JJPC is supported by a Canadian Institutes of Health Research (CIHR) postdoctoral fellowship. MAM receives a merit scholarship from the German National Academic Foundation - Studienstiftung des Deutschen Volkes and is supported by a grant from the EFCNI (European Foundation for the Care of the Newborn Infant). BT is supported by CIHR, the Canadian Lung Association, the Stem Cell Network and the Children's Hospital of Eastern Ontario Research Institute.

所有的程序是由渥太华大学的动物护理委员会（动物伦理协议OHRI-1696）的批准。动物保健是按照机构的指导进行。 1.肺间质干细胞的分离<ol><li>在50毫升管准备酶混合物:权衡30单位中性蛋白酶2500ü我胶原酶和500üDNA酶一，这些款项足以满足成年小鼠或大鼠小狗的肺部。在4℃直至使用上隔离和存储的天制备。 </li><li>牺牲幼鼠第13天通过腹膜内注射戊巴比妥钠（0.2毫升，65毫克/毫升）。用脚趾捏反射建立神志不清。动物的死亡是通过打开胸部保证，概述如下。 </li><li>通过用70％乙醇喷雾动物消毒皮肤，小心使用手术剪，开始在膜片和朝喙侧切割打开胸廓，非常小心地不损伤肺部。传播胸腔打开使用止血钳，或者切掉胸腔以提供进入胸腔。 </li><li>通过移除心脏抽血动物。要取出心脏，把握胸腺和心脏小镊子和手术剪刀剪这些东西拿走。紧接着，吸收血液用纱布，直到没有更多的血液出来切断的主动脉和肺动脉的。 </li><li>从胸腔取出肺脏如下:持用小镊子气管，然后断绝与上喙侧手术剪气管。同时轻轻拉动肺包出来的胸部，切掉任何结缔组织沿肋骨背侧释放肺部。 </li><li>通过沿与手术剪膜片切割切断从主动脉和食管肺部。现在，肺是完全从胸部自由用纱布轻轻除去任何残留的血液。 </li><li>取出气管和支气管与河畔gical剪刀和仔细的肺叶转移到含冷35毫升30％柠檬酸盐 - 磷酸盐 - 葡萄糖腺嘌呤（CPDA-1）抗凝50ml管（26.30克柠檬酸三钠二水合物，3.27克抗坏血酸一水合物，2.22加入磷酸一钠，磷酸二氢钾， 31.80克D-葡萄糖，在1L0.275克腺嘌呤纯化的 H 2 O;无菌过滤的磷酸盐缓冲盐水（PBS），使用在使用前0.22μm的膜滤器）以除去血液和碎屑的溶液。 </li><li>在30％CPDA-1 / PBS中的5分钟的冲洗后，肺转移到含有35毫升无菌磷酸盐缓冲盐水（PBS）（RT）下一个新的50ml管中，倒置轻轻除去柠檬酸盐。转移到含有管35毫升Dulbecco氏PBS +钠丙酮酸+葡萄糖（DPBS ++）（RT）。每次清洗步骤大约需要5分钟。 注意:DPBS +可商购订购或由如下21:氯化钙二水合物132.4毫克，氯化镁六水合物100毫克，氯化钾（无水）200米克，磷酸二氢钾（无水）200毫克，氯化钠（无水）8000毫克，磷酸氢二钠（无水）1,144.5毫克，D-葡萄糖1000mg的，丙酮酸钠36毫克在1L纯净H 2 0，pH值7.3。无菌过滤器使用在使用前0.22μm滤膜过滤该溶液。 </li><li>通过加入10ml DPBS + +（预热至37℃）并轻轻翻转直至溶解溶解酶混合物。 </li><li>转移肺部到一个新的50ml管中，并用手术刀切碎在管的壁的肺直到切碎。酶组合加入至组织（10毫升酶混合物/每成年小鼠/大鼠小狗肺），盖紧管和在38℃，轻轻摇动孵育（无论是在热混合器，振荡水浴，或水与常规的手动搅拌槽）。持续时间取决于组织类型:胎儿组织60分钟，幼年/成人组织90-120分钟，纤维化成人组织120-150分钟。 </li><li>通过螯合二价阳离子W停止消化第i 200微升乙二胺四乙酸（EDTA，0.15M，pH为7.4。无菌过滤器在使用前用0.22微米的膜过滤器的溶液）。 </li><li>加入20毫升5％胎牛血清（FBS）/ PBS中，并通过100微米的细胞滤网整个悬浮液传递到一个新的50ml管中。冲洗管和细胞过滤用10毫升5％FBS / PBS中，使总体积至40ml。 </li><li>离心在500 xg离心，室温下5分钟。 </li><li>除去上清液（沉淀应清晰可见），悬浮在40毫升5％FBS / PBS，重复离心步骤1.13。 </li><li>悬浮在4ml 5％的FBS / PBS（RT）和层小心以上3毫升密度梯度介质（1.073克/厘米3）在一个15毫升管14。以获得良好的相间，至关重要的是，在单细胞悬浮液的分层发生在以非常低的速度&gt; 45°角。 </li><li>在900 XG离心20分钟，中（5/10）加速，减速没有，19℃。 </li><li>收集存在于细胞中的terphase和重悬在10ml无菌PBS。 </li><li>离心机在500 XG 5分钟，21℃。 </li><li>去除上清，重悬细胞沉淀在10毫升预热完成了L-MSC培养基（最低基本介质鹰，阿尔法修改（αMEM），辅以每500毫升αMEM，20％体积/体积FBS 2毫摩尔L-谷氨酰胺和1％体积/体积青霉素/链霉素/两性霉素B. </li><li>重复离心步骤1.18。随后，去除上清，重悬在10毫升预热的新鲜培养基。 </li><li>算使用血球或自动细胞计数器和板以高密度的细胞，因为在此阶段的大多数细胞都没有塑料贴壁。确切的铺板密度取决于动物种类和年龄，例如成年小鼠肺的细胞以10 5个细胞/ cm 2接种，而从大鼠小狗肺细胞是足够致密以1-2接种密度点¯x 10 4个细胞/ cm 2的</sup&gt;。使用0.2毫升L-MSC培养基/ cm 2以下。 </li><li>培养L-的MSC在湿润的5％ 的 O 2，5％的CO 2气氛中于37℃。 </li><li>用PBS洗涤一次后更改初始播种后媒体24小时，每3天以后。细胞应该是80-90，直到％汇合，这需要3-5天，平均取决于原产地的动物养殖。较高的汇合将促进分化为成纤维细胞。 </li></ol> 2. CD146 +肺间质基质细胞的选择<ol><li>二抗磁珠涂料<ol><li>涂层在圆底磁珠用生物素化的次级抗体，在10微克的二级抗体/ 100微升磁珠的比率无菌的毫升无菌条件离心管。使用10微升的二级抗体每预期的0.5×10 6个细胞 ，并用最小100微升磁珠和10μg抗体。 注:一个比每珠使用量可能制造商之间差异tibodies，请查阅制造商的说明选择的珠子。对于这个协议，请参考表1对该协议的优化中使用的珠子和抗体。 </li><li>孵育在一个旋转样品混合器在30转/分钟（RPM），在室温30分钟。 </li><li>重悬珠子在1.5ml的无菌0.1％牛血清白蛋白（BSA）/ PBS中，并转移到3毫升圆底管（流式细胞术管）。冲洗冷冻管与另外1.5毫升0.1N％BSA / PBS和转移到3毫升管。 </li><li>放置在适当的磁体的管，并等待2分钟，直到所有的珠粒附着到管的后壁，该溶液仍然是清楚的。除去用3毫升0.1％BSA / PBS两次径流和重复洗涤。 </li><li>重悬在4℃下在3毫升0.1％BSA / PBS的珠和存储，避光。 5天之内使用。 </li></ol></li><li>贴标CD146 + L-干细胞<ol><li>收获使用温和的非胰蛋白酶溶液用PBS漂洗后的间充质细胞。 注意:卷取决于培养瓶的大小（0.3毫升/厘米2介质或PBS）中。 <ol><li>除去培养基并用无菌PBS冲洗细胞三次。 </li><li>添加温和非胰蛋白酶溶液的适当体积（ 见表 1）。使用表 1中的溶液时，在孵化器在37℃下，直到细胞已经分离，约10分钟。 </li><li>通过加入L- MSC培养基，和离心机在500×g下5分钟使酶失活。 </li></ol></li><li>在0.1％BSA / PBS上清，重悬细胞，并计数与血球或自动细胞计数器细胞。 注:0.1％的BSA的体积/ PBS取决于培养瓶的大小:瞄准5或10毫升0.1N％BSA / PBS。 </li><li>重复步骤2.2.2重悬在3ml 0.1％BSA / PBS（块未细胞的专用量特异性结合位点，并保持细胞存活）。保持细胞在冰上。 </li><li>准备3毫升圆底管与主抗CD146抗体，并保持在冰上。 注意:被添加到细胞中的专用量初级抗体的浓度取决于所使用的抗体。对建议的抗大鼠CD146抗体见表 1。 </li><li>从步骤2.2.3添加的细胞的总体积到管与初级抗体。附近以15rpm，4℃紧紧孵育上的旋转样品混合器30分钟。 </li><li>离心细胞，在500 xg离心，4℃5分钟，取出3毫升0.1％BSA / PBS上清，重悬细胞沉淀。重复离心步骤。 </li><li>重悬在含有按照2.1节中的说明制备并以15rpm，4℃下在旋转样品混合器孵育30分钟的第二抗体包被的珠子3 ml溶液的细胞。 </li></ol></li><li>选择CD146 + L-干细胞<ol><li>将装有磁铁的标记CD146 + L间充质干管。阳性细胞将被吸引到管子的背面。无需移动管或触摸珠，收获与阴性细胞上清液，用无菌巴斯德移液管（这些既可以收集或基于实验设计丢弃）慢。 </li><li>从磁体取出管和重悬细胞在3毫升10％的BSA / PBS中。重复步骤2.3.1中描述的选择过程。四次（Σ5选择步骤）。 </li><li>悬浮CD146 + L-的MSC在预热的L- MSC培养基并返回到磁铁。 </li><li>在一个适当大小的培养瓶（0.4毫升/厘米2）上清，悬浮在L-MSC培养基和板块。作为一个经验法则，板中的细胞大小相同培养瓶中从该细胞前珠选择解除。 CD146 + L-MSCs的数目，因此，铺板密度，将取决于源动物的年龄，物种和菌株。总是瞄准的〜5,000个细胞/ cm 2的接种密度。 注意:珠不会干扰细胞生长和将逐渐消失的细胞增殖。 </li></ol></li></ol> 3.冷冻细胞<ol><li>使用以下冷冻介质:60％pentastarch溶液（10％pentastarch在0.9％NaCl）中，20％的FBS，12.5％CPDA-1，2.5％的NaCl（0.9％），5％二甲基亚砜（DMSO）22。 注意:如果不是所有的成分都可用，含5％DMSO，30％FBS和65％αMEM一个解决方案是一个很好的选择。 </li><li>重悬的细胞以0.5×10 6个细胞/ ml，等分试样中的各1ml冷冻管并用冷冻集装箱冻结O / N在-80℃。 </li><li>转移到液氮贮罐的第二天。 </li></ol> 4.细胞解冻<ol><li>解冻细胞的小瓶在37℃水浴中5分钟。 </li><li>悬浮细胞在10ml预热的培养基，离心500 XG 5分钟，21℃。 </li><li>除去上清，悬浮于10毫升培养基中的细胞沉淀。在通风盖的细胞培养的种子细胞在0.2ml / cm 2的培养基中于5000个细胞/ cm 2的处理过的瓶（ 例如，15毫升的75cm 2的烧瓶中）。 </li><li>培养的细胞，在5％O 2，5％的CO 2，直至 80-90％汇合收获和/或实验性使用。过汇合将诱导分化。当5000个/ 平方厘米播种，L-干细胞将在培养3-4天达到80-90％汇合。 </li></ol>

The authors have nothing to disclose.

主要L-干细胞的分离和培养提供了一个机会，以更好地了解它们的功能和它们与其他细胞群在细胞水平上的互动，以及它们在肺的发育，健康和疾病的作用。这是特别重要的，因为在缺乏这些细胞的特定单个标记的使得它几乎不可能来研究这些细胞中的原位 。与所有主要的细胞群，应该记住，这些细胞更可能改变它们的性质较长它们保持在培养物19（在24中综述）。保持左旋的MSC中是尽可能接近其自然状态下的状态，这是非常重要的是，它们在5％O 2，5％CO 2中培养，在37℃湿润培养箱。在呼吸生理人们普遍，基于分压道 ​​尔顿定律，环境空气（21％O 2）的氧分压为160毫米汞柱，并下降到〜101毫米汞柱肺泡空域25-27。在成熟的肺的肺泡-动脉氧分压梯度为〜3-4毫米汞柱，但是这可能显著在患病的或未成熟的肺其中肺泡-动脉距离增大25,28增加。体内，所述间充质小生通常暴露于2-8％29,30的氧浓度。只有极少数的论文已实际测量的PO 2在肺，但一项研究测得这20例患者肺部，从23到656毫米汞柱的正常肺寻找范围PO 2，用 42.8毫米汞柱27,31的中位数。根据道尔顿定律，具有37℃的温度和5％O 2的加湿培养箱具有〜36毫米汞柱的PO 2，它完全落在正常肺组织测量的范围内，并且是非常接近的值。此外，低氧培养条件促进祖人口文化29维护。总之，我是很难说什么氧气浓度L-的MSCs在正常出生后发育或疾病过程中的不同阶段暴露在原地，但现在看来，5-10％O 2可能是最从生理上讲27,31准确。根据上面提到的信息，我们选择在我们的培养条件下保持5％ 的 O 2，我们发现，当这些条件被维持在体外的L的MSC将生长较快，显示较少的应力纤维，和具有较小的可能性自发分化或衰老。这将阻止自发分化或其他异常情况的因素是使用传代温和的非胰蛋白酶分解剂，传代L-干细胞，当他们达到80-90％汇合（密集条件下会促进成纤维细胞分化），并保持通道数低（&lt;P4）。在由Hoffmann和同事的研究中，胰蛋白酶显示出具有对L-干细胞功能产生不良影响，并表型，而免费的胰蛋白酶分解剂可以保持这种13。 为了优化在分离过程的产率，为精细地剁碎肺部，同时保持记住该组织不应使其干燥是很重要的。在酶促消化，重要的是将管搅拌频繁如果不使用加热摇动装置，如组织碎片会沉淀并不会得到最佳曝光给酶。另一个关键步骤是密度梯度分离:如果分层不这样做非常缓慢或以一角度&lt;45°，这将导致两种液体，而不是分层的混合，造成整个细胞群体的损失。此外，该密度梯度介质的密度是非常依赖于环境温度。任何其他的温度超过19°C（或RT），将导致次优或无密度梯度分离。 感兴趣至f这个协议的uture用户是，我们已经成功地从收获到24小时，隔离之前肺中分离活的L-的MSC，其前提是肺部直接存储在后收获冷L-MSC介质，并保持在4 C。这允许更灵活的试验计划，甚至是不同的实验室之间的肺部出货。产率是大致类似于新收获的肺，和细胞生长得同样好。 这将是可能的另一变形例中，是使用流式细胞仪代替磁珠选择，即使该方法受试者的细胞更应力作为过程需要更多的时间和作为细胞经受高的压力和速度，并且携带污染的风险如果不是在无菌条件下完成的。此处所用的磁珠不干扰细胞生长，并在培养数天消失。一种FACS或磁珠分选的偏好也可能取决于可用的设施给最终用户。此外，将有可能修改该协议从其他组织隔离驻地的MSC。在这种情况下，它将给酶定制到感兴趣的器官，作为基体组合物的器官之间变化是重要的。 只使用用于获得间充质细胞的密度梯度和随后的塑料粘附的一个重要的限制是，所得到的群体包含两个L-MSC和成纤维细胞。选择用于CD146 +细胞强烈补救这个警告，但尽管CD146 +选择过程和仔细的培养条件，L- MSCs的性质，并在体外细胞培养的影响，使其无法避免某种程度的分化成成纤维细胞。在一般的MSCs已经报道了包含多能的层次结构，其中，细胞进一步向下的层级慢慢直到它们成为最终分化成纤维细胞失去其潜能研究&lt;SUP&gt; 23。在所有的可能性，这也发生在L-MSC利基4，看来要体现在培养CD146 +人口CD146 +选择之后，已经一个通道细胞的大约40％已经失去了CD146的表达。此外，在该群体中只有80％的CD73和40％CD90表达。在某种程度上，这可以通过多能的MSC层次来解释。这将是可能的，CD146 +经受不对称的增殖，从而引发两个CD146阳性细胞和阴性，更加分化的，子细胞。肺的居民的MSC也可合理地预计将有一个稍微不同的表型和功能从骨髓MSC，其上CD73和CD90表达的定义是基于。国际学会细胞疗法已经认识到，并非所有的MSC表达的经典的MSC标记，包括CD73和CD90，以及表面标记表达不以限定的MSC 32的最佳方法。电子商务的〜CD146的+ L-MSC人口的70％xceptionally高效率CFU支持此。由于CD146 +人口进行相对比无序的间充质人口MSC特性更好，作者并没有进一步的特征的CD146 -人口。我们力争用干细胞来丰富我们的间充质人口和消耗成纤维细​​胞的他们，但我们并不认为这是唯一真正的L-MSC人口。 L-干细胞有可能很异类，无论是在起源，表型和功能。 虽然CD146被广泛认为是干细胞和周16,17,33,34多能的标志，非常知之甚少体内 CD146 + MSC人口。 CD146是一个细胞粘附分子，其为血管生成和血管内皮细胞的功能很重要，在内皮细胞，平滑肌细胞，周细胞，干细胞和T淋巴细胞15,16,35,36表示。最近几年，证据已经出现，干细胞居住在血管周围利基在多个器官，如CD146 +细胞共染色的MSC-CD73标记CD105和37。的确，培养的周细胞和干细胞具有非常相似的基因表达谱38，加强，干细胞是从周细胞4,39得到的图。虽然我们还没有进行免疫组化分析，以确定我们的CD146 + L-MSC人口的位置，这是非常有可能，他们也将设在血管周围的位置。未来的研究将需要验证，如果这是事实确实如此。这可能是我们的细胞群的一个子集是周细胞，尽管应该指出，所有细胞看起来表型CD146 +选择之后非常相似。 间充质基质细胞是众所周知的难以确定，由于缺乏一个包罗万象的标记。各种研究已经报道了隔离不同的方法L-MSC的，并已令人信服地表明，这些细胞群体彼此类似相对于MSC标志物表达。基于所述CD146 + L-MSC的表征如这里提出，所述CD146 + L-MSC人口非常类似于如由生长或使用干细胞相关表面标记如的Sca各种形式的FACS来实现先前报道的L- MSC群体-1，ABCG2和CD90。时相比长出左旋的MSC 13，CD146 + L-的MSC似乎不得不产生从单个细胞集落更大能力，这表明我们的方法产生了更丰富的多能的L- MSC群。相比基于CD90 7或血小板衍生的生长因子受体α（PDGFRα）40,41即隔离左旋的MSC方法时，这个优点也是如此，因为这两个表面标志物还选择用于更终末分化lipofibroblasts和肺泡/结构成纤维细胞和因此只能合理bË确定为基质细胞20,42。与此相反，这两种的Sca-1和ABCG2是有据可查的，与干性和MSC表达9,43-46关联标记。 ABCG2 + L-的MSC但是具有更内皮性质，因为它们对于CD31 46阳性99％，而在这里报导的方法中选择对通过聚蔗糖梯度和塑料粘附血管内皮细胞。相比McQualter和同事5,8的基于的Sca-1隔离协议我们的方法的主要优点是，它是广泛适用于除小鼠其它物种，其中的Sca-1，不能使用。 虽然这是非常具有挑战性的，大概是不可能的近了，从晚发展中国家或成人肺获得L-MSC的"纯"人口和维持其未分化状态，这里介绍的协议提供了一个快速，一致，可靠的方法获得的细胞群高度富集多能自我更新的肺水库IDENT干细胞。使用这种方法将有一个更加限定的L- MSC的在各种疾病模型和它们在显影肺角色的角色的研究。

间充质基质细胞（MSC）越来越多地认识到它们的治疗潜力在很宽范围的疾病，包括肺疾病。除了使用骨髓和外源细胞疗法脐带干细胞的，还增加居民组织的MSCs在修兴趣和再生潜能。在开发过程中，包括肺，间质发育是线索的重要来源，而居民干细胞是可能的候选人是在这个中心。此外，有证据表明居民的MSCs在成人疾病扰动，包括癌症1,2和纤维化3。这些居民组织干细胞研究中的主要障碍是缺乏这些细胞4的分离和鉴定明确的标记。干细胞抗原-1（SCA-1）在小鼠中作为用于各种组织的干细胞的标志物被鉴定，并且可用于L-的MSC 5的隔离，但不幸的是没有已知的其他物种6直向同源物。研究人员已经报道了各种不同的分离方法对L- MSC的从任肺组织或流体的分离。 - / CD45 - - / CD90 +细胞 7，CD31 - / CD45 - /上皮细胞粘附分子（EpCAM的） -这些从荧光激活细胞分选（FACS）为基础的方法选择用于CD31变化/的Sca-1 +细胞8，多药耐药转运ATP结合盒G（ABCG2）阳性细胞9或赫斯特33342染料外排10，塑料粘附11,12和迁移出组织糜13。 在这里所提出的方法的优点是数倍。通过使用温和酶消化和密度梯度14中，可以得到包含的MSC，但不包括上皮或内皮细胞的密度范围的所有细胞。随后塑料吸附步骤确保只有mesenchymal细胞粘附和留在文化，消除白细胞。最重要的是然而，CD146 +选择步骤允许消除成纤维细胞，因为这些细胞不表达CD146。细胞粘附分子，CD146的表达是积极与多能相关，并且因此是一个很好的标志物从间充质细胞群体15-19剔除成纤维细胞。这是通过使用CD90作为选择标记的优点，因为它不仅在干细胞，而且在lipofibroblasts 5,20表示。在这个协议中，我们已经明确选择磁珠选择，因为它是在细胞上温和，而且整个过程可在无菌条件下进行。相对于生长方法这种隔离方法的另一个重要优点是，它是比较快的，6-10天，而不是在一个月或更长时间的生长方法。初始分离后三到五天的间充质人口准备CD146 +塞莱<html> ction;后3到5天，CD146 +细胞准备用于实验或可以冷冻供以后使用。培养的下降时间，从而提高了电池的质量的MSC朝向延长体外培养 19成纤维细胞转分化。最后，由于该协议的性质，有可能通过简单地调节消化酶和孵育时间的选择选择合适的抗体，或甚至其他器官系统这种方法适用于其它物种。 这种隔离方法的详细方案在下面给出， 并在图1A和1B分别设置在隔离和CD146 +亚群的随后选择的示意图。此外，详细信息包含用于传代，冷冻和解冻这些细胞。 广告/ 53782 / 53782fig1.jpg"/&gt; 图1.肺间质细胞（A）和随后的CD146 +细胞的选择（B）的最小=分钟的隔离的示意图概览; EDTA =乙二胺四乙酸; 第二抗体=二级抗体; α-CD146抗体=初级抗CD146抗体; -ve细胞= CD146阴性细胞; +已经细胞= CD146阳性细胞。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53782/53782fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table border="0" cellpadding="0" cellspacing="0" width="664">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:175px;">Neutral Protease</td>
			<td style="width:120px;">Worthington Biochemical Corporation</td>
			<td style="width:61px;">LS02104</td>
			<td style="width:309px;">Prepare on day of isolation and store at 4&#176;C until use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:175px;">Collagenase I</td>
			<td style="width:120px;">Worthington Biochemical Corporation</td>
			<td style="width:61px;">LS004196</td>
			<td style="width:309px;">Prepare on day of isolation and store at 4&#176;C until use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">DNAse I</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:61px;">D5025</td>
			<td style="width:309px;">Prepare on day of isolation and store at 4&#176;C until use</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:175px;">Pentobarbital sodium (Euthanyl)</td>
			<td style="width:120px;">Bimeda-MTC Animal Health Inc, Dublin, Ireland</td>
			<td style="width:61px;">-</td>
			<td style="width:309px;">Use 0.2 mL for rat pups between 20-30g; larger animals may need more.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:175px;">Dulbecco&#8217;s PBS + Sodium-pyruvate + Glucose</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:61px;">14287-072</td>
			<td style="width:309px;">Very crucial to use this type of D-PBS, as the calcium and magnesium that are present in this D-PBS facilitate facilitate the enzymatic digestion</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:175px;">Ficoll-Paque PREMIUM (1.073 g/cm3)</td>
			<td style="width:120px;">GE Healthcare</td>
			<td style="width:61px;">17-5446-52</td>
			<td style="width:309px;">Density gradient media. To obtain a good interphase, it is crucial that the layering of the single cell suspension occurs at a &#62;45o angle at very low speed, and that the subsequent centrifugation is done at 19oC.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">&#945;MEM</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:61px;">M8042</td>
			<td style="width:309px;">Warm in 37oC water bath before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">200 mM L-Glutamine</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:61px;">25030-164</td>
			<td style="width:309px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">100x Antibiotic-Antimycotic</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:61px;">15240-062</td>
			<td style="width:309px;">Penicillin/Streptomycin/Fungizone</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">M-280 Dynabeads</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:61px;">11205D</td>
			<td style="width:309px;">Magnetic beads</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:175px;">biotinylated polyclonal rabbit-anti-mouse IgG</td>
			<td style="width:120px;">Dako, Agilent Technologies</td>
			<td style="width:61px;">E0464</td>
			<td style="width:309px;">Biotinylated secondary antibody that matches with the anti-rat CD146 antibody described below</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:175px;">DynaMag5-magnet</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:61px;">12303D</td>
			<td style="width:309px;">Magnet that is recommended for use with Dynabeads. NOTE: magnet should be chosen based on the manufacturer&#8217;s instructions of the magnet beads of choice.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:175px;">TrypLE express&#160;</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:61px;">12605-028</td>
			<td style="width:309px;">Gentle non-trypsin alternative, use 1 mL of TrypLE express/ 25cm2 surface area. After detachment, 10 mL L-MSC culture medium is sufficient to inactivate 3 mL TrypLE</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">anti-rat CD146 antibody</td>
			<td style="width:120px;">Lifespan Biosciences Inc.</td>
			<td style="width:61px;">C35841</td>
			<td style="width:309px;">12.5 &#956;l per 0.5 x 106 cells</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:175px;">Pentaspan (pentastarch solution)</td>
			<td style="width:120px;">Bristol-Myers Squibb Canada</td>
			<td style="width:61px;">-</td>
			<td style="width:309px;">Can be obtained through local blood donation services or hematology departments. Alternatively, one could use the PSI 20% Pentastarch solution (Preservation Solutions, PST001) diluted 1:1 with 0.9% NaCl.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Mr.Frosty freezing container</td>
			<td style="width:120px;">ThermoFisher Scientific</td>
			<td style="width:61px;">5100-0001</td>
			<td style="width:309px;">Improves viability when freezing L-MSCs overnight at -80oC</td>
		</tr>
	</tbody>
</table>

isolation of cd146+ resident lung mesenchymal stromal cells from rat lungs

间充质基质细胞（MSC）越来越多地认识到它们的治疗潜力在很宽范围的疾病，包括肺疾病。除了使用骨髓和外源细胞疗法脐带干细胞的，还增加居民组织的MSCs在修兴趣和再生潜能。此外，它们可能在正常器官发育的作用，并已被归因于疾病的作用，特别是那些患有纤维变性性质。这些居民组织干细胞研究中的主要障碍是缺乏对这些细胞的分离和鉴定明确的标记。这里所描述的隔离技术应用于肺间充质干居民（L-MSCS）的多个特征。一旦老鼠的牺牲，肺被删除，漂洗多次，以去除血。继手术刀机械分离，肺部使用I型胶原酶，中性蛋白酶和DNA酶I型的obtaine混合消化2-3小时D单细胞悬浮液，随后洗涤并铺在密度梯度介质（密度1.073克/毫升）中。离心后，从相间洗涤细胞并涂布在培养处理的烧瓶中。细胞是4-7天在生理5％O 2，5％CO 2的条件下培养。耗尽成纤维细胞（CD146 - ），并确保只有L-干细胞（CD146 +），CD146为+细胞阳性选择的人口通过磁珠选择执行。总之，这个过程可靠地产生主L-MSC的进一步的体外研究和操纵的群体。因为协议的性质，它可以很容易地转换为其他实验动物模型。

间充质干的，密度和贴壁的最可靠的物理特性二，在这个协议的第一部分是用来获取包含L-干细胞肺的间质细胞部分。虽然密度梯度相间将包括除了肺间质细胞的单核细胞和巨噬细胞，塑料粘附然后培养3-5天确保只有肺间质细胞保持。确实该细胞群表达了经典的MSC表面标记CD73，CD90和CD146，并为负标记CD34，CD45，主要组织相容性复合物II类（MHCII）-RT1B，CD11b和CD79A，表明不再有存在于任何白细胞细胞群体（图2A）。感兴趣的是，出CD146 +子集，一个范围的44.4-65.7％也是CD73 +和 CD90 +（数据未显示）。而且，该细胞群体是能够在菌落形成单位（CFU）的效率，在〜30％是远高于一般报道为骨髓MSC或者甚至其他的MSC类型（图2B），并沿三个经典MSC谱系（图2C）区分。 <img alt="图2" src="/files/ftp_upload/53782/53782fig2.jpg" /> 图2.酶消化，密度梯度分离，塑料贴壁后的MSC特征。（A）的MSC相关的表面标记的表达CD146，CD73和CD90是存在于塑料贴壁细胞群的一部分，而阴性白细胞标记的CD11b，CD79A ，CD34，CD45和MHCII，RT1B中几乎不表达。 （二）通过菌落有限稀释法（5个/厘米2）形成单位检测表明，塑料〜贴壁细胞30％的克隆能力，表明干细胞的。 （C）塑料贴CELLS有能力成骨基质的生产（红色）的，含有较高数的当与脂肪形成分化培养基诱导相比，非分化的控制以及形成软骨样含有软骨细胞（红色）球小脂质囊泡（红色）。数据被表示为平均值的（SEM）的平均值±标准误差。与通道1-3细胞生成的所有数据。 NM =负标记; CFU =菌落形成单位。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53782/53782fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 然而，主要的条件是，该人群可能包含的L- MSC和肺成纤维细胞的不同亚型，L型的MSC分化后代23。由于多能性是积极与CD146表达相关，和成纤维细胞应该没有这个标志的表达，CD146的亚群+邻正选择stensibly导致在被在L型的MSC高度富集的细胞群。这显然是由细胞群的比例更高证实表达的MSC相关联的表面标记（图3A），〜80％的相当高的集落形成潜力（图3B）和在特别是软骨细胞谱系更强分化响应（图3C）相比，是CD146选择之前所获得的总质人口。值得注意的是，这些L型的MSC形式只有极少数真正的脂肪细胞，但显示有更多的梭形细胞充满小脂质囊泡。这些可能反映了lipofibroblast血统这对肺的发育和肺泡Ⅱ型细胞同时支持至关重要。 CD146选择之后，更像是填充有大的脂囊泡的细胞的脂肪细胞可相比CD146选择之前被观察（图3C），但是大部分细胞仍然是lipofibroblasts状策含有小脂囊泡LLS。 <img alt="图3" src="/files/ftp_upload/53782/53782fig3.jpg" /> 后附加CD146 +磁珠选择图3的MSC特性。的CD146 +亚群的阳性筛选之后，这些细胞显示出的MSC相关的表面标记物的更高表达，CD73特别是（A）中，单细胞后一个相当高的CFU效率电镀（B）和用于特别是软骨细胞系，并在较小程度上成脂谱系（C）的更强的分化响应。数据被表示为平均值±SEM表示。与第3代细胞产生的所有数据。 NM =负标记; CFU =菌落形成单位。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53782/53782fig3large.jpg" target="_blank">请点击此处Ť</a>Ø查看此图的放大版本。

Watch this Scientific Journal Video about Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs at JoVE.com

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

这个协议描述了从大鼠获得原发性肺癌驻地间充质基质细胞，通过使用酶消化，密度梯度分离，塑料吸附和CD146 +磁珠选择的隔离技术。

CD146的分离<sup&gt; +</sup&gt;从大鼠肺组织驻地肺间质基质细胞

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

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Nanyang Technological University

Research

JoVE Journal

Biology

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

8.3K 次观看.  Ottawa Hospital Research Institute. 这个协议描述了从大鼠获得原发性肺癌驻地间充质基质细胞，通过使用酶消化，密度梯度分离，塑料吸附和CD146 +磁珠选择的隔离技术。 

视频: Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

科研

生物学

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

分离和富集大鼠间质干细胞（MSCS）及衍生的干细胞分离单细胞集落

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

对赫斯特的分离与表征低 CD45负小鼠肺间质干细胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

米色/布里特细胞的间质血管细胞的分离和分化

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

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

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 mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

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 ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

分离和富集人肺上皮祖细胞用于类器官培养

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...