Nous reconnaissons la contribution de Renata Sano à concevoir le protocole initial. Ad&#39;A. détient les bijoutiers pour les enfants (JFC) de la chaire de génétique et thérapie génique. Ce travail a été financé en partie par des subventions du NIH GM60905, DK52025 et CA021764, et les américains libanais syriens Associated Charities (ALSAC).

<html> Le protocole suivant est destiné à l&#39;isolation et la purification de MMA propres et de pierres précieuses du cerveau de souris Solutions nécessaire pour l&#39;isolement des mitochondries, MMA propres et de pierres précieuses Fractionnement pour obtenir brut préparation mitochondriale: Solution A: 0,32 M saccharose, 1 mM NaHCO 3, 1 mM de MgCl2, 0,5 mM de CaCl 2 + inhibiteurs de la protéase (ajouter frais, le cas échéant) Solution B: 0,32 M saccharose, 1 mM NaHCO 3 + inhibiteurs de la protéase (ajouter frais, le cas échéant) Isolation MMA propres: Moyen d&#39;isolement: 250 mM, HEPES 5 mM mannitol à pH 7,4, 0,5 mM EGTA, 0,1% de BSA Tampon gradient: 225 mM, HEPES 25 mM mannitol à pH 7,5, 1 mM EGTA, 0,1% de BSA (concentration finale) Isolation GEM: contenu &quot;&gt; GEM tampon d&#39;extraction: 25 mM HEPES pH 7,5, 0,15 M de NaCl, 1% Triton X-100 + inhibiteurs de la protéase (ajouter frais, le cas échéant) Tampon de solubilisation GEM: 50 mM Tris-HCl, pH 8,8, EDTA 5 mM, SDS 1% + Inhibiteurs de la protéase (ajouter frais, au besoin). 1. Le fractionnement subcellulaire Première étape: suppression des noyaux des cellules, non lysées et les débris cellulaires <ol><li> Euthanasier de la souris dans la chambre de CO 2. </li><li> Retirer immédiatement du cerveau, réduire de moitié, placez-le dans tubes de 2 ml sur de la glace et peser. </li></ol> Remarque: Conservez moitiés du cerveau séparé pendant toute la procédure. Ce qui suit s&#39;applique au traitement d&#39;une moitié du cerveau unique. <ol start="3"><li> Placer la moitié du cerveau dans un pré-broyeur réfrigéré 2 ml en verre Dounce tissus contenant 1 ml de solution froide A. Homogénéiser avec 15 coups au total d&#39;un pilon jeu important. </li><li> Transférer dans un tube de 15 ml faucon, sur la glace, et diluerhomogénats jusqu&#39;à 10 volumes p / v, par exemple 0,225 g à 2,25 ml. </li><li> Centrifuger l&#39;échantillon à 1400 xg pendant 10 min à 4 ° C. </li><li> Retirez délicatement le surnageant et transférer dans un 30 ml à fond rond tube à centrifuger en verre, sur la glace. Assurez-vous de ne pas perturber le culot. </li><li> Remettre en suspension le culot dans les mêmes 10 volumes de la solution A. Homogénéiser même dans le broyeur, avec 3-6 coups de pilon faible jeu, 1 ml à la fois. </li><li> Transférer dans un nouveau tube Falcon de 15 ml et centrifuger à 710 xg pendant 10 min à 4 ° C. Cela se traduit par une pastille de noyaux et les débris cellulaires. </li><li> Retirez délicatement le surnageant et la piscine avec le surnageant enregistré à l&#39;étape 1.6. </li></ol> 2. Deuxième étape: Isolement brut Mitochondries <ol><li> Centrifuger surnageants à 13800 xg pendant 10 min à 4 ° C. </li><li> Transférer le surnageant dans un tube Ultra-Clear Centrifugeuse Beckman, sur la glace </li><li> Resuspendre le culot dans 10 volumes de solution A. Homogénéiser même dans le broyeur, avec 3-6 coups de pilon faible jeu, 1 ml à la fois. </li><li> Centrifugeuse étape 2,1. </li><li> Répétez les étapes 02.02 à 02.04. </li><li> Le culot résultant est une fraction enrichie mitochondrial. Réunir les fractions surnageantes (cytosol et ER), couvrir avec du parafilm et garder sur la glace. </li><li> Remettre en suspension le culot avec 6 coups de pilon faible jeu, dans 4,8 ml / g (g est renvoyé au poids du cerveau d&#39;origine) de la solution B, dans un homogénéisateur frais. </li><li> À l&#39;aide de pipettes Pasteur en verre, préparer un gradient de saccharose discontinu dans un tube ultra-centrifugeuse Beckman dégagé, en ajoutant ensuite à la partie inférieure du tube de la manière suivante, soigneusement, en veillant à pas de bulles sont formées: Resupended Pellet (0,32 M saccharose) 3 mM Saccharose ml 850 (dans 1 mM NaHCO 3) 3 ml 1 M de saccharose (dans 1 mM NaHCO 3) 3 ml 1,2 M de saccharose (dans 1 mM NaHCO3) </li><li> Centrifuger à 82500 g pendant 2 h à 4 ° C. La séparation résulte à la fin de la pente se produire trois bandes et un culot: 1) la myéline et autres contaminants membranaires (0,32 M - 0,85 M d&#39;interface), 2) ER, Golgi, membranes plasmiques (0,85 M - 1 M d&#39;interface); 3) synaptosomes (1 M - 1,2 M d&#39;interface), 4) les mitochondries brut (pastille) qui est utilisé pour les étapes de purification ultérieures </li></ol> 3. Isolement des mitochondries associée à membrane du RE, MMA propres <ol><li> Reprendre le brut fraîchement mitochondries isolées à partir de la moitié du cerveau, en 2 Medium Isolation ml contenant des inhibiteurs de protéase fraîchement ajoutés. </li><li> Préparer un gradient de Percoll 30% avec le tampon Gradient 8 ml par demi-cerveau, et placer dans un tube Ultra-Clear Centrifugeuse Beckman. </li></ol> * Remarque: Assurez-tampon en gradient plus concentrée (1,43 fois) pour obtenir une concentration finale correcte après diluting Percoll à 30%. <ol start="3"><li> Suspension mitochondriale couche sur le dessus du gradient préparé lentement pour éviter les bulles. </li><li> Centrifuger à 95000 g pendant 30 min, 4 ° C </li><li><ol class="lroman"><li> Retirez la fraction lourde (bande inférieure) d&#39;abord avec une pipette Pasteur en verre et transférer dans un tube en verre à fond rond de frais, sur glace. </li><li> Retirer la fraction légère (bande supérieure) DEUXIÈME avec une autre pipette Pasteur en verre et transférer dans un tube séparé en verre à fond rond de frais, sur glace. </li></ol></li><li> Diluer les deux fractions collectées à l&#39;étape 3.5 avec 10 ml d&#39;isolation moyen et centrifuger à 6300 xg pendant 10 min à 4 ° C. </li><li> Jeter le surnageant de la fraction lourde obtenue à l&#39;étape 3.6, remettre en suspension le culot avec un autre moyen d&#39;isolation 10 ml et centrifuger à nouveau, comme à l&#39;étape 3.6. Le culot résultant sera la fraction pure mitochondriale. </li><li> Transférer le surnageant du Frac Lumièretion obtenue à l&#39;étape 3.6 à un tube Ultra-Clear Centrifugeuse Beckman, sur la glace. Jeter le culot. </li><li> Ajouter milieu d&#39;isolement suffisante pour le surnageant obtenu à l&#39;étape 3.8 à remplir le tube et centrifuger à 100.000 xg pendant 1 h à 4 ° C. Le culot résultant sera la fraction la SMMA. Retirez délicatement et jeter le surnageant. </li></ol> Remarque: A ce stade, vous pouvez également centrifuger à 100.000 xg pendant 1 h à 4 ° C, le surnageant sauvegardé à l&#39;étape 2.6. Le culot sera la fraction ER et le surnageant de la fraction cytosolique. 4. Extraction de glycosphingolipide enrichis en microdomaines GEM <ol><li> Lyse pures fractions mitochondries et / ou MAM dans 500 pi-1 ml de tampon d&#39;extraction pendant 20 min sur la glace. </li><li> Centrifuger lysats à 15.300 xg pendant 2 min à 4 ° C. </li><li> Recueillir des surnageants (Triton X-100 matières solubles) et re-centrifuger pelletspendant 2 min pour enlever les matières solubles. (Triton X-100 extrait Mitochondries et / ou Triton X-100 MMA propres extraits). </li><li> Solubiliser granulés à solubiliser tampon. Ce matériau solubilisé représente les fractions GEM et peut être utilisé pour une analyse ultérieure. </li></ol>

<ol>
	<li>Poburko, D., Kuo, K. H., Dai, J., Lee, C. H., van Breemen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organellar+junctions+promote+targeted+Ca2++signaling+in+smooth+muscle:+why+two+membranes+are+better+than+one.">Organellar junctions promote targeted Ca2+ signaling in smooth muscle: why two membranes are better than one.</a> Trends Pharmacol. Sci. 25 (1), 8-15 (2004).</li><li>Pani, B., Ong, H. L., Liu, X., Rauser, K., Ambudkar, I. S., Singh, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+rafts+determine+clustering+of+STIM1+in+endoplasmic+reticulum-plasma+membrane+junctions+and+regulation+of+store-operated+Ca2++entry+(SOCE.">Lipid rafts determine clustering of STIM1 in endoplasmic reticulum-plasma membrane junctions and regulation of store-operated Ca2+ entry (SOCE.</a> J. Biol. Chem. 283 (25), 17333-17340 (2008).</li><li>Levine, T., Rabouille, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum:+one+continuous+network+compartmentalized+by+extrinsic+cues.">Endoplasmic reticulum: one continuous network compartmentalized by extrinsic cues.</a> Curr. Opin. Cell Biol. 17 (4), 362-368 (2005).</li><li>Sano, R., Annunziata, I., Patterson, A., Moshiach, S., Gomero, E., Opferman, J., Forte, M., d'Azzo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM1-ganglioside+accumulation+at+the+mitochondria-associated+ER+membranes+links+ER+stress+to+Ca(2+)-dependent+mitochondrial+apoptosis.">GM1-ganglioside accumulation at the mitochondria-associated ER membranes links ER stress to Ca(2+)-dependent mitochondrial apoptosis.</a> Mol. Cell. 36 (3), 500-511 (2009).</li><li>Raturi, A., Simmen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Where+the+endoplasmic+reticulum+and+the+mitochondrion+tie+the+knot:+The+mitochondria-associated+membrane+(MAM).">Where the endoplasmic reticulum and the mitochondrion tie the knot: The mitochondria-associated membrane (MAM).</a> Biochim. Biophys. Acta. , (2012).</li><li>Giorgi, C., De Stefani, D., Bononi, A., Rizzuto, R., Pinton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+link+between+the+mitochondrial+network+and+the+endoplasmic+reticulum.">Structural and functional link between the mitochondrial network and the endoplasmic reticulum.</a> Int. J. Biochem. Cell Biol. 41 (10), 1817-1827 (2009).</li><li>d'Azzo, A., Tessitore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sano+Gangliosides+as+apoptotic+signals+in+ER+stress+response.">Sano Gangliosides as apoptotic signals in ER stress response.</a> Cell Death Differ. 13, 404-414 (2006).</li><li>Kroemer, G., Galluzzi, L., Brenner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+membrane+permeabilization+in+cell+death.">Mitochondrial membrane permeabilization in cell death.</a> Physiol. Rev. 87, 99-163 (2007).</li><li>Rizzuto, R., Brini, M., Murgia, M., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdomains+with+high+Ca2++close+to+IP3-sensitive+channels+that+are+sensed+by+neighboring+mitochondria.">Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria.</a> Science. 262, 744-747 (1993).</li><li>Lynes, E. M., Bui, M., Yap, M. C., Benson, M. D., Schneider, B., Ellgaard, L., Berthiaume, L. G., Simmen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitoylated+TMX+and+calnexin+target+to+the+mitochondria-associated+membrane.">Palmitoylated TMX and calnexin target to the mitochondria-associated membrane.</a> EMBO J. 31 (2), 457-470 (2011).</li><li>Fujimoto, M., Hayashi, T., Su, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cholesterol+in+the+association+of+endoplasmic+reticulum+membranes+with+mitochondria.">The role of cholesterol in the association of endoplasmic reticulum membranes with mitochondria.</a> Biochem. Biophys. Res. Commun. 417 (1), 635-639 (2012).</li><li>Grimm, 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+ER-mitochondria+interface:+the+social+network+of+cell+death.">The ER-mitochondria interface: the social network of cell death.</a> Biochim. Biophys. Acta. 1823 (2), 327-334 (2012).</li><li>Vance, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+synthesis+in+a+membrane+fraction+associated+with+mitochondria.">Phospholipid synthesis in a membrane fraction associated with mitochondria.</a> J. Biol. Chem. 265, 7248-7256 (1990).</li><li>Wieckowski, M. R., Giorgi, C., Lebiedzinska, M., Duszynski, J., Pinton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mitochondria-associated+membranes+and+mitochondria+from+animal+tissues+and+cells.">Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells.</a> Nat. Protoc. 4 (11), 1582-1590 (2009).</li><li>Pizzo, P., Viola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+lipid+rafts:+structure+and+function.">Lymphocyte lipid rafts: structure and function.</a> Curr. Opin. Immunol. 15 (3), 255-260 (2003).</li></ol>

Aucun des auteurs n&#39;a de conflit d&#39;intérêt à déclarer.

Les sites de contact entre les membranes intracellulaires ou organites entre la membrane plasmique des cellules plates-formes représentent dynamiques de signalisation pour les processus cellulaires de base. La caractérisation précise de leur fonction et la composition dans les deux conditions physiologiques et pathologiques nécessite des protocoles de purification fiables et reproductibles. Les méthodes décrites ici ont été spécifiquement optimisés par notre laboratoire pour l&#39;isolement et la purification ...

<table border="1"><tbody><tr><td> Nom de réactif / Matériel </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td></td><td></td><td></td><td> REACTIFS </td></tr><tr><td></td><td></td><td></td><td> Fractionnement </td></tr><tr><td> Saccharose </td><td> Fisher Scientific </td><td> S5-500 </td><td></td></tr><tr><td> Bicarbonate de sodium </td><td> Sigma-Aldrich </td><td> S-5761 </td><td></td></tr><tr><td> Le chlorure de magnésium, hexahydrate </td><td> Fisher Scientific </td><td> BP214-500 </td><td></td></tr><tr><td> Chlorure de calcium dihydraté </td><td> Sigma-Aldrich </td><td> C-5080 </td><td></td></tr><tr><td></td><td></td><td></td><td> MAM </td></tr><tr><td> D-Mannitol </td><td> Sigma-Aldrich </td><td> M9546-250G </td><td></td></tr><tr><td> Hepes </td><td> Fisher Scientific </td><td> BP310-500 </td><td></td></tr><tr><td> EGTA </td><td> Sigma-Aldrich </td><td> E4378-250G </td><td></td></tr><tr><td> BSA, Fraction V, choc thermique, lyophilisat </td><td> Roche </td><td> 03-116-964-001 </td><td></td></tr><tr><td> Percoll </td><td> GE </td><td> 17-0891-02 </td><td></td></tr><tr><td></td><td></td><td></td><td> GEM </td></tr><tr><td> Triton X-100 </td><td> Sigma-Aldrich </td><td> T9284-500 ml </td><td></td></tr><tr><td> Chlorure de sodium </td><td> Fisher Scientific </td><td> S271-3 </td><td></td></tr><tr><td> Base de tris </td><td> Roche </td><td> 03-118-142-001 </td><td></td></tr><tr><td> HCl </td><td> Fisher Scientific </td><td> A144S-500 </td><td></td></tr&gt; <tr><td> EDTA </td><td> Fisher Scientific </td><td> BP120-500 </td><td></td></tr><tr><td> Dodécyl sulfate de sodium (SDS) </td><td> Fisher Scientific </td><td> BP166-500 </td><td></td></tr><tr><td></td><td></td><td></td><td> Commun </td></tr><tr><td> Comprimés inhibiteurs de la protéase, Complete EDTA-libres </td><td> Roche </td><td> 11-873-580-001 </td><td></td></tr><tr><td></td><td></td><td></td><td> ÉQUIPEMENT </td></tr><tr><td> 2 ml Meuleuses Douce tissus tout en verre </td><td> Kimble Chase </td><td> 885300-0002 </td><td></td></tr><tr><td> 15 ml Polypropylène Tubes à centrifuger coniques, BD Falcon </td><td> BD </td><td> 352097 </td><td></td></tr><tr><td> 30 ml à fond rond Tubes à centrifuger en verre </td><td> Kimble Chase </td><td> 45500-30 </td><td></td></tr><tr><td> 15 ml à fond rond en verre CentriTubes Fuge </td><td> Kimble Chase </td><td> 45500-15 </td><td></td></tr><tr><td> Tubes d&#39;ultracentrifugation, Ultra-Clear, à paroi mince, 14x89 mm </td><td> Beckman-Coulter </td><td> 344059 </td><td></td></tr><tr><td> Parafilm </td><td> Cole-Parmer </td><td> PM996 </td><td></td></tr><tr><td> Disposable Pipettes Pasteur en verre borosilicate, 9 &quot; </td><td> Fisher Scientific </td><td> 13-678-20C </td><td></td></tr></tbody></table>

mitochondria-associated er membranes (mams) and glycosphingolipid enriched microdomains (gems): isolation from mouse brain

Organites intracellulaires sont des structures très dynamiques avec plus ou moins de forme et de composition, qui sont soumis à des cellules spécifiques indices intrinsèques et extrinsèques. Leurs membranes sont souvent juxtaposées à des sites de contact définies, qui deviennent des plaques tournantes pour l&#39;échange de molécules de signalisation et des composants membranaires 1,2,3,4. Les microdomaines membranaires inter-organites qui sont formées entre le réticulum endoplasmique (RE) et les mitochondries lors de l&#39;ouverture de l&#39;IP3 Ca 2 sensible à canal + sont connus comme les mitochondries associé-ER membranes ou SMMA 4,5,6. La composition en protéines / lipides et les propriétés biochimiques de ces sites de contact membranaire ont été étudiés en particulier par rapport à leur rôle dans la régulation intracellulaire de Ca 2 + 4,5,6. L&#39;ER sert le magasin principal de la concentration intracellulaire de Ca 2 +, et à ce titre régit une multitude de processus cellulaires en aval de Ca 2 + signalisation, y comprisuding pliage post-traductionnelle de protéine et protéine maturation7. Mitochondries, d&#39;autre part, maintenir l&#39;homéostasie du Ca 2 +, par mise en mémoire tampon Ca 2 + concentration en empêchant ainsi l&#39;ouverture de voies de l&#39;apoptose en aval du Ca 2 + 4,8 balourd. La nature dynamique des MMA rend sites idéaux pour disséquer les mécanismes de base cellulaires, y compris le Ca 2 + signalisation et la réglementation des mitochondriale de Ca 2 + de survie biosynthèse des lipides concentration, et le transport, le métabolisme énergétique cellulaire et 4,9,10,11,12. Plusieurs protocoles ont été décrits pour la purification de ces microdomaines de tissus du foie et des cellules cultivées 13,14. Les méthodes de prise en compte précédemment publiées, nous avons adapté un protocole pour l&#39;isolement des mitochondries et des MMA propres du cerveau de souris adulte. Pour cette procédure, nous avons ajouté une étape de purification supplémentaire, à savoir une extraction Triton X100, qui enables de l&#39;isolement du glycosphingolipide enrichi microdomaine (GEM) fraction des MMA. Ces préparations GEM partager des composants de plusieurs protéines avec des radeaux lipidiques et cavéoles, dérivées de la membrane plasmique ou d&#39;autres membranes intracellulaires, et sont proposés à fonctionner comme des lieux de rencontre pour le regroupement des protéines réceptrices et des interactions protéine-protéine 4,15.

Basé sur notre expérience avec l&#39;utilisation de ce protocole, nous pouvons en toute sécurité le recommande pour l&#39;isolement et la purification de la SMMA, les pierres précieuses et les fractions mitochondriales de cerveau de souris. La procédure décrite est hautement reproductible et cohérente. Dans la figure 1, nous montrons une image représentative de la façon dont la couche mitochondries et MMA propres pure sur un gradient Percoll (étape 3.4). Une définie, bande laiteuse contenant...

Watch this Scientific Journal Video about Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain at JoVE.com

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

Cette procédure illustre comment isoler du cerveau de souris adulte les membranes des mitochondries associées à l&#39;urgence ou la SMMA et les glycosphingolipides-fractions enrichies en microdomaines de MMA propres et préparations mitochondriales.

Les mitochondries associées à membranes ER (SMMA) et glycosphingolipides microdomaines enrichis (GEM): Isolement à partir de cerveau de souris

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

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Neuroscience

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

24.2K vues.  St Jude Children's Research Hospital. Cette procédure illustre comment isoler du cerveau de souris adulte les membranes des mitochondries associées à l'urgence ou la SMMA et les glycosphingolipides-fractions enrichies en microdomaines de MMA propres et préparations mitochondriales.

Vidéo: Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Isolement et culture de cellules endothéliales de la embryonnaire du cerveau antérieur

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

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Neurosciences

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Isolement des capillaires cérébraux du tissu de cerveau humain frais

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

Isolement et Extraction de l’ARN des neurones, les Macrophages et les cellules microgliales du cerveau de larves de poisson zèbre

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

Isolement des microglies spécifiques à la région de l'hémisphère cérébral d'une souris adulte pour le séquençage profond de l'ARN à une seule cellule

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Studying Pre-formed Fibril Induced &amp;#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

Étude de l’accumulation préforme induite d’α-synucléine par fibrile dans les neurones dopaminergiques de la souris embryonnaire primaire

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

Dissection et isolement de la glie murine de plusieurs régions du système nerveux central

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

Un modèle d’AVC photothrombotique enrichi en fibrine et sensible au tPA

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

Quantification de la modification des histones cérébrales de souris à l’aide de la cytométrie en flux

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

Pleins feux sur l’auteur : Améliorations de la recherche sur la régulation de l’expression génique

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

Quantification des modifications post-traductionnelles des histones globales à l’aide de la cytométrie en flux intranucléaire dans la microglie cérébrale isolée de souris

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...