这项工作是由美国国立卫生研究院HL109478（KNF）的支持。作者吉娜·金正日和乔安·泰勒承认并感谢他们的协助隔离和保持PASMC文化。

在西北大学的机构动物护理和使用委员会批准该协议。 1。从新生小鼠肺动脉隔离 - 第一天<ol><li>准备完成的M199媒体 - M199混合400毫升，用100毫升（最终浓度= 20％）热灭活的胎牛血清和5毫升（最终浓度= 1％），青霉素/链霉素。 </li><li>准备用5毫升（终浓度= 1％），青霉素/链霉素的无血清M199媒体-混合500毫升M199。 </li><li>准备PA琼脂糖 -混合0.05克的琼脂糖凝胶加0.05克的铁颗粒在10毫升无菌，无血清的M199媒体。 </li><li>准备的龙琼脂糖 -混合0.1克的琼脂糖在10毫升无菌，无血清的M199媒体。 </li><li>成无菌的，无血清的M199媒体准备的胶原酶 -混合胶原酶的终浓度为80 U / ml的无菌过滤器，然后该混合物。 </lI&gt; <li>设置所有的设备的时间提前，然后再实施安乐死鼠标。 </li><li>设置PA琼脂糖和肺琼脂糖煮沸前右安乐死鼠标。 </li><li>安乐死鼠标在异氟醚麻醉的jar过量，要小心，不要让鼠标小狗接触到异氟醚。最佳小区的产率时，收获细胞，以避免微血栓形成在流通死后死后立即获得。 </li><li>麻醉罐子取出鼠标，固定鼠标的操作板，消毒的鼠标，仪器仪表，用酒精和手套。 </li><li>手术刀放入刀片切开鼠标，从颈部到下腹部。 </li><li>打开鼠标的左侧腹部，肾脏暴露，并切断肾动脉，让鼠标抽血，从而避免在肺部的小血管血栓。 </li><li>用剪刀剪下沿着鼠标的左侧胸骨，打开胸腔。 </li><li>小心地用手术刀切割膜片两侧完全打开胸腔。使用镊子或针保持打开胸腔。 </li><li>坚持RV G½英寸与27针朝向PA灌注的RV / PA /用无菌PBS肺部直到肺部出现白色的（所有血液冲出肺循环）。根据鼠标的大小，这将需要约3-5毫升的PBS。重要的是先冲洗，以防止在小肺血管的凝块的形成。 </li><li>小心使用钝性分离暴露气管和线缝合下方。 </li><li>气管下方支撑将最小的镊子和进气管将地下24 angiocatheter的。将angiocatheter，如果放置的IV。 </li><li>固定拧在步骤1.16中与缝合入气管的angiocatheter。 </li><li>以水煮出来的开水，PA琼脂糖和反转拌匀。冷却阿加罗的本身的体温约在冰上旋流。琼脂糖需要留在溶液中，但不会这么热，它会损害细胞的PA。 </li><li>坚持RV一个27 G½英寸的针，的灌注的RV / PA / PA琼脂糖肺部直到肺部出现灰色。琼脂糖混合物的铁粒子将使肺部出现灰色的颜色。这将需要约3-5毫升PA的琼脂糖根据鼠标的大小。同样重要的是去慢慢注射速度过快，可能会导致泄漏到气道的铁。 </li><li>肺琼脂糖的开水和反转拌匀。如果是，冷却琼脂糖约体温通过在冰上旋流。琼脂糖需要留在溶液中的，但不这么热，会损坏细胞的肺。 </li><li>肺琼脂糖和连接注射器在气管的地下24 angiocatheter的。通过气管注入琼脂糖充分膨胀肺部。肺将EASIEr来剁碎（见后面的步骤）如果肺部得到适当充气。 </li><li>取出心脏肺集团，浸入到25毫升冰冷的PBS中，以固化琼脂糖（〜5分钟）。利用这段时间来清理手术区。 </li><li>对于新生小鼠（P7-P21），最佳PASMC产量来自2-3幼仔组合成一个批次的细胞。的PASMC偏好是最佳的细胞的健康和生长与其他细胞接触。如果隔离每一个鼠标，那么细胞是非常稀疏，不能很好地生长。当动物，收获和寒冷心脏肺集团的结合在一起，然后将切碎，一旦所有心脏肺集团已从动物。 </li><li>从这点起，移动到上述细胞培养无菌罩。 </li><li>将冷冻肺部进入10厘米的组织培养皿的盖子，小心地取出心脏，胸腺，气管，任何从肺部结缔组织。 </li><li>肺部移动到10厘米的组织培养皿的底部并添加约1毫升冰冷的无菌PBS。 </li><li>剁碎成非常小的碎片（1-2毫米）2手术刀肺部。 </li><li>打破通风报信灭菌的5毫升血清吸管。移液器剁碎肺浆到无菌的50毫升锥形管。使用额外的无菌冰冷的PBS洗板，以确保所有的肺组织进入50毫升锥形管。 </li><li>到磁铁上，将50毫升的锥形管。等待含铁组织移动到磁铁旁边的侧管。根据含铁船只肺组织的比例，船舶可能仍然会漂浮在这一点上。吸PBS缓慢而仔细地试图不吸以来浮动肺组织目前还不清楚它是否包含血管。 </li><li>将猪肺洗净沉淀3次，用5毫升无菌PBS，再次尝试，以尽量减少吸气量的肺组织。 </li><li>重悬肺颗粒在3毫升胶原酶，倒入60毫米组织培养皿中。别尽量吸取试剂;，肺块将坚持移液器的内部。 </li><li>使用额外的3毫升冲洗管浇筑后，倒到60毫米组织培养皿。 </li><li>放入37℃组织培养孵化1小时。 </li><li>移液器组织+胶原酶浆向上和向下一个15 G钝针5毫升注射器，直到所有的大肺片被打乱。该组织+胶原酶浆将是多云的在这一点上，没有明显的组织块。 </li><li>倒入一个新的无菌的50毫升锥形管，并附着到磁体上。 </li><li>用约5ml，以确保所有的组织已收集完整的M199媒体组织培养皿洗净，然后倒入到锥形管中。 </li><li>吸胶原酶+媒体上清。这时候会非常少的浮子，和一个紧凑的含铁颗粒将上侧的磁铁旁边的锥形管。 </li><li>洗净，用5毫升完整的MED保险业监督3倍。这是必需的，以钝化胶原酶。 </li><li>加入3毫升的完整的​​媒体，和混合上下几次含铁颗粒悬浮。 </li><li>倒入到35毫米组织培养皿中悬浮的颗粒。利用显微镜，会有小血管周围的血管腔中包含的铁颗粒漂浮的片段。 </li><li>在组织培养孵化器中的组织培养皿中（标记板零）放置过夜。细胞迁移到板零约50％的成纤维细胞和50％的肺动脉平滑肌细胞，平滑肌标记染色。此板被称为镀零点和随后的浓缩步骤之后最终被丢弃。 </li></ol> 2。从新生小鼠肺动脉隔离 - 第二天<ol><li>倒入板零上清到一个干净的50毫升锥形管媒体。 </li><li>吸媒体上清。这时会有几乎没有飞蚊症，和一个紧凑的铁CONTA伊宁颗粒将磁铁旁边的锥形管的一侧上形成。 </li><li>洗净，用5毫升完整的媒体3倍。 </li><li>加入3毫升的完整的​​媒体，和混合上下几次含铁颗粒悬浮。 </li><li>倒入35毫米组织培养皿。利用显微镜，左右浮动血管腔中包含的铁颗粒的小血管片段能够被看见。这将是板1（ 图1A）。 </li><li>将组织培养孵化器的组织培养皿中。 </li></ol> 3。从新生小鼠肺动脉隔离 - 第六天<ol><li>倒入媒体上清板块之一到一个干净的50毫升锥形管。多次重复上述步骤，确保所有驻留在容器器壁的平滑肌细胞迁移到细胞培养板有机会。有一个收益递减，每一步， 即每块板将有越来越少，细胞附着。 </li><li&gt;再供给板一个完整的M199媒体和地方进入组织培养孵化器。 </li><li>吸媒体上清。这个时候就会几乎没有浮子，和一个紧凑的含铁颗粒将形成上侧的锥形管，磁铁旁边。 </li><li>洗净，用5毫升完整的媒体3倍。 </li><li>加入3毫升的完整的​​媒体，和混合上下几次含铁颗粒悬浮。 </li><li>倒入35毫米组织培养皿。利用显微镜，左右浮动血管腔中包含的铁颗粒的小血管片段能够被看见。这将是板两种。 </li><li>将组织培养孵化器的组织培养皿中。 </li></ol> 4。从新生小鼠肺动脉隔离 - 第九天<ol><li>媒体上清液倒入到一个干净的组织培养皿板两个50毫升锥形管。 </li><li>再供给板两个完整的M199媒体，并放回到组织文化孵化器。 </li><li>吸媒体上清。这个时候就会几乎没有浮子，和一个紧凑的含铁颗粒将形成上侧的锥形管，磁铁旁边。 </li><li>洗净，用5毫升完整的媒体3倍。 </li><li>加入3毫升的完整的​​媒体和混合上下几次含铁颗粒悬浮。 </li><li>倒入35毫米组织培养皿。利用显微镜，左右浮动血管腔中包含的铁颗粒的小血管片段能够被看见。这将是第三板。 </li><li>将组织培养孵化器的组织培养皿中。 </li></ol> 5。从新生小鼠肺动脉隔离 - 第十三天<ol><li>吸板1-3媒体。 </li><li>轻轻用温水，无菌PBS洗细胞。 </li><li>加入胰蛋白酶（0.25-0.5毫升）胰蛋白酶，胰蛋白酶消化细胞，每块板的薄膜。细胞粘附和将需要在组织中的胰蛋白酶丘尔重约10分钟的孵化器抬离板。 </li><li>收获细胞脱落板分为5毫升的完整的​​媒体在50毫升锥形管连接到磁铁拉出任何残留的铁粒子。 </li><li>清洗每个板具有一个额外的0.5毫升的完整的​​媒体，该媒体，并添加50毫升锥形管。 </li><li>这个时候，采取媒体上清（不要吸），并丢弃的铁粒子颗粒。 </li><li>盘细胞在5毫升完整的M199培养基在60毫米的菜有融合细胞在几天之内。另外，板材10毫升完全M199媒体在一个10厘米的板，和肺动脉平滑肌细胞中的细胞将采取另外约1-2周达到汇合。 </li></ol> 6。从新生小鼠肺动脉隔离 - 第十四天<ol><li>减小介质的新鲜的完全培养基媒体，以除去残留的胰蛋白酶。会有肺动脉平滑肌细胞的组织培养皿中（ 图1B）上的薄人口。 </li></ol> 7。日常护理<ol><li>更改媒体新鲜完整的M199媒体，每周约2倍。 </li><li>当这些细胞分裂，约10分钟，在孵化器，在一部电影中的胰蛋白酶（0.25-0.5毫升）的细胞需要抬离板。使用胰蛋白酶膜允许的PASMC replating的无纺纱细胞。如果使用太多的胰蛋白酶，肺动脉平滑肌细胞必须被离心旋出的胰蛋白酶，或在的细胞不会readhere板。有时，在纺丝后的细胞时，它是很难得到的肺动脉平滑肌细胞再悬浮成完整的M199媒体。 </li><li>在实际观测中，孤立的肺动脉平滑肌细胞像平滑肌细胞4-5代可靠，但，隔离细胞染色平滑肌细胞标记通过7。十三天数首次汇集了60毫米或10厘米板作为第2代（电镀后，第一次接触到胰蛋白酶）。 </li></ol>

<ol>
	<li>Levin, D. L., Rudolph, A. M., Heymann, M. A., Phibbs, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+development+of+the+pulmonary+vascular+bed+in+fetal+lambs.">Morphological development of the pulmonary vascular bed in fetal lambs.</a> Circulation. 53, 144-151 (1976).</li><li>Walsh-Sukys, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+pulmonary+hypertension+of+the+newborn+in+the+era+before+nitric+oxide:+practice+variation+and+outcomes.">Persistent pulmonary hypertension of the newborn in the era before nitric oxide: practice variation and outcomes.</a> Pediatrics. 105, 14-20 (2000).</li><li>Khemani, E., 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=Pulmonary+artery+hypertension+in+formerly+premature+infants+with+bronchopulmonary+dysplasia:+clinical+features+and+outcomes+in+the+surfactant+era.">Pulmonary artery hypertension in formerly premature infants with bronchopulmonary dysplasia: clinical features and outcomes in the surfactant era.</a> Pediatrics. 120, 1260-1269 (2007).</li><li>Jobe, A. H., Bancalari, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchopulmonary+dysplasia.">Bronchopulmonary dysplasia.</a> Am. J. Respir. Crit. Care Med. 163, 1723-1729 (2001).</li><li>Mourani, P. M., Sontag, M. K., Younoszai, A., Ivy, D. D., Abman, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+utility+of+echocardiography+for+the+diagnosis+and+management+of+pulmonary+vascular+disease+in+young+children+with+chronic+lung+disease.">Clinical utility of echocardiography for the diagnosis and management of pulmonary vascular disease in young children with chronic lung disease.</a> Pediatrics. 121, 317-325 (2008).</li><li>Check, 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=Fetal+growth+restriction+and+pulmonary+hypertension+in+premature+infants+with+bronchopulmonary+dysplasia.">Fetal growth restriction and pulmonary hypertension in premature infants with bronchopulmonary dysplasia.</a> J. Perinatol. , (2013).</li><li>Farrow, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperoxia+increases+phosphodiesterase+5+expression+and+activity+in+ovine+fetal+pulmonary+artery+smooth+muscle+cells.">Hyperoxia increases phosphodiesterase 5 expression and activity in ovine fetal pulmonary artery smooth muscle cells.</a> Circ. Res. 102, 226-233 (2008).</li><li>Aschner, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+nitric+oxide+synthase+gene+transfer+enhances+dilation+of+newborn+piglet+pulmonary+arteries.">Endothelial nitric oxide synthase gene transfer enhances dilation of newborn piglet pulmonary arteries.</a> Am. J. Physiol. 277, 371-379 (1999).</li><li>Cornfield, D. N., Stevens, T., McMurtry, I. F., Abman, S. H., Rodman, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+hypoxia+increases+cytosolic+calcium+in+fetal+pulmonary+artery+smooth+muscle+cells.">Acute hypoxia increases cytosolic calcium in fetal pulmonary artery smooth muscle cells.</a> Am. J. Physiol. 265, 53-56 (1993).</li><li>Bailly, K., Ridley, A. J., Hall, S. M., Haworth, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoA+activation+by+hypoxia+in+pulmonary+arterial+smooth+muscle+cells+is+age+and+site+specific.">RhoA activation by hypoxia in pulmonary arterial smooth muscle cells is age and site specific.</a> Circ. Res. 94, 1383-1391 (2004).</li><li>Black, S. M., DeVol, J. M., Wedgwood, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+fibroblast+growth+factor-2+expression+in+pulmonary+arterial+smooth+muscle+cells+involves+increased+reactive+oxygen+species+generation.">Regulation of fibroblast growth factor-2 expression in pulmonary arterial smooth muscle cells involves increased reactive oxygen species generation.</a> Am. J. Physiol. Cell Physiol. 294, 345-354 (2008).</li><li>Cogolludo, A., Moreno, L., Lodi, F., Tamargo, J., Perez-Vizcaino, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+maturational+shift+from+PKCzeta+and+voltage-gated+K++channels+to+RhoA/Rho+kinase+in+pulmonary+vasoconstriction.">Postnatal maturational shift from PKCzeta and voltage-gated K+ channels to RhoA/Rho kinase in pulmonary vasoconstriction.</a> Cardiovasc. Res. 66, 84-93 (2005).</li><li>Wedgwood, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+regulates+extracellular+superoxide+dismutase+activity+and+expression+in+neonatal+pulmonary+hypertension.">Hydrogen peroxide regulates extracellular superoxide dismutase activity and expression in neonatal pulmonary hypertension.</a> Antiox. Signal. 15, 1497-1506 (1089).</li><li>Farrow, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+oxidant+stress+increases+PDE5+activity+in+persistent+pulmonary+hypertension+of+the+newborn.">Mitochondrial oxidant stress increases PDE5 activity in persistent pulmonary hypertension of the newborn.</a> Respir. Physiol. Neurobiol. 174, 272-281 (2010).</li><li>Chester, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cinaciguat,+a+soluble+guanylate+cyclase+activator,+augments+cGMP+after+oxidative+stress+and+causes+pulmonary+vasodilation+in+neonatal+pulmonary+hypertension.">Cinaciguat, a soluble guanylate cyclase activator, augments cGMP after oxidative stress and causes pulmonary vasodilation in neonatal pulmonary hypertension.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 301, 755-764 (2011).</li><li>Konduri, G. G., Bakhutashvili, I., Eis, A., Gauthier, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+voltage+gated+potassium+channel+responses+in+a+fetal+lamb+model+of+persistent+pulmonary+hypertension+of+the+newborn.">Impaired voltage gated potassium channel responses in a fetal lamb model of persistent pulmonary hypertension of the newborn.</a> Pediatr. Res. 66, 289-294 (2009).</li><li>Olschewski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+K(Ca)+channel+to+membrane+potential+and+O2+sensitivity+is+decreased+in+an+ovine+PPHN+model.">Contribution of the K(Ca) channel to membrane potential and O2 sensitivity is decreased in an ovine PPHN model.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 283, L1103-L1109 (2002).</li><li>Aslam, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+attenuate+lung+injury+in+a+murine+model+of+neonatal+chronic+lung+disease.">Bone marrow stromal cells attenuate lung injury in a murine model of neonatal chronic lung disease.</a> Am. J. Respir. Crit. Care Med. 180, 1122-1130 (2009).</li><li>Balasubramaniam, V., 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=Bone+marrow-derived+angiogenic+cells+restore+lung+alveolar+and+vascular+structure+after+neonatal+hyperoxia+in+infant+mice.">Bone marrow-derived angiogenic cells restore lung alveolar and vascular structure after neonatal hyperoxia in infant mice.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 298, L315-L323 (2010).</li><li>de Visser, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphodiesterase-4+inhibition+attenuates+pulmonary+inflammation+in+neonatal+lung+injury.">Phosphodiesterase-4 inhibition attenuates pulmonary inflammation in neonatal lung injury.</a> Eur. Respir. J. 31, 633-644 (2008).</li><li>de Visser, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sildenafil+attenuates+pulmonary+inflammation+and+fibrin+deposition,+mortality+and+right+ventricular+hypertrophy+in+neonatal+hyperoxic+lung+injury.">Sildenafil attenuates pulmonary inflammation and fibrin deposition, mortality and right ventricular hypertrophy in neonatal hyperoxic lung injury.</a> Respir. 10, 30 (2009).</li><li>Ladha, F., 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=Sildenafil+improves+alveolar+growth+and+pulmonary+hypertension+in+hyperoxia-induced+lung+injury.">Sildenafil improves alveolar growth and pulmonary hypertension in hyperoxia-induced lung injury.</a> Am. Respir. Crit. Care Med. 172, 750-756 (2005).</li><li>Farrow, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+hyperoxia+increases+mitochondrial+oxidation+and+increases+phosphodiesterase+5+activity+in+fetal+pulmonary+artery+smooth+muscle+cells.">Brief hyperoxia increases mitochondrial oxidation and increases phosphodiesterase 5 activity in fetal pulmonary artery smooth muscle cells.</a> Antioxid. Redox Signal. 17, 460-470 (2012).</li><li>Waypa, G. 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=Hypoxia+triggers+subcellular+compartmental+redox+signaling+in+vascular+smooth+muscle+cells.">Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells.</a> Circ. Res. 106, 526-535 (2010).</li><li>Waypa, G. 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=Superoxide+Generated+at+Mitochondrial+Complex+III+Triggers+Acute+Responses+to+Hypoxia+in+the+Pulmonary+Circulation.">Superoxide Generated at Mitochondrial Complex III Triggers Acute Responses to Hypoxia in the Pulmonary Circulation.</a> Am. J. Respir. Crit. Care Med. 187, 424-432 (2013).</li><li>Marshall, C., Mamary, A. J., Verhoeven, A. J., Marshall, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+artery+NADPH-oxidase+is+activated+in+hypoxic+pulmonary+vasoconstriction.">Pulmonary artery NADPH-oxidase is activated in hypoxic pulmonary vasoconstriction.</a> Am. J. Respir. Cell Mol. Biol. 15, 633-644 (1996).</li><li>Farrow, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+Dismutase+and+Inhaled+Nitric+Oxide+Normalize+Phosphodiesterase+5+Expression+and+Activity+in+Neonatal+Lambs+with+Persistent+Pulmonary+Hypertension.">Superoxide Dismutase and Inhaled Nitric Oxide Normalize Phosphodiesterase 5 Expression and Activity in Neonatal Lambs with Persistent Pulmonary Hypertension.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 299, L109-L116 (2010).</li></ol>

作者宣称，他们有没有竞争经济利益。

在这个手稿中，我们描述了在第一次的PASMC在P7，P14，和P21小鼠的隔离。为了做到这一点，通过琼脂糖凝胶和0.2μM铁颗粒的浆料注入到PA的RV。由于小尺寸的铁粒子，它们无法通过肺毛细血管床，因此沉积在小PA。肺部膨胀，除去游离。含铁的容器内被拉出使用磁铁的溶液。最终，该船舶被镀成的组织培养皿中，细胞迁移的船只，到培养板。的初始板的细胞是成纤维细胞和肺动脉平滑肌细胞的混合物...

肺动脉高压是正常期间宫内寿命时间作为气体交换的主要器官，胎盘在子宫内的心输出量只有10％被循环通过肺血管床。类似全身的压力，由于肺血管阻力升高，肺动脉压力。随着妊娠的进展，有快速增长的小PA在肺部，准备对胎儿的肺血流量的急剧增加，发生在出生1。当正常的围产期过渡失败，在近期和足月，其结果是持续性肺动脉高压（PPHN）的新生儿。 PPHN是由许多不同的相关病症引起的一组临床综合征。然而，所有这些婴儿有着共同的病理生理功能，如肺血管阻力升高，低​​氧血症，由右至左分流血流跨持续胎儿连接，如动脉导管未闭或阿门未闭。 PPHN每千名活产和影响2-6，传达的死亡率，以及显着的短期和长期患病2 8-10％的风险。此外，极低出生体重早产儿可能会由于其潜在的肺部疾病发展肺动脉高压。最常见的潜在肺疾病的早产儿支气管肺发育不良（BPD）。虽然BPD的整体风险与胎龄和出生体重，目前还不清楚为什么一个子集，这些婴儿的发展显着的肺动脉高压，以及如何适当地对待这些婴儿。差的结果，包括住院时间延长，死亡率增加，是常见的3-6。 从历史上看，从健康动物绵羊胎儿肺动脉平滑肌细胞或猪胎儿肺动脉平滑肌细胞已被用来研究信号通路参与在正常出生后肺血管过渡。这些通常是孤立的第五代性PAÑ ​​绵羊或猪胎儿的交付实施安乐死之前，任何自发呼吸7-9。此外，一些研究者已经分离并利用PASMC从年龄稍大的自主呼吸的羔羊和仔猪在3天，2周，4周10-12。最近，一些团体已经成功地分离和利用PASMC孤立羔羊PPHN检查紊乱信号通路在疾病状态13-17。这些细胞已被证明是一个有价值的工具来检查信号通路是至关重要的正常和病变的近期和长期的肺血管。然而，他们不给洞察信号通路的影响，早产儿肺动脉高压。他们也不可以看出，在小鼠疾病模型的遗传操作的可能性。 长期以来，大鼠和小鼠模型已被用于模型BPD以及最近正被用于模型肺HYpertension造成BPD 18-22。新生大鼠是诱人的工作，由于其较大的规模，但他们也遭受缺乏基因改造的潜力。转基因动物已被广泛用于研究特定基因的目标，整个动物生理学新生小鼠的影响，但至今没有任何人曾成功分离PASMC从这些小老鼠。通过隔离PASMC，可以得到更多的信息，关于如何在应对环境刺激和/或遗传修饰，特别是在肺动脉平滑肌途径改变。此外，现场PASMC可以成像实时的，，检查快速变化的关键信号分子，如钙和活性氧23-25。最近，我们描述了使用的变化的技术，Marshall 等从成年小鼠肺动脉平滑肌细胞成功地分离出26用于隔离大鼠肺动脉平滑肌细胞23,25,26。我们现在已经适应了ND扩展这种技术小老鼠7-21日龄（P7，P14，P21）。到这个新的PASMC隔离技术的主要限制是，它要求多鼠标，以产生足够的用于实验的细胞，细胞的生长非常缓慢，这是主平滑肌细胞的特征。尽管有这些限制，我们相信这种技术来隔离新生小鼠PASMC将允许增强调查的关键信号通路参与了肺动脉高压的发展，并表示在这一领域的一个显着的进步。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Bard Parker surgical blade handle</td>
			<td>BD</td>
			<td>371030</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel surgical blades #10 (sterile)</td>
			<td>Miltex</td>
			<td>4-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (3 ml and 5 ml, sterile)</td>
			<td>BD</td>
			<td>309657 and 306646</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles (27 G, sterile)</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>Angiocatheter (24 G, sterile)</td>
			<td>BD</td>
			<td>381412</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoject blunt cannula (15 G)</td>
			<td>Kendall</td>
			<td>SWD202314Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures</td>
			<td>Fisher Scientific</td>
			<td>NC9782896</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynal magnet particle collector</td>
			<td>Invitrogen</td>
			<td>120-01D</td>
			<td>This is a critical tool for the protocol.</td>
		</tr>
		<tr>
			<td>Tissue culture plates (35 mm, 60 mm, and 10 cm, sterile)</td>
			<td>BD</td>
			<td>353001, 353004, and 353003</td>
			<td>Any brand of tissue culture plates will be fine.</td>
		</tr>
		<tr>
			<td>Iris Scissors (4 &#189; inch stainless steel)</td>
			<td>American Diagnostic Corporation</td>
			<td>3424</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (4 inch stainless steel)</td>
			<td>Quick Medical</td>
			<td>L5-5004</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Mediatech</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>M199 media</td>
			<td>Mediatech</td>
			<td>10-060-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>VWR</td>
			<td>TX16777-164NWU</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone/Thermo Scientific</td>
			<td>SH3091003</td>
			<td>Heat inactivate at 55 &#176;C for 45 min. For consistency in results, lot match all serum and obtain from same vendor.</td>
		</tr>
		<tr>
			<td>Iron particles (iron (II, III) oxide powder)</td>
			<td>Aldrich Chemical Company</td>
			<td>#31,006-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase (made to 80 U/ml)</td>
			<td>Sigma</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Butlet Schein</td>
			<td>NDC 11695-6776-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse TE2000-U with a Cascade Photometrics 12-bit camera</td>
			<td>Nikon</td>
			<td>TE2000-U</td>
			<td>Any good light microscope will be fine to observe PASMC in culture.</td>
		</tr>
		<tr>
			<td>Anti-desmin antibody</td>
			<td>Sigma</td>
			<td>D-8281</td>
			<td>Use at 1:200 dilution for immunostaining.</td>
		</tr>
		<tr>
			<td>Anti-smooth muscle myosin</td>
			<td>Biomedical Technologies</td>
			<td>BT-562</td>
			<td>Use at 1:2,000 dilution for immunostaining.</td>
		</tr>
		<tr>
			<td>Rhodamine-red anti-rabbit secondary</td>
			<td>Molecular Probes/Invitrogen</td>
			<td>R-6394</td>
			<td>Use at 1:200 dilution for immunostaining.</td>
		</tr>
		<tr>
			<td>Nikon Eclipse TE-300 fluorescent microscope and Cool Snap digital camera</td>
			<td>Nikon</td>
			<td>TE300</td>
			<td>Any good epifluorescence microscope will be fine for immunostaining.</td>
		</tr>
		<tr>
			<td>Cyclic nucleotide phosphodiesterase assay kit</td>
			<td>Enzo Life Sciences</td>
			<td>BML-AK800-0001</td>
			<td>This is the only colorimetric PDE enzyme activity assay available.</td>
		</tr>
		<tr>
			<td>Sildenafil</td>
			<td>Sigma</td>
			<td>PZ-0003</td>
			<td>A PDE5-selective inhibitor is required for the PDE enzyme activity to determine specificity of cGMP hydrolysis.</td>
		</tr>
	</tbody>
</table>

isolation of pulmonary artery smooth muscle cells from neonatal mice

肺动脉高压是在婴幼儿的发病率和死亡率的一个重要原因。在历史上，一直存在重大研究信号通路参与血管平滑肌收缩的肺动脉平滑肌细胞从胎羊。虽然羊长期肺动脉高压的一个很好的模型，他们都非常昂贵，缺乏发现在小鼠遗传操作的优势。相反，该​​系统是一个重大的限制无法从小鼠分离的PASMC。在这里，我们描述了的的小鼠肺动脉平滑肌细胞的原代培养的隔离P7，P14，和P21小鼠使用先前所描述的技术，Marshall 等的变形例26中 ，曾用于隔离大鼠PASMC。这些鼠肺动脉平滑肌细胞代表了一种新的工具在新生儿期的信号转导途径的研究。简单地说，淤浆的0.5％（重量/体积）的琼脂糖+通过右心室（RV）的M199培养基中的0.5％的铁粒子注入肺血管床。该铁颗粒是直径为0.2μM，并不能穿过肺毛细血管床。因此，铁肺小动脉（PA）递交。琼脂糖，取出游离肺膨胀。含铁船舶用磁铁被拉下来。胶原酶（80单位/毫升）处理，并进一步解离后，将容器放入一个组织培养皿中的M199培养基含有20％胎牛血清（FBS）和抗生素（M199完全培养基）中，以允许到培养皿中的细胞迁移。初始板的细胞，成纤维细胞和肺动脉平滑肌细胞是50-50混合物。因此，下拉过程重复多次，以实现更纯净的的PASMC人口和消除任何残余的铁。平滑肌细胞的身份被确认通过免疫组化平滑肌肌球蛋白和结。

隔离期间和之后，肺动脉平滑肌细胞的光镜和平滑肌细胞标志物免疫组化检查。通过光镜，早在该协议中，PASMC看到小铁容器（ 图1A）迁移到组织培养皿。池板后一通三13天，然后不再被看作是那些已经在最后的池步拉出铁颗粒。相反，人口肺动脉平滑肌细胞在组织培养皿（ 图1B）。 基于免疫染色，最初的细胞迁移含铁船舶约50％的成纤维细胞和50％?...

Watch this Scientific Journal Video about Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice at JoVE.com

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

我们已经开发了一种新的和可重复的技术来隔离为P7作为年轻的小鼠原代培养的肺动脉平滑肌细胞（PASMC），从而使更好地参与新生儿平滑​​肌细胞的收缩和舒张的信号转导通路的研究。

肺动脉平滑肌细胞从新生小鼠隔离

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

isolation-of-pulmonary-artery-smooth-muscle-cells-from-neonatal-mice

Research

JoVE Journal

Biology

18.2K 次观看.  Northwestern University Feinberg School of Medicine. 我们已经开发了一种新的和可重复的技术来隔离为P7作为年轻的小鼠原代培养的肺动脉平滑肌细胞（PASMC），从而使更好地参与新生儿平滑​​肌细胞的收缩和舒张的信号转导通路的研究。

视频: Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

焦距钙 2 +</sup瞬态检测平滑肌

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

科研

生物学

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

人脐动脉平滑肌细胞的分离（HUASMC）

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

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.

从新生小鼠的肺血管内皮细胞的分离和培养

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

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

骨骼肌的线粒体分离

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

分离，培养和移植肌卫星细胞

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.

新生儿肝外胆管的隔离

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

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.

鼠阀门内皮细胞的分离

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

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.

鼠冠状动脉血管平滑肌细胞的分离

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

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog 3H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2&#8217; deoxyuridine (BrdU) and 5-ethynyl-2&#8242;-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites.

Proliferation is a critical part of cellular function, and a common readout used to assess potential toxicity of new drugs. Measuring proliferation is, therefore, a frequently used assay in cell biology. Here we present a simple, versatile method of measuring proliferation that can be used in adherent and non-adherent cells.

使用点击化学测量血管平滑肌细胞增殖

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease ...