感谢L. Rinaldi，S.Schäffer，D. Reitz，H. Thomas和A. Weber的技术支持。作者还希望感谢Dr. E. Martinson博士对手稿进行广泛的阅读和语言编辑。该研究由Giessen Anschubsfinanzierung大学授予M. Aslam和D.Gündüz授予的支持。

调查符合美国国家卫生研究院出版的"实验动物护理和使用指南"（NIH出版号85-23 1985），并得到了吉森大学当地伦理委员会的批准。在本研究中使用体重200-250g的成年雄性Wistar大鼠。 高压灭菌<ol><li>在进行手术至少一天之前，将所有手术器械，移液管吸头和巴斯德吸管进行高压灭菌，以在121℃的隔离程序中使用30分钟。 </li></ol> 2.媒体和解决方案的准备注意:步骤2.1-2.4中的解决方案可以在隔离前一周准备并储存在4°C，但在隔离当天的步骤2.5-2.8中制备溶液。缓冲液和介质的完整配方见表1 。将所有培养基和溶液加热至37°C水浴开始准备之前。 <ol><li>通过将表1中列出的化学物质在室温（RT）下溶解在4.5L H 2 O中来制备鲍威尔培养基。用2.0N NaOH将pH调节至7.4，使体积达到5.0L。无菌过滤溶液，并将其储存在4℃。 </li><li>通过将M199粉末培养基溶解在4.5升H 2 O中来制备肌酸，L-肉毒碱和牛磺酸（CCT）培养基。将根据表格的HEPES粉末加入培养基中并混合15分钟。根据表1添加肌酸，肉碱，牛磺酸和Ara-C。混合均匀，用2.0 N NaOH将pH值调节至7.4，使体积达到5.0 L.无菌过滤器并在4℃下储存。 </li><li>通过向培养基M199中加入10％胎牛血清（FCS）和2％青霉素/链霉素（Pen / Strep），为成纤维细胞制备细胞培养基。 DMEM同样适用于M199，也可用于成纤维细胞培养。 </li><li>准备MV2细胞培养e培养基用于内皮细胞，根据制造商的说明书将补充混合物的所有内容物添加到基础MV2培养基中。取5 mL等分试样，并在水浴中温热至37°C。 </li><li>通过将0.05g牛血清白蛋白（BSA）溶解在50mL Hanks平衡盐溶液（HBSS）中并向其中加入0.5mL的200mM乙二胺四乙酸（EDTA）溶液来制备内皮细胞分离缓冲液。将其无菌过滤并将其储存在4°C。 </li><li>通过加入0.1mL 200mM至9.9mL PBS并将其储存在4℃来制备内皮细胞洗涤缓冲液。 </li><li>通过将27毫克胶原酶溶解在5毫升的鲍威尔培养基中并加入12.5μL的100mM CaCl 2溶液来制备胶原酶溶液。在水浴中将其温热至37°C。 </li><li>通过加入12.5μL，25μL和120μL的100mM CaCl 2溶液来制备增加的Ca 2+修复溶液1,2和3用于心肌细胞分别离子至6mL，6mL和12mL的Powell培养基。 </li><li>通过在每个35-mm细胞培养皿中加入1mL含有层粘连蛋白的预包衣培养基，在分离前一天预先涂覆心肌细胞的细胞培养皿。将它们在细胞培养箱中37℃孵育过夜。 </li></ol> 3.磁珠的制备<ol><li>将0.5 mL内皮细胞分离缓冲液置于1.5 mL样品管中。 </li><li>加入5μL（2×10 6 ）的平板小鼠IgG磁珠并充分混合。 </li><li>将管放在磁性架上1分钟。珠将粘附到管的壁朝向磁体。用1 mL移液管小心清除上清液。 </li><li>重复洗涤两次，最后重新悬浮在50μL内皮细胞分离缓冲液中，并保存在4°C直到使用。 </li></ol> Langendorff灌注系统的准备<ol><li>清洁t他Langendorff系统灌注2升蒸馏水。 </li><li>通过系统循环50 mL鲍威尔培养基5 min，弃去培养基。 </li><li>加入80 mL新鲜鲍威尔培养基。通过从气瓶中用玻璃巴斯德吸管鼓泡，持续地将其与"碳霉素"一起灌注。让介质循环通过系统。该系统现在可以灌输心脏（ 图1A ）。 </li></ol> 5.解剖大鼠<ol><li>将大鼠放在通风柜中的5L干燥器中，并通过排气口加入1.5mL的4-5％异氟烷，并关闭盖子。 注意:将动物放在干燥器内的高架多孔板上，以避免动物与液体异氟烷直接接触。 </li><li>等到大鼠失去盖子反射（通常〜5分钟），然后将大鼠从干燥器中取出。通过颈椎脱位安乐死老鼠。 </li><li>切开腹部皮肤和下面的肌肉水平线在中间使用一把锋利的剪刀。此后，将肌肉垂直切割至大鼠胸骨，打开胸部。与肺一起取出心脏，立即将器官置于冰冷的0.9％NaCl溶液中。 </li><li>在一把剪刀的帮助下取出肺，然后将其丢弃。清洁任何结缔组织的心脏，并在头臂主干的分支尾部切开主动脉。 </li></ol>心脏灌注和胶原酶消化<ol><li> 通过 Langendorff灌注系统的主动脉（第4节），使用两对薄的弯曲镊子安装心脏。为了确保以逆行方式灌注心肌，将灌注系统的插管的末端置于第一主动脉分支和主动脉瓣之间。用鳄鱼夹固定主动脉。 </li><li>用手术缝合线固定心脏，取出鳄鱼夹（ 图1B ）。 </li><li>打开套管的阀门，使灌注液（35 mL）逐滴通过心脏（60滴/ min; 5 mL / min），以清洗任何残留的血液。在这个阶段放弃任何流通。 注意:避免在灌注的任何阶段形成气泡，因为这将导致不完全消化和细胞分离过程失败。 </li><li>将悬挂的心脏放置在收集漏斗（心室; 图1B ）中，并启动蠕动泵，这将使灌注介质从收集漏斗再循环到储存器（ 图1A ）。 </li><li>将胶原酶溶液（从步骤2.8）加入到灌注培养基中。用循环胶原酶溶液灌注心脏30分钟。 注意:此步骤对于优化细胞产量非常关键。灌注时间取决于胶原酶的活性，这可能因批次而异。测试3-4批胶原酶以找到具有opti的胶原酶活动不良那么可以购买大量最适合的批次，这些批次足够至少一年可重复的结果。 </li><li>在消化时间结束时，停止灌注，使用一把剪刀从Langendorff系统中取出心脏，并将其放在玻璃培养皿中。 </li><li>切除并丢弃两个心房，并清除任何残留的脂肪组织。借助两对镊子仔细剥离心包膜，以避免上皮细胞的污染。 注意:如果需要，可以用胶原酶进一步消化心包，以获得纯心脏上皮细胞。在本研究中没有给出详细的方案，因为这超出了本议定书的范围。 </li><li>用一把剪刀将心脏放在中间，放入组织切碎机中。使心脏2-3次，并从Langendorff灌注中将切碎的组织重新悬浮在12 mL消化缓冲液中。 注意:不要砍h过量过量，因为这会导致死亡心肌细胞比例较高。 </li><li>将切碎的组织转移到15mL含胶原酶的解离介质中，并使其在水浴中消化5-10分钟，偶尔混合通过使用5mL一次性塑料移液管重新悬浮。 </li><li>在消化期结束时，通过100μm筛子过滤细胞悬浮液。丢弃组织碎屑和未消化的物质。 </li><li>在Langendorff灌注结束时，立即用至少1升蒸馏水冲洗灌注系统。通过将100mL的0.1N NaOH再循环30分钟来对系统进行清洗，最后用2-3L蒸馏水冲洗该系统。通过冲洗空气干燥系统，并用对分膜覆盖开口。 </li></ol> 7.大鼠心脏细胞分离<ol><li> 心肌细胞的分离培养 <ol><li>将细胞悬浮液以25 xg离心1分钟（无Brakes）以沉淀心肌细胞。也可以将心肌细胞在室温下静置10分钟而不进行离心。 </li><li>借助于一次性塑料移液管，在新的50mL管中，小心地去除含有内皮细胞和成纤维细胞的上清液。 </li><li>在Ca 2+ 6mL溶液1中重悬心肌细胞沉淀（步骤2.9）。允许细胞1分钟调节至低Ca 2+浓度环境，并以25 xg再次离心细胞悬浮液1分钟（无制动）。 </li><li>弃去上清液并将细胞沉淀重悬于6mL Ca 2+溶液2中（步骤2.9）。允许细胞1分钟调节至中等浓度的Ca 2+ ，最后加入12 mL的Ca 2+溶液3（步骤2.9）给细胞。在一次性塑料移液器的帮助下混合。 </li><li>以25 xg离心细胞悬浮液1分钟。 </li><li>弃去上清液，并将细胞重悬于20 mL预处理液中受热CCT培养基。将1mL细胞悬浮液加入到用层粘连蛋白预包被的20个无菌35mm细胞培养皿中（步骤2.9）中，并使细胞在无CO 2的细胞培养培养箱中附着2小时。 注意:心肌细胞也可以在二氧化碳环境下培养;然而，在这种情况下，应使用碳酸氢盐缓冲液系统的介质。 </li><li> 2小时后，用新的2 mL CCT培养基更换培养基，以去除任何死细胞。 注意:细胞可以进行任何计划的实验，并且可以在典型的细胞培养箱（不含CO 2 ）中培养长达3天，而不会明显损失活细胞。典型的健康的棒状心肌细胞显示在图2A中 。 </li></ol></li><li> 内皮细胞和成纤维细胞的分离 <ol><li>将含有内皮细胞和成纤维细胞的上清液（步骤7.1.2）以250×g离心10分钟。 </li><li>丢弃supe在2 mL样品管中将细胞沉淀重悬于1.5 mL内皮细胞分离缓冲液中。 </li><li>向细胞悬浮液中加入3μg（1:500）小鼠抗大鼠CD31抗体，并在4℃温育30分钟，并进行端对端旋转。 注意:建议用最佳稀释滴定抗体。该步骤应在4℃下进行，在室温下，抗体经内皮细胞的快速内化将导致低产率。 </li><li>将先前制备的（第3部分）泛抗小鼠IgG珠添加到细胞悬浮液中，并在4℃下以端对端旋转孵育20分钟。 注意:或者，可以将小鼠抗大鼠CD31抗体预连接到磁珠上，然后加入细胞悬液。然而，这将增加珠与细胞悬浮液的孵育时间，并且可能导致非内皮细胞的非特异性结合增加，因此增加其他细胞类型的污染。 </li><li>一个在抗体孵育时间结束时，将含有细胞悬浮液的管放置在磁性架中，并允许2分钟将附着于内皮细胞的磁珠沉积到管侧。 </li><li>用1 mL移液管小心地取出剩余的细胞悬浮液（主要含有成纤维细胞），并加入到含有10mL成纤维细胞培养液的10cm细胞培养皿中（步骤2.3）。将其置于含有5％CO 2的典型细胞培养箱中。步骤7.3继续。 </li><li>使用含有内皮细胞的磁珠，向管中加入1 mL洗涤缓冲液。 </li><li>盖住管子，将其从磁性架上取下，并通过轻轻摇动4〜5次，仔细重新悬挂珠子。将管再次放置在磁性架上，使珠子沉淀在管壁上1分钟，并小心地取出洗涤缓冲液。 </li><li>重复洗涤步骤5 - 7次以摆脱任何污染的n内皮细胞。 </li><li>将附着在珠上的内皮细胞重悬于1mL MV2内皮细胞培养基中，加入12孔板的单孔中，并在CO 2细胞培养箱中孵育。 </li><li>允许细胞附着过夜，并在第二天，然后每2-3天更换培养基，直到细胞变得半融合（通常为5-7天）。 注意:分离7-10天之后3-4天和融合内皮细胞的典型非汇合内皮细胞分别显示在图3A和3B中。然后将细胞进行胰蛋白酶消化（第7.4节）并接种到T25细胞培养瓶中。 </li></ol></li><li> 成纤维细胞的洗涤和培养（从7.2.6步继续） <ol><li>培养1小时后，通过加入适量体积的预热的PBS将培养基吸出并洗涤附着于细胞培养皿的成纤维细胞。吸出PBS并重复step 2-3次，最后加入预热新鲜成纤维细胞培养液。 </li><li>第二天用预先加热的PBS（如上一步）洗涤附着的细胞，并加入预热的新鲜培养基。每2-3天更换一次。在分离的第2天，成纤维细胞的典型表型如图4A所示 。 注意:成纤维细胞通常在5-7天内半融合，然后准备进行胰蛋白酶消化（第7.4节），并用于计划实验。通过这个程序，我们通常获得95-99％的纯成纤维细胞。分离第7天的成纤维细胞的典型表型如图4B所示 。 </li></ol></li><li> 内皮细胞和成纤维细胞的胰蛋白酶消化 <ol><li>从细胞培养皿吸出培养基，并加入足够的预热PBS以覆盖细胞。 </li><li>吸出PBS并加入足够的胰蛋白酶-EDTA溶液（分别为0.05％和0.02％）以覆盖细胞并置于37℃孵育5分钟。 </li><li>通过用1毫升移液管的帮助，将细胞上下移液重悬细胞，并通过加入10％FCS停止胰蛋白酶作用。 </li><li>通过在室温下250xg离心10分钟来沉淀细胞悬液。 </li><li>将细胞重悬于适量体积的预热的细胞培养基中。 </li></ol></li></ol> 8.内皮细胞表征注意:血管内皮细胞的纯度通过Vil Willebrand因子（vWF）的Dil-Ac-LDL摄取和免疫染色测定。通过荧光或共焦显微镜观察细胞（ 图3C-3E ）。 <ol><li> Dil-Ac-LDL摄取内皮细胞 <ol><li>对内皮细胞进行胰蛋白酶消化（步骤7.4），并在12孔板（约10×10 4个细胞/ cm 2 ）的玻璃盖玻片上培养它们过夜。 </li><li>吸出培养基，并用1 mL的pre-w冲洗细胞武装HBSS。 </li><li>吸入HBSS并加入含有10μg/ mL Dil-Ac-LDL的1mL内皮细胞培养基，并将细胞置于37℃培养箱中4小时。 </li><li>吸出培养基并用预热的PBS冲洗细胞三次，并用1mL的4％多聚甲醛（PFA;溶解在PBS中）固定。用PFA在37℃下孵育20分钟。 注意:PFA具有腐蚀性;在处理PFA溶液时，始终使用耐酸手套。 </li><li>吸出PFA，用1 mL PBS将12孔板上的盖玻片上的细胞冲洗3次，并在室温下用核染色溶液TO-PRO（10μM）孵育1小时。 注意:如果适用于显微镜的过滤器，也可以使用4&#39;，6-二脒基-2-苯基吲哚（DAPI;1μg/ mL）。在这种情况下孵育时间应减少到10分钟。 </li><li>用1 mL PBS在12孔板上的盖玻片上冲洗细胞，抽出PBS，重复3次。 </li><li>拿c超过12孔板，并将其安装在带有一滴安装介质的玻璃滑块上。细胞准备在荧光/共焦显微镜下显现。对于Dil-Ac-LDL，对于TO-PRO使用530-560nm的激发滤光片，对于TO-PRO使用640nm，对于DAPI使用340-380nm。 </li></ol></li><li> Von Willebrand（vWF）染色 <ol><li>如步骤8.1.1所述培养内皮细胞。 </li><li>吸出培养基并用1mL预热的HBSS冲洗细胞;然后如步骤8.1.4所述用4％PFA固定。 </li><li>吸出PFA，用12mL平板的盖板上的细胞用1mL PBS冲洗两次，并用1mL的0.1％Triton X-100（在PBS中）将细胞在室温下透化20分钟。 </li><li>吸出透化溶液，并用封闭溶液（PBS中5％BSA / 5％FCS）孵育细胞1小时。 </li><li>用兔抗vWF抗体（1:100）在4℃下在潮湿室中孵育细胞过夜。添加一个盖玻片没有the一抗作为阴性对照。 </li><li>用1 mL PBS冲洗12孔板盖玻片上的细胞3次，并用驴抗兔Alexa Fluor-488抗体（1:500）和核染色溶液TO-PRO（10μM）孵育细胞1小时在RT。 </li><li>用1 mL PBS冲洗12孔板盖玻片上的细胞，抽出PBS，重复3次。 </li><li>将盖玻片从12孔板中取出，并将其安装在带有一滴安装介质的玻璃载玻片上。细胞准备在荧光/共焦显微镜下显现。对于vWF使用490nm的激发滤光片，对于核染色使用640nm的激发滤光片。 </li></ol></li></ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+of+calcium+stable+adult+cardiac+myocytes.">Culturing of calcium stable adult cardiac myocytes.</a> J Mol Cell Cardiol. 14 (7), 397-412 (1982).</li><li>Xu, X., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+adult+rat+heart+myocytes.">Primary culture of adult rat heart myocytes.</a> J Vis Exp. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+metabolism+in+cultured+adult+rat+cardiac+fibroblasts:+response+to+angiotensin+II+and+aldosterone.">Collagen metabolism in cultured adult rat cardiac fibroblasts: response to angiotensin II and aldosterone.</a> J Mol Cell Cardiol. 26 (7), 809-820 (1994).</li><li>Shahzad, T., 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=Mechanisms+involved+in+postconditioning+protection+of+cardiomyocytes+against+acute+reperfusion+injury.">Mechanisms involved in postconditioning protection of cardiomyocytes against acute reperfusion injury.</a> J Mol Cell Cardiol. 58, 209-216 (2013).</li><li>G&#252;nd&#252;z, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+Stabilizes+Microvascular+Endothelial+Barrier+Function+via+Phosphatidylinositol+3-Kinase/Akt-Mediated+Rac1+Activation.">Insulin Stabilizes Microvascular Endothelial Barrier Function via Phosphatidylinositol 3-Kinase/Akt-Mediated Rac1 Activation.</a> Arterioscler.Thromb.Vasc.Biol. 30, 1237-1245 (2010).</li><li>Lipps, 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=N-terminal+fragment+of+cardiac+myosin+binding+protein-C+triggers+pro-inflammatory+responses+in+vitro.">N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro.</a> J Mol Cell Cardiol. 99, 47-56 (2016).</li><li>Abdallah, Y., 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=Interplay+between+Ca2++cycling+and+mitochondrial+permeability+transition+pores+promotes+reperfusion-induced+injury+of+cardiac+myocytes.">Interplay between Ca2+ cycling and mitochondrial permeability transition pores promotes reperfusion-induced injury of cardiac myocytes.</a> J Cell Mol Med. 15 (11), 2478-2485 (2011).</li><li>G&#252;nd&#252;z, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ticagrelor+on+endothelial+calcium+signalling+and+barrier+function.">Effect of ticagrelor on endothelial calcium signalling and barrier function.</a> Thromb Haemost. , (2016).</li><li>G&#252;nd&#252;z, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+effects+of+ATP+and+adenosine+on+barrier+function+of+rat+coronary+microvasculature.">Opposing effects of ATP and adenosine on barrier function of rat coronary microvasculature.</a> J Mol.Cell Cardiol. , (2012).</li></ol>

作者没有任何声明。

在本文中，描述了用于分离和培养心肌细胞，内皮细胞和成纤维细胞的可重复方案。该方案描述了与仅仅一种细胞类型相反的心脏组织， 即心肌细胞，内皮细胞和成纤维细胞的主要细胞类型的同时和高质量分离。分离过程中的关键步骤是适当消化心脏组织。如果消化不完全，仍然可以获得大量的肌细胞，但其质量非常差，并且它们大多是超收缩和圆形的（ 图2B ），并且在实验...

长期以来，啮齿动物模型被用作扩大对健康和疾病心血管生理学理解的工具。 1虽然这些动物模型可以让我们了解器官水平的疾病的病理生理学，并且分析用于治疗心血管疾病的各种药理学药物的药代动力学和药效学，了解心血管疾病发展的分子机制以及特定的细胞类型需要使用体外细胞培养模型。为此，已经开发了来自心血管系统的不同永生细胞系;然而，新鲜分离的原代细胞在生理和功能上与活组织和生物体更相关。 心脏是含有所有主要类型的心血管细胞的通用器官ystem，大鼠心脏仍然是一种常用的心血管生理学理解模型。在过去几十年中，已经描述了将各种细胞类型与心脏组织隔离的不同方法。然而，这些方法仅关注一种特定细胞类型的分离，导致不能再用于细胞分析的其他类型细胞的丧失。这里描述了优化的方案，其能够同时且高质量地分离主要细胞类型的心脏组织，即心肌细胞，内皮细胞和成纤维细胞。所有这些细胞类型可用于不同的实验装置8,9,10以及来自相同动物的细胞 - 细胞相互作用的分析。

<table border="0" cellpadding="0" cellspacing="0" width="717">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">anti-vWF</td>
			<td style="width:133px;">Santa Cruz Biotech.</td>
			<td style="width:119px;">SC-14014</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride</td>
			<td style="width:133px;">Merck</td>
			<td style="width:119px;">102378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carnitin</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">C0283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Collagenase type II</td>
			<td style="width:133px;">Worthington</td>
			<td style="width:119px;">LS004176</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Creatin</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">C0780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">D-Glucose</td>
			<td style="width:133px;">Merck</td>
			<td style="width:119px;">108342</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Dil-Ac-LDL</td>
			<td style="width:133px;">Thermo Scientific</td>
			<td style="width:119px;">L3484</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">EDTA Solution (0.2 M)</td>
			<td style="width:133px;">Biochrome AG</td>
			<td style="width:119px;">L2113</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Embeding solution</td>
			<td style="width:133px;">Citiflour</td>
			<td style="width:119px;">AF1-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Endothelial cell medium MV2</td>
			<td style="width:133px;">PromoCell</td>
			<td style="width:119px;">C-22022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Foetal calf serum (FCS)</td>
			<td style="width:133px;">Biochrome AG</td>
			<td style="width:119px;">S0615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Gentamicin</td>
			<td style="width:133px;">Serva Chemicals</td>
			<td style="width:119px;">47991</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">HEPES</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Isoflurane</td>
			<td style="width:133px;">Abbott</td>
			<td style="width:119px;">TU 061219</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Laminin&#160;</td>
			<td style="width:133px;">Roche/Sigma</td>
			<td style="width:119px;">11243217001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">M199 medium</td>
			<td style="width:133px;">Thermo Scientific</td>
			<td style="width:119px;">11150059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">M199 medium (Powder)</td>
			<td style="width:133px;">Biochrome AG</td>
			<td style="width:119px;">T061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Magnesium sulphate</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">63138</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Mouse anti-rat CD31 antibody (TLD-3A12)</td>
			<td style="width:133px;">Thermo Scientific</td>
			<td style="width:119px;">MA1-81051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">NaCl solution (0.9%), Sterile</td>
			<td style="width:133px;">B. Braun</td>
			<td style="width:119px;">30820080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Pan mouse IgG beads (Dynabeads)</td>
			<td style="width:133px;">Thermo Scientific</td>
			<td style="width:119px;">11041</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Paraformaldehyde (PFA) 4% Solution</td>
			<td style="width:133px;">Santa Cruz Biotech.</td>
			<td>sc-281692</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin-Streptomycin</td>
			<td style="width:133px;">Thermo Scientific</td>
			<td style="width:119px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Phosphate buffer saline (PBS) 1x&#160;</td>
			<td style="width:133px;">PAN-Biotech</td>
			<td style="width:119px;">P04-36500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Plastic consumables</td>
			<td style="width:133px;">Greiner Bio-One</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Potassium Chloride</td>
			<td style="width:133px;">Merck</td>
			<td style="width:119px;">4933</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Potassium dihydrogen phosphate</td>
			<td style="width:133px;">Merck</td>
			<td style="width:119px;">7873</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sodium Chloride</td>
			<td style="width:133px;">Merck</td>
			<td style="width:119px;">6404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sodium Hydroxide Solution (2 N)</td>
			<td style="width:133px;">Merck</td>
			<td style="width:119px;">109136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sterile filtration system</td>
			<td style="width:133px;">Thermo Scientific</td>
			<td style="width:119px;">5660020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Taurine</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">T8691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">TO-PRO</td>
			<td style="width:133px;">Thermo Scientific</td>
			<td style="width:119px;">T3605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Trypsin-EDTA Solution (10X)</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4174</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Water, Sterile</td>
			<td style="width:133px;">B. Braun</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous isolation of high quality cardiomyocytes, endothelial cells, and fibroblasts from an adult rat heart

大鼠是用于心血管研究的重要动物模型，大鼠心脏细胞常规用于体外分析心血管疾病进展的分子机制，如心脏肥大，纤维化和动脉粥样硬化。尽管已经对从心血管系统开发永生化细胞系以了解这些细胞机制进行了多次成功的尝试，但是对于这些研究，原代细胞提供了更加自然和接近于体内环境。因此，在特定细胞类型上工作的不同实验室已经制定了分离感兴趣的大鼠心脏细胞的各种类型的方案。然而，允许隔离多个单元格类型的协议丢失。这里描述了优化的方案，其允许从单一制剂中分离高质量的主要心脏细胞类型（心肌细胞，内皮细胞和成纤维细胞），并使得该r用于细胞分析。这样可以最有效地利用可用资源，这可以节省时间并降低研究成本。

<html>分离程序导致可用于计划实验的70-80％可行的棒状条纹心肌细胞（ 图2A和2C ）的产量。在我们的实验室中，心肌细胞通常用于分析Ca 2+信号。 图5A显示了在加载Fura-AM（5μM）的心肌细胞中对缺血/再灌注反应的细胞内钙[Ca 2+ ] i振荡。 图5B显示加入ATP（100μM）后内皮细胞和成纤维细胞中[Ca <s...

Watch this Scientific Journal Video about Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart at JoVE.com

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

已经开发并描述了用于从大鼠心脏分离不同心脏细胞类型的几种方案。在这里，描述了优化的方案，其允许从单一制剂分离高质量的主要心脏细胞类型（心肌细胞，内皮细胞和成纤维细胞），降低了实验成本。

从成年大鼠心脏同时分离高质量心肌细胞，内皮细胞和成纤维细胞

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

simultaneous-isolation-high-quality-cardiomyocytes-endothelial-cells

Research

JoVE Journal

Biology

13.2K 次观看.  University Hospital Giessen. 已经开发并描述了用于从大鼠心脏分离不同心脏细胞类型的几种方案。在这里，描述了优化的方案，其允许从单一制剂分离高质量的主要心脏细胞类型（心肌细胞，内皮细胞和成纤维细胞），降低了实验成本。 

视频: Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

成年大鼠心脏心肌细胞的原代培养

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

科研

生物学

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

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 ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

胚胎心室血管内皮细胞的分离

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

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, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

分离培养和成年小鼠心肌细胞转导

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

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

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

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

成人小鼠心脏干细胞的分离、鉴定及分化

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

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心脏纤维细胞和肌纤维细胞的分离与表征

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and &#945;-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.

The goal of this work is to develop a method to reproducibly isolate cardiomyocytes from the adult heart and measure DNA content and nucleation.

从固定心脏分离心肌细胞进行免疫细胞化学和倍体分析

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably ...