Name: isolation of mouse coronary endothelial cells
Uploaded: 2016-07-03T13:00:00.000+00:00
Duration: 7 min 36 s
Description: Watch this Scientific Journal Video about Isolation of Mouse Coronary Endothelial Cells at JoVE.com

Este trabalho foi apoiado pela concessão do National Institutes of Health (HL115578 para A. Makino).

A pesquisa em ratos foi aprovado pelo Comitê Institucional de Animal Care and Use (IACUC) da Universidade do Arizona e foi realizada de acordo com o Instituto Nacional de Saúde (NIH) orientações. NOTA: Seções 1, 2 e 3 devem ser realizadas um dia antes do experimento, como a instalação. 1. Soluções Preparando <ol><li> tampão de CD31 (50 ml) <ol><li> Adicionar 50 ml de 1 x tampão fosfato salino (PBS) a um tubo de 50 ml. Pesar 0,05 g de Albumina de Soro Bovino (BSA) e 0,037 g de ácido etilenodiaminotetracético (EDTA) e adicionar ao tubo de 50 ml e colocar o tubo para rodar durante cerca de 1 hora até dissolver o pó. Ajustar o pH para 7,4, filtrar através de um filtro de 0,2 um para um novo tubo de 50 ml e armazenar a 4 ° C até à sua utilização. </li></ol></li><li> Solução Salina Equilibrada de Hank (HBSS) com HEPES (500 ml) <ol><li> Adicionar 1,1915 g de HEPES fazer HEPES 10 mM em HBSS solução e misturar bem. Medir o pH e ajustar para 7,4. Filtrar e armazenar a 4C. </li></ol></li><li> A solução de Kreb 1x (1 L) <ol><li> Adiciona-se 100 ml de solução de Krebs × 10 [Tabela 1] para se dobrar de água destilada em um balão de 1 L. Pesar 2,1 g de bicarbonato de sódio e 2 g de D-glucose e adiciona-se ao balão. Misture e ajustar o volume total de 1 L. </li><li> Solução de bolha com 95% de O2 / 5% de CO 2 gasoso durante 30 minutos e solução de filtro no interior do exaustor. Armazenar a 4 ° C e manter em gelo durante o experimento. </li></ol></li></ol> 2. Preparar as esferas magnéticas <ol><li> Adicionar 1 ml de tampão de CD31 em cada tubo de 1,5 ml (um tubo por amostra). Misture as ovelhas anti-ratos contas de IgG e adicione a quantidade adequada a cada amostra [Tabela 1]. Agite os tubos vigorosamente 30 vezes e colocá-los no prato magnético durante 1 min de agitação. Abrir os tubos, enquanto no prato e despejar as soluções dentro de um balde. </li><li> Adicionar 1 ml de tampão de CD31 em cada tubo. Adicionar 2 mL de rato anti-CD31 anticorpo a cada tubo e girar tubos O / N a 4 ºC. </li></ol> 3. A autoclavagem <ol><li> Autoclave todas as ferramentas de cirurgia e ponteiras. Corte as pontas de pontas de pipeta de modo que há dicas com 4 diâmetros diferentes, de mais vasto para mais estreito. diâmetros aproximados para as pontas são de 3 mm, 2,5 mm, 2 mm e 1,5 mm, respectivamente. Cortar as pontas e a autoclave dias antes com 30 min de esterilização a uma temperatura de 121 ºC. NOTA: As secções 4-11 deve ser levada a cabo no dia da experiência. </li></ol> 4. Soluções Preparando <ol><li> Prepare tampão de lavagem contendo 2% de ferro-fortificada Calf Serum (IFCS) em M199. Para geração de imagens, preparar 30 ml / mouse. </li><li> Preparar a solução de enzima (10 mL / amostra). Pesar 0,6 unidade / ml de Dispase e 1 mg / mL de colagenase tipo II para um tubo de 50 ml. Adicionar M199 e girar o tubo a 4 ºC durante 15 min. Filtra-se através de filtro de 0,2 um para um novo tubo de 50 ml inside o capô e manter a 4 ° C até o uso. </li><li> Prepare HBSS / HEPES com heparina. Adicionar 10 ml de HBSS com 10 mM de HEPES em 15 ml de tubo. Adicionar 100 ul de heparina para o tubo e manter a 4 ° C até à sua utilização. </li></ol> 5. lavagem das pérolas <ol><li> Retire os tubos com contas do rotator a 4 ºC e trazer para a capa. Colocar os tubos na placa magnética e agitar durante 1 min. Despejar a solução e adicionar 1 ml de tampão de CD31 em cada tubo. </li><li> Pegue os tubos fora da placa magnética e agitar vigorosamente 30 vezes. Colocar os tubos em volta da placa magnética e repetir para um total de 3 lavagens. Após a conclusão, colocar os tubos de volta à 4C e continuar girando até o uso. </li></ol> 6. Dissecção <ol><li> Injectar os ratinhos intraperitonealmente com 0,1 ml de heparina para evitar a coagulação do sangue. Esperar 10 minutos, em seguida, anestesiar os ratos usando injeção intraperitoneal de 0,01 ml de pentobarbital. Verifique se o mouse está sentindodor, apertando o pé. </li><li> Posicione o mouse em uma placa de poliestireno com a cabeça para cima e mantenha os braços com pinos. Use uma pinça e difíceis-CUT-tesoura para cortar a pele na região torácica superior até a garganta. </li><li> Fazer um corte no centro da caixa torácica logo acima do diafragma. Cortar o osso para os lados e para cima para a garganta para fazer um triângulo expor o coração e os pulmões. Cortar a aorta logo acima do diafragma. </li><li> Segure o timo com uma pinça sem puxar e usar uma tesoura para cortar qualquer tecido conjuntivo com cuidado para remover pulmões e coração juntos. tecidos lugar em um copo com 1 × Kreb de, agitar com uma pinça e transferir para 6cm de prato com 1 × Kreb de. </li><li> Retire os lobos pulmonares, utilizando tesouras e pinças. Inserir um cateter estéril de 20 G para a aorta. Lave o sangue no sistema circulatório coronária com HBSS / heparina. Fazer um pequeno corte no ventrículo, agitar o coração e transferir para um tubo de amostra de M199. Mantenha os tubos em gelo. </li></ol><p<html> class = &quot;jove_title&quot;&gt; 7. digestão do tecido <ol><li> Transferir o coração a um disco de 6 cm cheia com 10 ml de solução de enzima e mediu-lo em pequenos pedaços com uma tesoura. Coloque os pratos na incubadora a 37 ºC durante 15 min. Agitar materiais digeridos por pipetagem cima e para baixo 30 vezes através de uma ponta com uma extremidade cortada para permitir que o tecido a passar por mais fácil. </li><li> Coloque-o de volta na 37 ºC incubadora por mais 15 min. Repita agitação três vezes, cada vez com pontas de pipeta de maiores aberturas para mais estreito e 15 minutos de incubação entre cada. </li><li> Depois da última 15 min de incubação, colher amostras para fora e para dentro do capô. Pipetar solução cima e para baixo 30 vezes utilizando ponta da pipeta com a abertura mais estreita e passar através de um filtro de 40 | iM de células para um novo tubo de 50 ml. </li><li> Lava-se a placa com 10 ml de tampão de lavagem, utilizando uma pipeta de 10 ml e transferir para filtro de células. Colocar os tubos de 50 ml de centrífuga e centrifugação a 400 g durante 10 min à temperatura ambiente. </li></ol>itle &quot;&gt; 8. de lavar roupa e conjugação com grânulos <ol><li> Remover o sobrenadante sem perturbar o sedimento. Lavar as células com 5 ml de tampão de lavagem por duas vezes. </li><li> Obter um dos anteriormente preparados tubos de 1,5 ml com grânulos e trabalho em um tubo de cada vez. Coloque um tubo de 1,5 ml na placa magnética, agitar 3 vezes e abri-lo. </li><li> Após a última centrifugação, remover tanto sobrenadante quanto possível, sem perturbar o sedimento. Dissociar os aglomerados de células sacudindo vigorosamente. </li><li> Adicionar 1 ml de tampão de lavagem para o tubo de 50 mL e misturar as células com uma pipeta de 1 ml. Despejar a solução a partir do tubo de 1,5 ml e adicionar 1 ml de suspensão de células a partir do tubo de 50 mL para os grânulos. Flick os tubos de 30 vezes, em seguida, gire a 4 ºC durante 30 minutos. </li></ol> 9. Preparação para Cultura de Células / Imagem <ol><li> Prepare câmaras para as células plaqueamento. Adicionar solução de gelatina (5% w / v, 300 mL) para a câmara bem para cobrir a superfície e incuba-se durante 30 min a 37 ºC. Após a incubação, REMgelatina ove e secar a superfície. </li><li> Retire os tubos com contas / células após 30 min de rotação e colocar na placa magnética no interior do exaustor. Agita-se durante 2 min, despejo e adicionar 1 ml de tampão de lavagem. </li><li> Tome tubos para fora da placa, agitar vigorosamente 30 vezes em seguida, agite 30 vezes. Colocá-los de volta na placa magnética e agitar durante 1 min. Repetir 4 vezes, para um total de 5 lavagens, mas com 1 min de agitação sobre a placa. </li><li> Após a última lavagem, retirar um tubo e trabalhar em cada um tubo de cada vez. Agite 30 vezes em seguida, agite 30 vezes. Coloque-o sobre a placa magnética e agitar durante 1 min. Despejar e adicionar 1 ml de 20% de mídia IFCS CE (pré-aquecido a 37 ºC). </li><li> Agita-se vigorosamente 30 vezes em seguida, agite 30 vezes. Ajuste pipeta para 500 mL e pipeta cima e para baixo 10 vezes. Transferir 500 ul a cada câmara. </li><li> Coloque a 37 ºC incubadora e deixar O / N. No dia seguinte, lave as células com 10% de media FBS CE. </li></ol> 10. Preparação para Blot As amostras (BM) ocidentais <ol><li> Takum e os tubos com contas / células após 1 hora de rotação. Colocá-los na placa magnética e agitar por 2 min. Despejar o sobrenadante e adicionar 1 ml fria HBSS. Agita-se vigorosamente 30 vezes em seguida, agite 30 vezes. Repita duas vezes para 3 lavagens, mas com 1 min de agitação sobre a placa. </li><li> Depois da última lavagem, colocar tubos na placa magnética e agitar durante 1 min. Remover o tampão usando uma pipeta e lisar as células de WB utilizando um método previamente descrito 11,12. lisado foi recolhido normalmente contém cerca de 30 ug de proteína. </li></ol> 11. Imagem <ol><li> Lave as células 3 vezes com a mídia, com volume final de 250 ul de uma câmara. Adicionar 1,5 mL de AcLDL-Dil para as células. A partir deste ponto, manter as células no escuro. </li><li> Incubar por 3,5 horas a 37 ° C. Adicionar 1,5 uL de Hoechst, misturar bem e incubar durante 30 min a 37 células ˚C.Wash com PBS e fixar com 4% de paraformaldeído (PFA) durante 20 min à TA. </li><li> Lavar duas vezes com PBS e bloquear com 5% de BSA-PBS, durante 30 min à temperatura ambiente. Lavar duas vezes com 1% de BSA-PBS. Diluir BS-lectina-FITC utilizando 1% de BSA-PBS a uma concentração de 2 ug / ml e adicionar 300 ul a cada câmara. </li><li> Coloque a câmara no agitador orbital, à temperatura ambiente durante 30 min. Lavar com PBS 3 vezes. </li><li> Visualizar as células sob o microscópio de fluorescência com o tempo de exposição de 200 ms para FITC (coloração com lectina, um marcador CE, excitação / emissão a 488 nm / 519 nm), 30 ms para DAPI (Hoechst, uma coloração, de excitação / emissão nuclear a 358 nm / 461 nm), e 300 ms para TRITC (coloração AcLDL, um marcador CE, excitação / emissão a 557 nm / 576 nm). Adquirir imagens usando uma lente objetiva de 20X. </li></ol>

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	<li>Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+functions.">Endothelial cell functions.</a> J Cell Physiol. 196, 430-443 (2003).</li><li>Sumpio, B. E., Riley, J. T., Dardik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+in+focus:+endothelial+cell.">Cells in focus: endothelial cell.</a> Int J Biochem Cell Biol. 34, 1508-1512 (2002).</li><li>Cines, D. B., 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=Endothelial+cells+in+physiology+and+in+the+pathophysiology+of+vascular+disorders.">Endothelial cells in physiology and in the pathophysiology of vascular disorders.</a> Blood. 91, 3527-3561 (1998).</li><li>Deanfield, J. E., Halcox, J. P., Rabelink, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+function+and+dysfunction:+testing+and+clinical+relevance.">Endothelial function and dysfunction: testing and clinical relevance.</a> Circulation. 115, 1285-1295 (2007).</li><li>Jin, Y., Liu, Y., Antonyak, M., Peng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+vascular+endothelial+cells+from+murine+heart+and+lung.">Isolation and characterization of vascular endothelial cells from murine heart and lung.</a> Methods Mol Biol. 843, 147-154 (2012).</li><li>Makino, A., Platoshyn, O., Suarez, J., Yuan, J. X., Dillmann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downregulation+of+connexin40+is+associated+with+coronary+endothelial+cell+dysfunction+in+streptozotocin-induced+diabetic+mice.">Downregulation of connexin40 is associated with coronary endothelial cell dysfunction in streptozotocin-induced diabetic mice.</a> Am J Physiol Cell Physiol. 295, C221-C230 (2008).</li><li>Marelli-Berg, F. M., Peek, E., Lidington, E. A., Stauss, H. J., Lechler, R. I. <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+endothelial+cells+from+murine+tissue.">Isolation of endothelial cells from murine tissue.</a> J Immunol Methods. 244, 205-215 (2000).</li><li>van Beijnum, J. R., Rousch, M., Castermans, K., vander Linden, E., Griffioen, A. W. <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+endothelial+cells+from+fresh+tissues.">Isolation of endothelial cells from fresh tissues.</a> Nat Protoc. 3, 1085-1091 (2008).</li><li>Nolan, D. J., 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=Molecular+signatures+of+tissue-specific+microvascular+endothelial+cell+heterogeneity+in+organ+maintenance+and+regeneration.">Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.</a> Dev Cell. 26, 204-219 (2013).</li><li>Sobczak, M., Dargatz, J., Chrzanowska-Wodnicka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+pulmonary+endothelial+cells+from+neonatal+mice.">Isolation and culture of pulmonary endothelial cells from neonatal mice.</a> J Vis Exp. , (2010).</li><li>Makino, A., Dai, A., Han, Y., Youssef, K. D., Wang, W., Donthamsetty, R., Scott, B. T., Wang, H., Dillmann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=O-GlcNAcase+overexpression+reverses+coronary+endothelial+cell+dysfunction+in+type+1+diabetic+mice.">O-GlcNAcase overexpression reverses coronary endothelial cell dysfunction in type 1 diabetic mice.</a> Am J Physiol Cell Physiol. 309, C593-C599 (2015).</li><li>Sasaki, K., Donthamsetty, R., Heldak, M., Cho, Y., Scott, B. T., Makino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VDAC:+old+protein+with+new+roles+in+diabetes.">VDAC: old protein with new roles in diabetes.</a> Am J Physiol Cell Physiol. 303, C1055-C1060 (2012).</li></ol>

The authors have nothing to disclose.

As células endoteliais mais utilizados na pesquisa são células humanas endoteliais da veia umbilical (HUVECs) por causa de sua conveniência e facilidade de cultivo. No entanto, uma grande quantidade de pesquisa requer a disponibilidade de um modelo fisiológico atingido pelo isolamento de células endoteliais específicas de outros órgãos 10. As células endoteliais formam uma população muito reduzida das células no tecido do coração; seu isolamento e cultura pode revelar-se muito difícil. <p ...

modelos de ratinho de várias doenças cardiovasculares e desordens metabólicas suportar mudanças fisiológicas e moleculares que são semelhantes aos encontrados em pacientes. Além disso, a alteração genética de ratinhos é uma ferramenta poderosa que nos permite investigar o papel patogénico de genes específicos no desenvolvimento e progressão de doenças. Hoje em dia, o gene-knock-in específico de células-tipo ou ratinhos -out podem ser facilmente gerados em muitos laboratórios e a medição de ARNm e os níveis de proteína em tipos específicos de células é o primeiro passo para determinar se os ratos foram verdadeiramente geneticamente modificado. O endotélio é uma camada única fina de células endoteliais (ECs) que reveste a parede vascular interior. As células endoteliais desempenham um papel crítico na regulação da tensão vascular, da permeabilidade vascular, e nova formação vascular 1,2. A disfunção endotelial é uma característica de muitas condições patológicas e alterações na função endotelial pode conduzir a vários carrosdoenças cardiovasculares 3,4. É, assim, importante para estudar a função das células endoteliais em condições fisiológicas e fisiopatológicas. Existem várias maneiras de isolar ECs 5-8 e o método de isolamento tem que ser optimizado dependendo de qual tecidos e espécies serão utilizados. Isto é porque CEs a partir de diferentes tecidos apresentam um elevado nível de heterogeneidade no que diz respeito aos seus marcadores de superfície e expressão da proteína 9. isolamento de células endoteliais pode muitas vezes ser um desafio e requer algum grau de treinamento e prática. Uma vez alcançada, o método de isolamento proposto neste protocolo revela-se estável e eficiente. O objetivo geral deste método é a obtenção de rato ECs coronariana (MCECs) de alta qualidade e quantidade. Embora a quantidade de células recolhidas a partir de um coração é menos do que as células recolhidas a partir de outros tecidos, utilizando outras técnicas, esta técnica ainda proporciona apostaqualidade ter. A pureza das células endoteliais na população resultante é maior do que 90%. Assim, essa técnica seria o ideal para aplicações que dependem de células endoteliais puramente isolados, como imagens de células.

<table><tbody><tr><td>BSA</td><td>Sigma</td><td>A3311-10G</td><td></td></tr><tr><td>EDTA</td><td>Fisher</td><td>BP120-500</td><td></td></tr><tr><td>HBSS</td><td>Fisher</td><td>SH3026801</td><td></td></tr><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H4034-500G</td><td></td></tr><tr><td>Sodium Bicarbonate&amp;nbsp;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>D-(+)-Glucose&amp;nbsp;</td><td>Sigma</td><td>G7021-1KG</td><td></td></tr><tr><td>DynaBeads pan mouse IgG beads&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11035</td><td></td></tr><tr><td>Rat anti-mouse CD31 Antibody</td><td>BD Biosciences</td><td>550274</td><td></td></tr><tr><td>IFCS&amp;nbsp;</td><td>Fisher</td><td>SH30072.04</td><td></td></tr><tr><td>M199</td><td>Fisher</td><td>MT10060-CV</td><td></td></tr><tr><td>Dispase (Neutral Protease)</td><td>Worthington Biochemical Corp./Fisher</td><td>LS02109</td><td></td></tr><tr><td>Collagenase Type II&amp;nbsp;</td><td>Worthington Biochemical Corp./Fisher</td><td>LS004176</td><td></td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Fisher</td><td>BP381-1</td><td></td></tr><tr><td>Igepal</td><td>Sigma</td><td>I3021-50 ml</td><td></td></tr><tr><td>Phosphatase inhibitor cocktail</td><td>Sigma</td><td>P0044-1ml</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma</td><td>P8340-1 ml</td><td></td></tr><tr><td>Heparin&amp;nbsp;</td><td>Sigma</td><td>H3149-100KU</td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Fisher/Mediatech</td><td>MT35010CV</td><td></td></tr><tr><td>D-Valine</td><td>Sigma</td><td>V1255-5G</td><td></td></tr><tr><td>EGS</td><td>Corning</td><td>356006</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Fisher/Mediatech</td><td>MT-30-002-CI</td><td></td></tr></tbody></table>

isolation of mouse coronary endothelial cells

As células endoteliais estão alinhados na parede interna dos vasos sanguíneos e desempenham um papel importante na regulação do tónus vascular, da permeabilidade vascular, e nova formação vascular. disfunção das células endoteliais está implicada no desenvolvimento e progressão de muitas doenças cardiovasculares, incluindo doença cardíaca isquémica. Para examinar a função e caracterização de células endoteliais coronárias, o isolamento de células é o primeiro passo e exige elevado grau de pureza e a quantidade para conduzir experiências subsequentes. Este protocolo descreve um método eficiente para isolar ratinho adulto células endoteliais coronárias. O coração do rato é dissecado e picada em pedaços pequenos. Após a digestão do coração utilizando colagenase e dispase II, as células são lavadas e incubadas com esferas magnéticas que são conjugados com o anticorpo anti-CD31. As contas com células endoteliais são lavadas várias vezes e está pronto para usar em várias aplicações, incluindo imagens e experime biológica molecularNTS. Isolamento eficiente rende aproximadamente 10 4 células por um coração com mais de 90% de pureza.

O isolamento de células endoteliais coronárias de um coração de rato normalmente produz cerca de 10 4 células com mais de 90% de pureza com uma forma de paralelepípedos. Para uma população pura de células endoteliais, o cuidado deve ser tomado ao longo do protocolo para garantir um ambiente estéril livre de qualquer contaminação. O aparecimento de células alongadas ou células alargada dentro da população indica contaminação com outros tipos de células, as c?...

Watch this Scientific Journal Video about Isolation of Mouse Coronary Endothelial Cells at JoVE.com

Isolation of Mouse Coronary Endothelial Cells

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Isolamento de células de camundongo Coronária endoteliais

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

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JoVE Journal

Biology

11.1K visualizações.  University of Arizona, Tucson. This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Vídeo: Isolation of Mouse Coronary Endothelial Cells

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

Isolamento das Células Endocárdicas e Coronárias da Parede Livre Ventricular do Coração de Rato

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

Biologia do Desenvolvimento

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolamento e cultura de células endoteliais pulmonares de camundongos Neonatal

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Biologia

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

Isolamento de células endoteliais Microvascular do principal músculo esquelético murino

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Neurociência

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

Isolamento de tubos endoteliais microvasculares de Artérias Rato Resistência

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Isolamento de Células Endoteliais Primárias de Pulmão de Camundongo

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

Imunologia e Infecção

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

O isolamento de células endoteliais de Murino Válvula

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

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.

Isolamento e cultura primária de células de camundongo aórtica endoteliais

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

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.

Isolamento de Murino vasculares coronários células musculares lisas

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

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Isolamento e Caracterização de Células Endoteliais Hemorlicais do Fígado Primário do Rato

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in the endothelium at the transcriptomic and epigenomic levels, resulting in proatherogenic responses. Recently, single-cell RNA sequencing (scRNAseq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATACseq) studies were performed to determine the transcriptomic and chromatin accessibility changes at a single-cell resolution using the mouse partial carotid ligation (PCL) model. As endothelial cells (ECs) represent a minor fraction of the total cell populations in the artery wall, a luminal digestion method was used to obtain EC-enriched single-cell preparations. For this study, mice were subjected to PCL surgery to induce d-flow in the left carotid artery (LCA) while using the right carotid artery (RCA) as a control. The carotid arteries were dissected out two days or two weeks post PCL surgery. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single cells or single nuclei were obtained. These single-cell and single-nuclei preparations were subsequently barcoded using a 10x Genomics microfluidic setup. The barcoded single-cells and single-nuclei were then utilized for RNA preparation, library generation, and sequencing on a high-throughput DNA sequencer. Post bioinformatics processing, the scRNAseq and scATACseq datasets identified various cell types from the luminal digestion, primarily consisting of ECs. Smooth muscle cells, fibroblasts, and immune cells were also present. This EC-enrichment method aided in understanding the effect of blood flow on the endothelium, which could have been difficult with the total artery digestion method. The EC-enriched single-cell preparation method can be used to perform single-cell omics studies in EC-knockouts and transgenic mice where the effect of blood flow on these genes has not been studied. Importantly, this technique can be adapted to isolate EC-enriched single cells from human artery explants to perform similar mechanistic studies.

We present a method for isolating endothelial cells and nuclei from the lumen of mouse carotid arteries exposed to stable or disturbed flow conditions to perform single-cell omics experiments.

Isolamento de células endoteliais do lúmen de artérias carótidas de camundongos para experimentos multi-ômicos unicelulares

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in ...