这项工作是由德意志研究联合会（DFG），弗里茨·蒂森基金会和查理特/ MDC补助。

1。动物麻醉和活摘器官<ol><li>麻醉的小鼠腹腔注射注射氯胺酮（200 mg / kg体重）和甲苯噻嗪（20 mg / kg体重）。 </li><li>抗凝治疗注射250 IU肝素的ip为避免血液凝固和血栓形成。 </li><li>等待直到达到深昏迷状态，其特点是通过反射消失。轻轻地接触角膜反射的尾巴捏或飞行试验，检查无反射，角膜反射测试。 </li><li>鼠标上手术台转移，并将其固定在仰卧位。 </li><li>切开皮肤和腹壁剑突下和执行翻盖开胸切到双方沿肋弓，随后切断肋骨在腋前线内侧，肋骨向上偏转。 </li><li>打开心包，找到大血管。轻轻地按压心脏的尾鳍，以更好地显示主动脉。使用镊子夹住主动脉。 </li><li>在凹部的一对单峰，将心脏<html> cissors和解剖与一个单一的切断所有连接的容器。请一定要保持一个足够大的一部分，Langendorff离体主动脉插管。 </li><li>立即切除心脏转移用冰冷和预充氧溶液1（的解决方案，请参阅表1）填充到陪替氏培养皿。 </li></ol> 2。制备心脏和Langendorff灌流<ol><li>导管插入主动脉与的1.8F钢套管。请一定要避免空气栓塞。 </li><li>注视主动脉插管与1毫升溶液1与手术用缝合线和冲洗冠状动脉。 </li><li>连接套管与Langendorff装置。 </li><li>请确保开胸的Langendorff插管时间不超过120秒，以避免延长心肌缺血/再灌注损伤。 </li><li>灌注心脏用10毫升的Ca 2 +免费的解决方案（4毫升/分钟）。 </li><li>灌注心脏与胶原酶溶液3的8分钟（4毫升/分钟）。 </li></ol>乐“&gt; 3显微离解心腔。 <ol><li>转移心脏成预先温热的100毫米培养皿中含有低Ca 2 +溶液4。 </li><li>主动脉和其他非心脏组织，用剪刀小心地取出，并将其丢弃。 </li><li>独立的心房和心室，并继续与各室分开。保持细胞浸渍在溶液4。使用少量（小于5毫升）。 </li></ol> 4。进一步解离的心肌细胞<ol><li>心房心肌细胞： <ol><li>个性化房心肌细胞心房转移到一个单独的预热的100-mm的培养皿中，用细镊子轻轻拉出，除了通过分离组织。确保几乎完全分离的组织。 </li><li>使用1毫升吸移管，用火抛光的放大塑料移液管尖，5分钟，使细胞悬浮在1ml的溶液5。 </li><li>从碎片中分离出细胞，通过使用细胞过滤器（200微米筛目大小）。 </li> &lt;李&gt;加入5毫升溶液5的细胞悬浮液，并离心2分钟，在室温下在16×g离心。 </li><li>下面的步骤操作下的细胞培养罩。弃去上清液并将其再悬浮的颗粒在5毫升的溶液6。 </li><li>通过重力在一个15毫升的试管10分钟后沉淀，离心1分钟，在室温下在16×g离心。除去上清液。重新悬浮细胞根据其数量在1-5毫升的溶液6。 </li></ol></li><li>心室肌细胞： <ol><li>解剖左心室感兴趣区域的细钳子在5毫升的溶液4。 </li><li>挂起轻轻移液直到大部分的细胞中分离的细胞。转移后的细胞溶液（200微米网目尺寸）过滤到50ml管中，加入25毫升的体积。 </li><li>离心2分钟，在室温下在16×g离心。 </li><li>下面的步骤操作下的细胞培养罩。移除上清液，沉淀重新悬浮在25毫升溶液6和允许沉降10分钟的细胞。 </li><li>计数细胞，除去上清液，添加25 - 50毫升溶液6上的细胞。 </li></ol></li></ol> 5。制备心肌细胞电生理，生化，如果研究<ol><li>电生理学研究中，保持在50毫升的试管中的溶液6中的心肌细胞和抑制沉淀。 </li><li>对于生化研究， 例如 ，钙成像，在层粘连蛋白涂层的细胞培养皿中（终浓度为20μg/ ml的层粘连蛋白在PBS中）的板肌细胞。 </li><li>对于免疫荧光染色制备细胞培养皿中板与玻璃盖玻片和涂有层粘连蛋白溶液（最终浓度为50μg/ ml的层粘连蛋白在PBS中）。 <ol><li>电镀前肌细胞中取出的解决方案。板的细胞的细胞密度和控制通过使用显微镜。 </li><li>让肌细胞坚持以盖玻片1小时，在37°C在2％CO 2，删除解决方案一个标准的染色程序协议，并立即开始。 </li><li>与固色剂， 例如 4％PFA在PBS（pH7.5）中孵育10分钟，在室温下，并按照与3的PBS洗涤步骤，每次5分钟。 </li><li>至细胞透性，并抑制非特异性结合的抗体孵育的心肌细胞含10％血清，0.2％BSA的PBS中，在室温下的30分钟，0.3％的Triton。 </li><li>初级抗体孵育1小时，在37℃下洗如前所述。 </li><li>孵育1小时，在室温下与第二抗体。染液核和α-辅肌动蛋白的利用DAPI荧光染料标记的鬼笔环肽（Invitrogen公司）的Alexa Fluor 488。 </li><li>洗涤后，盖玻片转移仔细硅烷处理过的显微镜载玻片和嵌入细胞的荧光安装介质。 </li></ol></li></ol> 6。细胞电<ol><li>对频率进行全细胞膜片钳记录shly分离心房和心室的心肌细胞在室温下。 </li><li>健康出现心肌转移到充满了限定体积的细胞外灌流液灌注室。 117 mM氯化钠，4 mM KCl中，1mM的KH 2 PO 4，4毫摩尔的NaHCO 3，1.7毫摩尔MgCl 2，3 mM的氯化钴，10mM的HEPES，10mM葡萄糖，和0.02 mM的河豚毒素（TTX），（pH值7.4） 。使用NaOH溶液调节pH值。 </li><li>使用正确的修补程序的夹紧设备（ 即 IX71倒置显微镜，一个Multiclamp的700B放大器，一个Digidata 1440A采集系统和PC，pClamp10.3软件（Molecular Devices公司））。 </li><li>使用补丁移液管电阻为3-5MΩ时充满了细胞内液含130毫米氯化钾，2毫摩尔MgCl 2，11 MM HEPES，EGTA 11毫米，5毫米的Na 2 ATP，0.4毫米的Na 2 GTP，5毫米的Na 2 CP和4.9毫摩尔的CaCl 2（pH7.2）中。使用KOH溶液pH值调节。 </li><li>唤起外向钾电流杜环4.5秒电压测试电位-60和+50 mV的10 mV递增，从20毫秒后，预脉冲至-40 mV的保持电位为-70 mV之间。 </li><li>确保泄漏电流&lt;100 pA的。 </li><li>对于解剖不同的K +电流的使用特异性抑制剂，如4 - 氨基吡啶（4-AP，ICN Biomedicals，尔湾，加利福尼亚州，美国），2 Heteropodatoxin（HpTx2; Alomone）或四乙基铵（TEA，Sigma公司）。联合解决方案应编制外沐浴液，并通过微尖“基线”的录音后，直接应用到最接近的可能的细胞附近。平衡应该被允许的“毒”录音的前2-3分钟。 </li><li>对于分析，在各个细胞的细胞尺寸（全细胞的膜电容）的电流幅值正常化。脱机使用pClamp10.3软件（Molecular Devices公司）或类似的软件进行数据分析。 </li></ol>

<ol>
	<li>Abbott, G. W., Goldstein, S. A., Sesti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+all+voltage-gated+potassium+channels+use+MiRPs?.">Do all voltage-gated potassium channels use MiRPs?.</a> Circ. Res. 88, 981-983 (2001).</li><li>Abbott, G. W., Sesti, F., Splawski, I., Buck, M. E., Lehmann, M. H., Timothy, K. W., Keating, M. T., Goldstein, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiRP1+forms+IKr+potassium+channels+with+HERG+and+is+associated+with+cardiac+arrhythmia.">MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia.</a> Cell. 97, 175-187 (1999).</li><li>Barry, D. M., Trimmer, J. S., Merlie, J. P., Nerbonne, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+voltage-gated+K++channel+subunits+in+adult+rat+heart.+Relation+to+functional+K++channels?.">Differential expression of voltage-gated K+ channel subunits in adult rat heart. Relation to functional K+ channels?.</a> Circ. Res. 77, 361-369 (1995).</li><li>Benjamin, E. J., Wolf, P. A., D'Agostino, R. B., Silbershatz, H., Kannel, W. B., Levy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+atrial+fibrillation+on+the+risk+of+death:+the+Framingham+Heart+Study.">Impact of atrial fibrillation on the risk of death: the Framingham Heart Study.</a> Circulation. 98, 946-952 (1998).</li><li>Bou-Abboud, E., Nerbonne, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+correlates+of+the+calcium-independent,+depolarization-activated+K++currents+in+rat+atrial+myocytes.">Molecular correlates of the calcium-independent, depolarization-activated K+ currents in rat atrial myocytes.</a> J. Physiol. 517 (Pt 2), 407-420 (1999).</li><li>Gaborit, N., Steenman, M., Lamirault, G., Le, M. N., Le, B. S., Lande, G., Leger, J., Charpentier, F., Christ, T., Dobrev, D., Escande, D., Nattel, S., Demolombe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+atrial+ion+channel+and+transporter+subunit+gene-expression+remodeling+associated+with+valvular+heart+disease+and+atrial+fibrillation.">Human atrial ion channel and transporter subunit gene-expression remodeling associated with valvular heart disease and atrial fibrillation.</a> Circulation. 112, 471-481 (2005).</li><li>Guo, W., Xu, H., London, B., Nerbonne, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+transient+outward+K++current+diversity+in+mouse+ventricular+myocytes.">Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes.</a> J. Physiol. 521 (Pt 3), 587-599 (1999).</li><li>Lundby, A., Ravn, L. S., Svendsen, J. H., Hauns, S., Olesen, S. P., Schmitt, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCNE3+mutation+V17M+identified+in+a+patient+with+lone+atrial+fibrillation.">KCNE3 mutation V17M identified in a patient with lone atrial fibrillation.</a> Cell Physiol. Biochem. 21, 47-54 (2008).</li><li>Ma, K. J., Li, N., Teng, S. Y., Zhang, Y. H., Sun, Q., Gu, D. F., Pu, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+KCNQ1+current+by+atrial+fibrillation-associated+KCNE4+(145E/D)+gene+polymorphism.">Modulation of KCNQ1 current by atrial fibrillation-associated KCNE4 (145E/D) gene polymorphism.</a> Chin Med. J. (Engl.). 120, 150-154 (2007).</li><li>McCrossan, Z. A., Abbott, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MinK-related+peptides.">The MinK-related peptides.</a> Neuropharmacology. 47, 787-821 (2004).</li><li>Pongs, O., Schwarz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ancillary+subunits+associated+with+voltage-dependent+K++channels.">Ancillary subunits associated with voltage-dependent K+ channels.</a> Physiol. Rev. 90, 755-796 (2010).</li><li>Ravn, L. S., Aizawa, Y., Pollevick, G. D., Hofman-Bang, J., Cordeiro, J. M., Dixen, U., Jensen, G., Wu, Y., Burashnikov, E., Haunso, S., Guerchicoff, A., Hu, D., Svendsen, J. H., Christiansen, M., Antzelevitch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain+of+function+in+IKs+secondary+to+a+mutation+in+KCNE5+associated+with+atrial+fibrillation.">Gain of function in IKs secondary to a mutation in KCNE5 associated with atrial fibrillation.</a> Heart Rhythm. 5, 427-435 (2008).</li><li>Ravn, L. S., Hofman-Bang, J., Dixen, U., Larsen, S. O., Jensen, G., Haunso, S., Svendsen, J. H., Christiansen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+of+97T+polymorphism+in+KCNE5+to+risk+of+atrial+fibrillation.">Relation of 97T polymorphism in KCNE5 to risk of atrial fibrillation.</a> Am. J. Cardiol. 96, 405-407 (2005).</li><li>Roepke, T. K., Abbott, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacogenetics+and+cardiac+ion+channels.">Pharmacogenetics and cardiac ion channels.</a> Vascul. Pharmacol. 44, 90-106 (2006).</li><li>Roepke, T. K., Kontogeorgis, A., Ovanez, C., Xu, X., Young, J. B., Purtell, K., Goldstein, P. A., Christini, D. J., Peters, N. S., Akar, F. G., Gutstein, D. E., Lerner, D. J., Abbott, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+deletion+of+kcne2+impairs+ventricular+repolarization+via+disruption+of+I(K,slow1)+and+I(to,f).">Targeted deletion of kcne2 impairs ventricular repolarization via disruption of I(K,slow1) and I(to,f).</a> FASEB J. 22, 3648-3660 (2008).</li><li>Sesti, F., Abbott, G. W., Wei, J., Murray, K. T., Saksena, S., Schwartz, P. J., Priori, S. G., Roden, D. M., George, A. L., Goldstein, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+polymorphism+associated+with+antibiotic-induced+cardiac+arrhythmia.">A common polymorphism associated with antibiotic-induced cardiac arrhythmia.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 10613-10618 (2000).</li><li>Temple, J., Frias, P., Rottman, J., Yang, T., Wu, Y., Verheijck, E. E., Zhang, W., Siprachanh, C., Kanki, H., Atkinson, J. B., King, P., Anderson, M. E., Kupershmidt, S., Roden, 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=Atrial+fibrillation+in+KCNE1-null+mice.">Atrial fibrillation in KCNE1-null mice.</a> Circ. Res. 97, 62-69 (2005).</li><li>Yang, Y., Xia, M., Jin, Q., Bendahhou, S., Shi, J., Chen, Y., Liang, B., Lin, J., Liu, Y., Liu, 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=Identification+of+a+KCNE2+gain-of-function+mutation+in+patients+with+familial+atrial+fibrillation.">Identification of a KCNE2 gain-of-function mutation in patients with familial atrial fibrillation.</a> Am. J. Hum. Genet. 75, 899-905 (2004).</li><li>Zeng, Z. Y., Pu, J. L., Tan, C., Teng, S. Y., Chen, J. H., Su, S. Y., Zhou, X. Y., Zhang, S., Li, Y. S., Wang, F. Z., 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=[The+association+of+single+nucleotide+polymorphism+of+slow+delayed+rectifier+K++channel+genes+with+atrial+fibrillation+in+Han+nationality+Chinese].">[The association of single nucleotide polymorphism of slow delayed rectifier K+ channel genes with atrial fibrillation in Han nationality Chinese].</a> Zhonghua Xin. Xue. Guan. Bing. Za Zhi. 33, 987-991 (2005).</li><li>Zhang, M., Jiang, M., Tseng, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=minK-related+peptide+1+associates+with+Kv4.2+and+modulates+its+gating+function:+potential+role+as+beta+subunit+of+cardiac+transient+outward+channel?+Circ.">minK-related peptide 1 associates with Kv4.2 and modulates its gating function: potential role as beta subunit of cardiac transient outward channel? Circ.</a> Circ. Res. 88, 1012-1019 (2001).</li></ol>

我们什么都没有透露。

随着越来越多的发展遗传工程小鼠心脏功能和心肌病变相关的基因缺失株的研究也有一个专门的方法来研究在体外的特定基因缺失的影响越来越大的兴趣。在我们的实验室中，我们研究一个家庭的钾通道附属亚基对心肌复极的角色。的KCNE基因包括一个家庭的5个基因（KCNE1 - 5），发挥重要的作用在人的心室和心房复极11，15。KCNEs是一个跨膜域千伏通道辅助亚基，...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td></tr><tr><td>河豚毒素</td><td> Alomone </td></tr><tr><td> 4 - 氨基吡啶</td><td> ICN Biomedicals </td></tr><tr><td> Heteropodatoxin 2 </td><td> Alomone </td></tr><tr><td>四乙基</td><td>西格玛化学品</td></tr><tr><td>胶原酶2 </td><td>沃辛顿</td></tr></tbody></table>

isolation and kv channel recordings in murine atrial and ventricular cardiomyocytes

KCNE基因编码的钾离子通道Kv通道α亚单位形成异源复合物的辅助亚基修改其功能特性的一个小型的家庭。 KCNE基因的突变已发现的心律失常，如长QT综合征和/或心房纤维颤动患者。但是，确切的分子，导致这些疾病的病理生理仍然是难以捉摸的。在以前的研究中，大多的疾病，导致这些基因突变的电生理特性研究中的异源表达系统，我们不能确定是否可以直接翻译成原生的心肌细胞的影响。因此，在我们的实验室中，我们使用了不同的方法。我们直接的影响研究中的KCNE基因缺失孤立的基因敲除小鼠的心肌细胞电生理-一个独特的技术，我们在这个问题上的杂志的可视化实验描述。从遗传学的心或LLY的设计KCNE小鼠迅速切除，主动脉插管Langendorff装置由安装在。心肌组织中的游离Ca 2 +的约束，EGTA，由一个专门的低Ca 2 +缓冲液胶原酶逆行灌注的冠状动脉和心肌细胞的分离，然后实现。心房，然后可以自由的右心室壁和左心室的显微技术分离。钙然后慢慢地在洗涤过程中的多个步骤，包括乳鼠心肌细胞。健康的外表，没有自发性收缩的心房和心室肌细胞，然后立即进行电生理分析内前6个小时后，隔离膜片钳技术或其他生化分析。

从遗传工程小鼠的功能研究特定基因在体外的兴趣成年小鼠心肌细胞的分离已经成为一个强大的工具，以进一步了解心脏的病理生理机制。这种方法是目前使用的只有一小，但越来越多的全球基础科学实验室。但是，隔离成人心室鼠心肌细胞的可能会非常棘手，需要做彻底和反复的由经验丰富的手。 图1显示了新鲜分离的典范心房和心室的心肌细胞。心室肌细胞，我们建议只使...

Watch this Scientific Journal Video about Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes at JoVE.com

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

Kv通道功能障碍有关的心律失常。为了研究的分子机制，我们利用一个系统的协议，用于隔离的心房和心室的心肌细胞钾通道附属亚基基因敲除小鼠等心律失常。乳鼠心肌细胞，就可以立即用于细胞电生理研究，生化或免疫荧光法（IF）检测。

在小鼠的心房和心室肌细胞的分离及钾离子通道的录音

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

isolation-kv-channel-recordings-murine-atrial-ventricular

Research

JoVE Journal

Biology

13.3K 次观看.  Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine (MDC). Kv通道功能障碍有关的心律失常。为了研究的分子机制，我们利用一个系统的协议，用于隔离的心房和心室的心肌细胞钾通道附属亚基基因敲除小鼠等心律失常。乳鼠心肌细胞，就可以立即用于细胞电生理研究，生化或免疫荧光法（IF）检测。

视频: Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

科研

生物学

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

分离新生小鼠心肌文化和

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

重组脂膜的结构和功能研究的钾离子通道

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

鼠心肌细胞的分离及生理分析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

隔离成年小鼠心肌细胞的细胞信号传导和文化及<em&gt;在体外</em&gt;心肌肥厚

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

培养HL-1心房肌细胞单层的电压和钙双通道光映射

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

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 ...

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.