Reconhecemos a contribuição de Renata Sano na concepção do protocolo inicial. Ad&#39;A. detém os joalheiros para Crianças (JFC) cadeira dotada de Genética e Terapia Gênica. Este trabalho foi financiado em parte pelo NIH GM60905, DK52025 e CA021764, e os americanos libaneses sírios Charities Associados (ALSAC).

<html> O protocolo que se segue destina-se ao isolamento e purificação de MAMs e gemas de cérebro de rato Soluções necessárias para o isolamento de mitocôndrias, MAMs e pedras preciosas O fracionamento de obter preparação mitocondrial crua: Solução A: 0,32 M de sacarose, 1 mM de NaHCO 3, 1 mM de MgCl2, 0,5 mM de CaCl 2 + inibidores de protease (adicionar fresco, conforme necessário) Solução B: 0,32 M de sacarose, 1 mM de NaHCO 3 Inibidores da Protease + (adicionar fresco, conforme necessário) Isolamento MAMs: Meio de isolamento: 250 mM de manitol, 5 mM de HEPES pH 7,4, 0,5 mM de EGTA, 0,1% BSA Gradiente de tampão: 225 mM de manitol, 25 mM HEPES pH 7,5, 1 mM de EGTA, 0,1% BSA (concentração final) Isolamento GEMs: conteúdo &quot;&gt; Tampão de Extracção GEM: 25 mM HEPES pH 7,5, 0,15 M NaCl, 1% Triton X-100 Os inibidores de protease + (adicionar fresco, conforme necessário) Tampão GEM solubilizante: 50 mM Tris-HCl, pH 8,8, EDTA 5 mM, SDS a 1% + inibidores da protease (adicionar fresco, conforme necessário). 1. Fracionamento subcelular Primeiro Passo: Remoção de Núcleos, células não lisadas e restos celulares <ol><li> Euthanize do mouse em câmara de gás 2. </li><li> Remover imediatamente cérebro, reduzir pela metade, coloque em tubos de 2 ml em gelo e pesar. </li></ol> Nota: Mantenha as metades do cérebro separado durante todo o procedimento. O seguinte aplica-se ao tratamento de um meio único cérebro. <ol start="3"><li> Cérebro lugar metade em um 2 ml de pré-refrigerados de vidro moedor de tecido Dounce contendo 1 ml de solução A. frio Misturar com 15 tacadas no total de um pilão grande folga. </li><li> Transferir para um tubo Falcon de 15 ml, em gelo, e diluahomogeneizados até 10 volumes w / v, por exemplo 0,225 g a 2,25 ml. </li><li> Centrifugar a 1.400 xg amostra durante 10 min a 4 ° C. </li><li> Cuidadosamente remover o sobrenadante e transferir para um tubo de centrífuga de 30 ml de fundo redondo de vidro, em gelo. Certifique-se de não perturbar o sedimento. </li><li> Ressuspender o sedimento em 10 volumes dos mesmos de uma solução. Homogeneizar no mesmo moinho, com 3-6 movimentos de um pilão de pequena folga, 1 ml de cada vez. </li><li> Transferir para um tubo de 15 ml fresco falcon e centrifugar a 710 xg durante 10 min a 4 ° C. Isso resulta em um pellet de núcleos e restos celulares. </li><li> Cuidadosamente remover o sobrenadante e piscina com o sobrenadante guardado no passo 1.6. </li></ol> 2. Segunda Etapa: Isolamento mitocôndrias bruto <ol><li> Centrifugar os sobrenadantes a 13.800 xg durante 10 min a 4 ° C. </li><li> Transferir o sobrenadante para um tubo de centrífuga Ultra-Clear Beckman, em gelo </li><li> Ressuspender o sedimento em 10 volumes de solução A. Homogeneizar no mesmo moinho, com 3-6 movimentos de um pilão de pequena folga, 1 ml de cada vez. </li><li> Centrifugar o passo 2.1. </li><li> Repita os passos 2,2-2,4. </li><li> O sedimento resultante é uma fracção enriquecida mitocondrial. Piscina os sobrenadantes (citosol e ER), cubra com parafilme e manter em gelo. </li><li> Ressuspender o sedimento com 6 golpes de um pilão de pequena folga, em 4,8 ml / g (g é referida ao peso do cérebro original) de solução B, em um homogeneizador de fresco. </li><li> Utilizando pipetas de Pasteur de vidro, preparar um gradiente de sacarose descontínuo de um tubo de centrífuga Ultra-Clear Beckman, adicionando subsequentemente a parte inferior do tubo, como se segue, com cuidado, assegurando que nenhum bolhas formam-se: Resupended Pellet (0,32 M de sacarose) 3 ml de sacarose mM 850 (em 1 mM de NaHCO3) 3 ml de sacarose 1 M (em 1 mM de NaHCO3) 3 ml de sacarose 1,2 M (em 1 mM NaHCO3) </li><li> Centrifugar a 82.500 xg durante 2 horas a 4 ° C. A separação resultante, no final do gradiente irá produzir três bandas e uma pelota: 1) de mielina e de contaminantes de membrana outros (0,32 M - 0,85 M de interface), 2) ER, Golgi, as membranas plasmáticas (0,85 M - 1 M de interface); 3) sinaptossomas (1 M - 1,2 M de interface), 4) bruto mitocôndrias (pellet), que é utilizada para os passos de purificação subsequentes </li></ol> 3. Isolamento de mitocôndrias associada membrana ER, MAMs <ol><li> Ressuspender o bruto fresco mitocôndrias isoladas a partir de uma metade do cérebro, em 2 ml de meio de isolamento contendo inibidores de protease recentemente adicionados. </li><li> Prepare um gradiente de Percoll 30% com Gradiente ml de buffer de 8 por metade do cérebro, e colocar em um tubo de centrífuga Ultra-Clear Beckman. </li></ol> * Nota: Certifique-tampão gradiente mais concentrado (1,43 vezes) para alcançar a concentração final correta depois diluting Percoll a 30%. <ol start="3"><li> Suspensão mitocondrial camada em cima do gradiente preparado lentamente para evitar bolhas. </li><li> Centrifugar a 95.000 xg durante 30 min, 4 ° C. </li><li><ol class="lroman"><li> Remover a fracção pesada (banda inferior) PRIMEIRO com uma pipeta de Pasteur de vidro e transferir para um tubo de vidro de fundo redondo de fresco, em gelo. </li><li> Remova a fracção leve (banda superior) SEGUNDA com outra pipeta de Pasteur de vidro e transferir para um tubo de vidro separado de fundo redondo, fresco, em gelo. </li></ol></li><li> Diluir ambas as fracções recolhidas no passo 3.5 com 10 ml meio de isolamento e centrifugar a 6300 xg durante 10 min a 4 ° C. </li><li> Descartar o sobrenadante a partir da fracção pesada obtida no passo 3.6, ressuspender o sedimento com um outro meio de isolamento de 10 ml e centrifugar de novo, como no passo 3.6. O sedimento resultante será a fracção pura mitocondrial. </li><li> Transferir o sobrenadante do Frac Luzção obtida no passo 3.6 para um tubo de centrífuga Ultra-Clear Beckman, em gelo. Descartar o sedimento. </li><li> Adicionar meio de isolamento suficientes para o sobrenadante obtido no passo 3.8 para encher o tubo e centrifuga-se a 100.000 xg durante 1 h, 4 ° C. O sedimento resultante será a fracção MAMs. Remova cuidadosamente e elimine o sobrenadante. </li></ol> Nota: Nesta etapa, você também pode centrifugar a 100.000 xg durante 1 hora, 4 ° C, o sobrenadante salvo no passo 2.6. O sedimento será a fracção sobrenadante ER e a fracção citosólica. 4. Extração de glicoesfingolipídeos enriquecidas microdomínios, Gemas <ol><li> Lisar as fracções puras mitocôndrias e / ou MAM em 500 ml-1 ul de tampão de extracção durante 20 minutos em gelo. </li><li> Centrifugar os lisados ​​a 15300 xg durante 2 min a 4 ° C. </li><li> Recolher os sobrenadantes (Triton X-100 o material solúvel) e re-centrifugação de pelotasdurante 2 minutos para remover o restante material solúvel. (Triton X-100 As mitocôndrias extraído e / ou Triton X-100 MAMs extraídos). </li><li> Solubilizar pelotas em solubilizantes Buffer. Este material solubilizado representa as fracções de gema e pode ser usado para análise posterior. </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>

Nenhum dos autores tem quaisquer conflitos de interesse a declarar.

Os locais de contacto entre as membranas intracelulares ou entre organelas e da membrana plasmática de células representam dinâmicas plataformas de sinalização para os processos celulares básicos. A caracterização exata de sua função e composição em ambas as condições fisiológicas e patológicas requer protocolos de purificação confiáveis ​​e reprodutíveis. Os métodos descritos aqui foram especificamente optimizados pelo nosso laboratório para o isolamento e purificação dos MAMs e as suas gema...

<table border="1"><tbody><tr><td> Nome do reagente / Material </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td></td><td></td><td></td><td> REAGENTES </td></tr><tr><td></td><td></td><td></td><td> Fracionamento </td></tr><tr><td> Sacarose </td><td> Fisher Scientific </td><td> S5-500 </td><td></td></tr><tr><td> Bicarbonato de sódio </td><td> Sigma-Aldrich </td><td> S-5761 </td><td></td></tr><tr><td> Cloreto de magnésio, hexa-hidratada </td><td> Fisher Scientific </td><td> BP214-500 </td><td></td></tr><tr><td> O cloreto de cálcio hidratado, </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-Manitol </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, Fracção V, de choque de calor, liofilizado </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> Cloreto de Sódio </td><td> Fisher Scientific </td><td> S271-3 </td><td></td></tr><tr><td> Tris Base </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> Dodecil sulfato de sódio (SDS) </td><td> Fisher Scientific </td><td> BP166-500 </td><td></td></tr><tr><td></td><td></td><td></td><td> Comum </td></tr><tr><td> Inibidores de protease Tablets, completa EDTA-livres </td><td> Roche </td><td> 11-873-580-001 </td><td></td></tr><tr><td></td><td></td><td></td><td> EQUIPAMENTOS </td></tr><tr><td> 2 ml Douce todo em vidro Grinders Tecido </td><td> Kimble perseguição </td><td> 885300-0002 </td><td></td></tr><tr><td> 15 ml de polipropileno cónicas Tubos Centrífuga, BD Falcon </td><td> BD </td><td> 352097 </td><td></td></tr><tr><td> 30 ml de fundo redondo tubos de centrífuga de vidro </td><td> Kimble perseguição </td><td> 45500-30 </td><td></td></tr><tr><td> 15 ml de fundo redondo de vidro CentriTubos Fuge </td><td> Kimble perseguição </td><td> 45500-15 </td><td></td></tr><tr><td> Tubos de ultracentrifugação, ultra-claro, Thinwall, mm 14x89 </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> Descartável vidro borosilicato Pasteur Pipetas, 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

Organelas intracelulares são estruturas altamente dinâmicas com diferentes forma e composição, que estão sujeitos a células específicas sinais intrínsecos e extrínsecos. As membranas são freqüentemente justapostas em locais de contato definidas, que se tornam centros de troca de moléculas de sinalização e componentes de membrana 1,2,3,4. As inter-organellar microdomínios de membrana que são formados entre o retículo endoplasmático (ER) e das mitocôndrias durante a abertura do IP3-sensitive Ca 2 + canal são conhecidos como as mitocôndrias associado-ER membranas ou mams 4,5,6. A composição de proteína / lípido e propriedades bioquímicas dos sítios de contacto de membrana têm sido extensivamente estudadas em particular em relação ao seu papel na regulação da Ca 2 + intracelular 4,5,6. O ER serve como o armazenamento primário do Ca 2 + intracelular, e, nessa qualidade regula uma variedade de processos celulares a jusante de sinalização de Ca 2 +, including pós-traducional de dobragem de proteína e proteína maturation7. As mitocôndrias, por outro lado, manter a homeostase de Ca2 +, por tamponação citosólica concentração de Ca2 + que impede a abertura de vias apoptóticas jusante de Ca 2 + 4,8 desequilíbrio. A natureza dinâmica das MAMs torna locais ideais para dissecar mecanismos básicos celulares, incluindo Ca 2 + de sinalização e regulação da mitocondrial Ca 2 + sobrevivência concentração biossíntese de lipídios e transporte, metabolismo energético celular e 4,9,10,11,12. Vários protocolos têm sido descritas para a purificação dessas microdomínios de tecido do fígado e células de cultura 13,14. Tomando métodos anteriormente publicados em consideração, nós adaptamos um protocolo para o isolamento de mitocôndrias e MAMs a partir do cérebro de rato adulto. Para este procedimento, nós adicionamos uma etapa de purificação extra, ou seja, uma extração de Triton X100, que enables o isolamento da fracção microdomínio enriquecido glicosfingolípidos (GEM) dos MAMs. Estas preparações GEM partes componentes de proteínas com várias cavéolas e lipídico jangadas, derivadas a partir da membrana plasmática ou outras membranas intracelulares, e são propostos para funcionar como pontos de recolha para a agregação de proteínas receptoras e de interacções proteína-proteína 4,15.

Com base em nossa experiência com o uso deste protocolo podemos seguramente recomendar para o isolamento e purificação de MAMs, Gemas e frações mitocondriais de cérebro de rato. O processo como descrito é altamente reprodutível e consistente. Na Figura 1 mostra uma imagem representativa da camada de forma pura e mitocôndrias MAMs em um gradiente de Percoll (passo 3.4). Uma banda, definida leitoso contendo purificado mitocôndrias (Mito-P) segrega na parte inferior do tubo de ultracentrífuga, e...

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

Este procedimento ilustra como isolar a partir do cérebro do rato adulto membranas mitocôndrias associadas ER ou mams e as fracções enriquecidas glicoesfingolipídeos-microdomínio de MAMs e preparações mitocondriais.

Mitocôndrias associadas Membranas ER (MAMs) e glicoesfingolipídeos microdomínios enriquecidos (GEMS): Isolamento de cérebro de rato

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

mitochondria-associated-er-membranes-mams-glycosphingolipid-enriched

Research

JoVE Journal

Neuroscience

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

24.2K visualizações.  St Jude Children's Research Hospital. Este procedimento ilustra como isolar a partir do cérebro do rato adulto membranas mitocôndrias associadas ER ou mams e as fracções enriquecidas glicoesfingolipídeos-microdomínio de MAMs e preparações mitocondriais.

Vídeo: 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.

Isolamento e cultura de células endoteliais da Embryonic posencéfalo

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

Neurociência

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.

Isolamento dos capilares cerebrais do tecido do cérebro humano fresco

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.

Isolamento e extração de RNA de neurônios, macrófagos e Microglia de Zebrafish Larval cérebros

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.

Isolamento da Microglia específica da Região de um hemisfério cerebral de camundongo adulto para sequenciamento profundo de RNA unicelular

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.

Estudando o acúmulo de α-sinucleína induzida por fibril pré-formado em neurônios de dopamina de cérebro médio do camundongo embrionário primário

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.

Dissecção e isolamento da glia murina de múltiplas regiões do sistema nervoso 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.

Modelo de AVC fototrombótico enriquecido com fibrina e sensível ao tPA

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

Quantificação de modificação de histonas cerebrais de camundongos usando citometria de fluxo

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.

Autor em destaque: Aprimoramentos na pesquisa de regulação da expressão gênica

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.

Quantificação de modificações pós-traducionais globais de histona utilizando citometria de fluxo intranuclear em microglia cerebral isolada de camundongos

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