Seale, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRDM16+controls+a+brown+fat/skeletal+muscle+switch.">PRDM16 controls a brown fat/skeletal muscle switch.</a> Nature. 454, 961-967 (2008).</li><li>Petrovic, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+peroxisome+proliferator-activated+receptor+gamma+(PPARgamma)+activation+of+epididymally+derived+white+adipocyte+cultures+reveals+a+population+of+thermogenically+competent,+UCP1-containing+adipocytes+molecularly+distinct+from+classic+brown+adipocytes.">Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) activation of epididymally derived white adipocyte cultures reveals a population of thermogenically competent, UCP1-containing adipocytes molecularly distinct from classic brown adipocytes.</a> The Journal of Biological Chemistry. 285, 7153-7164 (2010).</li><li>Coulter, A. A., Bearden, C. M., Liu, X., Koza, R. A., Kozak, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+fat+interacts+with+QTLs+controlling+induction+of+Pgc-1+alpha+and+Ucp1+during+conversion+of+white+to+brown+fat.">Dietary fat interacts with QTLs controlling induction of Pgc-1 alpha and Ucp1 during conversion of white to brown fat.</a> Physiological Genomics. 14, 139-147 (2003).</li><li>Klingenspor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold-induced+recruitment+of+brown+adipose+tissue+thermogenesis.">Cold-induced recruitment of brown adipose tissue thermogenesis.</a> Experimental Physiology. 88, 141-148 (2003).</li><li>Cinti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adipose+organ.">The adipose organ.</a> Prostaglandins, leukotrienes, and essential fatty acids. 73, 9-15 (1016).</li><li>Ghorbani, M., Himms-Hagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+of+brown+adipocytes+in+white+adipose+tissue+during+CL+316,243-induced+reversal+of+obesity+and+diabetes+in+Zucker+fa/fa+rats.">Appearance of brown adipocytes in white adipose tissue during CL 316,243-induced reversal of obesity and diabetes in Zucker fa/fa rats.</a> International Journal of Obesity and Related Metabolic Disorders: Journal of the International Association for the Study of Obesity. 21, 465-475 (1997).</li><li>Hansen, J. B., Kristiansen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+circuits+controlling+white+versus+brown+adipocyte+differentiation.">Regulatory circuits controlling white versus brown adipocyte differentiation.</a> The Biochemical Journal. 398, 153-168 (2006).</li><li>Ohno, H., Shinoda, K., Spiegelman, B. M., Kajimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PPARy+agonists+induce+a+white-to-brown+fat+conversion+through+stabilization+of+PRDM16+protein.">PPARy agonists induce a white-to-brown fat conversion through stabilization of PRDM16 protein.</a> Cell metabolism. 15, 395-404 (2012).</li><li>Lee, Y. H., Petkova, A. P., Mottillo, E. P., Granneman, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+identification+of+bipotential+adipocyte+progenitors+recruited+by+beta3-adrenoceptor+activation+and+high-fat+feeding.">In vivo identification of bipotential adipocyte progenitors recruited by beta3-adrenoceptor activation and high-fat feeding.</a> Cell Metabolism. 15, 480-491 (2012).</li><li>Cinti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transdifferentiation+properties+of+adipocytes+in+the+adipose+organ.">Transdifferentiation properties of adipocytes in the adipose organ.</a> American Journal of Physiology. Endocrinology and Metabolism. 297, E977-E986 (2009).</li><li>Barbatelli, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emergence+of+cold-induced+brown+adipocytes+in+mouse+white+fat+depots+is+determined+predominantly+by+white+to+brown+adipocyte+transdifferentiation.">The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation.</a> American Journal of Physiology. Endocrinology and Metabolism. 298, E1244-E1253 (2010).</li><li>Petrovic, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+peroxisome+proliferator-activated+receptor+gamma+(PPARgamma)+activation+of+epididymally+derived+white+adipocyte+cultures+reveals+a+population+of+thermogenically+competent,+UCP1-containing+adipocytes+molecularly+distinct+from+classic+brown+adipocytes.">Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) activation of epididymally derived white adipocyte cultures reveals a population of thermogenically competent, UCP1-containing adipocytes molecularly distinct from classic brown adipocytes.</a> The Journal of Biological Chemistry. 285, 7153-7164 (2009).</li><li>Rong, J. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+mitochondrial+biogenesis+is+suppressed+in+db/db+and+high-fat+diet-fed+mice+and+improved+by+rosiglitazone.">Adipose mitochondrial biogenesis is suppressed in db/db and high-fat diet-fed mice and improved by rosiglitazone.</a> Diabetes. 56, 1751-1760 (2007).</li><li>Wilson-Fritch, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+remodeling+in+adipose+tissue+associated+with+obesity+and+treatment+with+rosiglitazone.">Mitochondrial remodeling in adipose tissue associated with obesity and treatment with rosiglitazone.</a> The Journal of Clinical Investigation. 114, 1281-1289 (2004).</li><li>Sell, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisome+proliferator-activated+receptor+gamma+agonism+increases+the+capacity+for+sympathetically+mediated+thermogenesis+in+lean+and+ob/ob+mice.">Peroxisome proliferator-activated receptor gamma agonism increases the capacity for sympathetically mediated thermogenesis in lean and ob/ob mice.</a> Endocrinology. 145, 3925-3934 (2004).</li><li>Fukui, Y., Masui, S., Osada, S., Umesono, K., Motojima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+thiazolidinedione,+NC-2100,+which+is+a+weak+PPAR-gamma+activator,+exhibits+potent+antidiabetic+effects+and+induces+uncoupling+protein+1+in+white+adipose+tissue+of+KKAy+obese+mice.">A new thiazolidinedione, NC-2100, which is a weak PPAR-gamma activator, exhibits potent antidiabetic effects and induces uncoupling protein 1 in white adipose tissue of KKAy obese mice.</a> Diabetes. 49, 759-767 (2000).</li><li>Vernochet, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C/EBPalpha+and+the+corepressors+CtBP1+and+CtBP2+regulate+repression+of+select+visceral+white+adipose+genes+during+induction+of+the+brown+phenotype+in+white+adipocytes+by+peroxisome+proliferator-activated+receptor+gamma+agonists.">C/EBPalpha and the corepressors CtBP1 and CtBP2 regulate repression of select visceral white adipose genes during induction of the brown phenotype in white adipocytes by peroxisome proliferator-activated receptor gamma agonists.</a> Molecular and Cellular Biology. 29, 4714-4728 (2009).</li><li>Tai, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+nuclear+receptor+peroxisome+proliferator-activated+receptor+gamma+promotes+brown+adipocyte+differentiation.">Activation of the nuclear receptor peroxisome proliferator-activated receptor gamma promotes brown adipocyte differentiation.</a> The Journal of Biological Chemistry. 271, 29909-29914 (1996).</li><li>Sugii, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PPARgamma+activation+in+adipocytes+is+sufficient+for+systemic+insulin+sensitization.">PPARgamma activation in adipocytes is sufficient for systemic insulin sensitization.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 22504-22509 (2009).</li></ol>

Les auteurs déclarent n&#39;avoir aucun conflit d&#39;intérêts financiers.

Nous présentons ici un système cellulaire fiable pour étudier le développement des cellules beige / brite dans les adipocytes en culture primaire chez la souris. Par rapport à plusieurs lignées cellulaires immortalisées disponibles, ce système est susceptible d&#39;offrir une importance accrue au brunissement de la graisse blanche in vivo. Même si l&#39;étude de ces adipocytes primaires offre certains avantages, il existe aussi quelques limitations et les préoccupations q...

<html> L&#39;obésité est dramatiquement dans le monde augmente et est maintenant considéré comme l&#39;un des problèmes les plus graves pour la santé publique 1. Cette condition est liée à un déséquilibre par rapport à l&#39;apport énergétique de dépenses et les résultats de l&#39;excès d&#39;énergie stockée sous forme de lipides dans le tissu adipeux blanc (WAT). WAT élargie est associée à une augmentation de masse corporelle et de poids, tandis que le tissu adipeux brun est capable de dissiper l&#39;énergie en excès pour produire de la chaleur. Ainsi BAT peut fonctionner comme une protection contre le froid et l&#39;obésité 2,3. Ceci est réalisé par le découplage du transport des électrons dans les mitochondries par la protéine découplante 1 (UCP1). Cette protéine est considérée comme une caractéristique de thermogenèse sans frisson dans la MTD 3. Plusieurs études de ces dernières années ont révélé que les humains adultes ont fonctionnel 4-8 BAT et, par conséquent, la manipulation des MTD chez l&#39;homme peut être une intervention thérapeutique potentielle dans la lutte contre l&#39;obésité et ses maladies associées. jove_content «preuves&gt; actuelles indiquent que les deux types d&#39;adipocytes bruns existent chez les rongeurs;« classique »ou« pré-existant &quot;graisse brune se développe pendant la période prénatale et forme dédiée dépôts adipeux brun dans la région inter-scapulaire et d&#39;autres tissus périphériques D&#39;autre part. , un &quot;inductible&quot; sous forme de graisse brune (que l&#39;on appelle les cellules brite ou beige) se développe pendant la phase post-natale et semble intercalées dans les tissus adipeux blancs. Les deux types d&#39;adipocytes bruns sont également séparés par différentes origines développementales. Alors que la pré-existant adipocytes bruns proviennent de myoblastsic-Myf5 comme précurseurs, les inductibles brite / beige cellules intercalées dans WAT proviennent d&#39;une lignée non Myf5 9,10. Par ailleurs, les voies de régulation de ce type de cellule est susceptible d&#39;être différente de la brune Myf5 dérivé adipocytes 11. Le développement des cellules beiges (ie &quot;brunissement&quot; de la graisse blanche) peuvent être activés en réponse à l&#39;exposition au froid chronique et àβ3-adrénergiques agonistes ou agonistes PPARy chez les adultes 12-14. Les cellules beige / brite sont susceptibles d&#39;être une cible thérapeutique prometteuse pour la manipulation de la balance énergétique globale et peut potentiellement faire partie du traitement de l&#39;obésité; par conséquent, il est important de comprendre les mécanismes moléculaires précis et voies de signalisation par laquelle des signaux environnementaux contrôlent le développement de beige cellules. Pour comprendre le contrôle moléculaire du brunissement de la graisse blanche, des expériences in vitro sont mieux adaptés comme la différenciation des pré-adipocytes se déroule plutôt de manière asynchrone et il est difficile de détecter les cellules dans 15 situ. Bien que les études sur le développement des adipocytes ont jusqu&#39;ici été effectuées principalement sur des lignées cellulaires telles que les cellules 3T3-L1, 3T3-F442A ou Hib1, ces lignées cellulaires semblent manquer de la signature moléculaire des cellules beiges. D&#39;autre part, les adipocytes primaires isolées à partir de WAT sous-cutanée sont les plus susceptibles de récapituler les process de brunissement de la graisse blanche dans une cellule autonome de la mode. Ici, nous fournissons un protocole pour une isolation efficace de la fraction stroma-vasculaire du tissu adipeux et pour induire le brunissement de la graisse blanche en réponse à des agonistes PPARy. La rosiglitazone a été montré pour être un médiateur particulièrement efficace de brunissement dans ces cellules. Comme suggéré précédemment 16, ce système cellulaire peut être utilisé pour servir un système cellulaire fiable pour étudier le développement des cellules beige / brite.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Réactif </td></tr><tr><td> Collaginase D </td><td> Roche </td><td> 11088874103 </td><td></td></tr><tr><td> Dispase II </td><td> Roche </td><td> 04942078001 </td><td></td></tr><tr><td> CaCl 2 </td><td></td><td></td><td></td></tr><tr><td> Milieu DMEM </td><td> Pêcheur </td><td> 10017-CV </td><td> Avec 2,5 g / l de glucose et L-glutamine sans pyruvate de sodium </td></tr><tr><td> Insuline </td><td></td><td></td><td></td></tr><tr><td> T3 (3,3 &#39;,5-triiodo-L-thyronine) </td><td> Sigma </td><td> T-2877 </td><td></td></tr><tr><td> Indomethacin </td><td> Sigma </td><td> I-7378 </td><td></td></tr><tr><td> Dexaméthasone </td><td> Sigma </td><td> D-1756 </td><td></td></tr><tr><td> IBMX </td><td> Sigma </td><td> Je-5879 </td><td></td></tr><tr><td> La rosiglitazone </td><td> Sigma </td><td> R-2408 </td><td></td></tr><tr><td></td><td></td><td></td><td> Équipement </td></tr><tr><td> Plats revêtus de collagène </td><td> BD </td><td> 354450 </td><td> Plaques de 10 cm </td></tr><tr><td> 70 um filtre </td><td> BD Falcon </td><td> 352350 </td><td> Cellule filtre, nylon 70 um 1/EA </td></tr></tbody></table>

isolation and differentiation of stromal vascular cells to beige/brite cells

Adipocytes bruns ont la possibilité de découpler la chaîne respiratoire dans les mitochondries et dissiper l&#39;énergie chimique sous forme de chaleur. Développement d&#39;UCP1-positifs adipocytes bruns dans les tissus adipeux blancs (que l&#39;on appelle les cellules beige ou brite) est fortement induite par une variété de facteurs environnementaux tels que l&#39;exposition au froid ou chronique par des agonistes PPARy, par conséquent, ce type de cellule a un potentiel en tant que cible thérapeutique pour traitement de l&#39;obésité. Bien que la plupart des lignes adipocytes immortalisés ne peut pas résumer le processus de «brunissement» de la graisse blanche dans la culture, les adipocytes primaires isolés de la fraction vasculaire du stroma dans le tissu sous-cutané adipeux blanc (WAT) fournir un système fiable cellulaire pour étudier le contrôle moléculaire du développement des cellules beige / brite . Nous décrivons ici un protocole pour une isolation efficace des pré-adipocytes primaires et pour induire la différenciation des cellules beige / brite en culture. L&#39;effet de brunissement peut être évaluée par l&#39;expression de la graisse brune sélective marqueteurs tels que UCP1.

Brunissement des adipocytes primaires sont accessibles par la mesure de l&#39;expression d&#39;ARNm de Ucp1 et d&#39;autres gènes de graisse brune spécifique ou sélectif par QRT-PCR. La figure 1 présente des données d&#39;expression de gènes dans inguinales WAT-adipocytes primaires dérivés. Les cellules ont été induites à se différencier en présence de deux doses différentes de la rosiglitazone à 50 nM et 500 nM, respectivement. Un sous-ensemble de cellules a été traité avec d...

Watch this Scientific Journal Video about Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells at JoVE.com

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Préadipocytes primaires blanches isolées à partir de tissus adipeux blancs chez la souris peuvent être différenciées en cellules beige / brite. Présenté ici est un système modèle cellulaire fiable pour étudier la régulation moléculaire de &quot;brunissement&quot; de graisse blanche.

L&#39;isolement et la différenciation des cellules stromales vasculaires de cellules Beige / Brite

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

isolation-differentiation-stromal-vascular-cells-to-beigebrite

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38.4K vues.  University of California, San Francisco. Préadipocytes primaires blanches isolées à partir de tissus adipeux blancs chez la souris peuvent être différenciées en cellules beige / brite. Présenté ici est un système modèle cellulaire fiable pour étudier la régulation moléculaire de "brunissement" de graisse blanche.

Vidéo: Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Isolement et animale expansion sans sérum du cordon ombilical humain dérivé cellules stromales mésenchymateuses (CSM) et des cellules endothéliales progénitrices Colony Forming (CPEF)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

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Biologie

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induction et analyse des épithéliale à mésenchymateuses transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

L&#39;isolement et la culture primaire de cellules endothéliales aortiques de souris

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Isolement de CD146<sup&gt; +</sup&gt; Cellules Resident pulmonaire mésenchymateuses stromales de Rat Poumons

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

Isolement de murin coronaire vasculaires cellules de muscle lisse

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Néphrotoxine microinjection dans Zebrafish au Modèle lésion rénale aiguë

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Une interface utilisateur graphique pour le suivi assisté par logiciel de la concentration de protéines dans les protrusions cellulaires dynamiques

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Isolement, caractérisation et différenciation des cellules souches cardiaques du coeur de souris adulte

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

Isolement, prolifération et différenciation des cellules souches adipeuses dérivées du macaque rhésus

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Isolement de myocytes auriculaires et ventriculaires murins de haute qualité pour des mesures simultanées des transitoires Ca2+ et du courant calcique de type L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...