作者感谢Pooneh巴盖尔博士和Erika韦斯科特博士在准备稿件的编辑意见。这项工作是由国家心脏，肺和美国国立卫生研究院血液研究所在获奖号码R37-HL041026和R01-HL086483到SSS和F32-HL107050到MJS支持。此内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

1。制备设备：流动室，移液器和解决方案<ol><li>流室：使用流室与底部24毫米×50毫米的玻璃盖玻片superfuse孤立的内皮管（ 见图1A）。前装配该室，与两个3％的HCl和70％的乙醇洗涤盖玻片，漂洗用超纯水，并用氮气干燥气体。 </li><li>移液器：三种不同类型的移液器在协议期间使用：插管，研制，并钉扎。同时插管和钉扎移液器可以在实验的前一天进行，但研磨移液管应，因为它需要在容器的直径的知识，以大小的管腔适当的日子。 <ol><li>插管移液器：为了冲洗红细胞从管腔，使用微量拉马以己之长，较细的一端拉硼硅玻璃毛细管。中断与镊子的外径端部〜30％乐SS比动脉（〜50-80微米），以及火抛光笔尖光滑（参见图1B）。是有帮助的角度也将移液管的尖端至约45°。 </li><li>研磨移液器：要机械地分离的平滑肌细胞和外膜的内皮管，由硼硅玻璃毛细管作研制过程移液器。制备毛细管如上，并打破端与钳。提示：使用玻璃刻痕装置，以使其更易于清洁破坏的磨碎玻璃移液管的较厚的壁。打破移液器上的实验中，约10-20微米比从其中内皮管将被隔离的容器的外径大的日子中确定的内直径。火抛光的断头，直到顺利（参见图1C）。 </li><li>钢钉移液器：为稳定流室中的内皮管，使移液管钉来固定内皮吨宇部对着所述腔室的底部的盖玻片。拉玻璃毛细管中相同的方式作为插管移液器。然后断开与钳两端的直径约为100μm，而火抛光，直到提示是密封的，圆润光滑（ 见图1D）。注意：如果穿针吸管尖不完全密封，流​​体将进入腔和可能混淆实验结果。 </li></ol></li><li>解决方案：准备在超纯所有的解决方案（18.2MΩ）H 2 O和通过0.2微米的过滤消毒的。确保解决方案的渗透压为285-300毫渗量之间。解决方案保持良好的约两周贮存于4℃时， <ol><li>剥离液：在腹壁上动脉的解剖过程中使用的盐溶液是由（以毫摩尔/升）：137氯化钠，氯化钾5，1的MgCl 2，10 N-2-羟乙基哌嗪-N&#39;-2-乙磺酸（ HEPES），10葡萄糖，0.01硝普钠（SNP）的ð0.1％牛血清白蛋白（BSA），pH为7.4。 NO供体SNP包括帮助松弛平滑肌细胞，从而使他们更容易导管插入动脉和研磨过程中促进平滑肌细胞去除。 </li><li>解离液：内皮管的解离过程中使用的盐溶液是由（以毫摩尔/升）：137氯化钠，氯化钾5，1的MgCl 2，10 HEPES，10葡萄糖，2的CaCl 2，和0.1％BSA，用的pH为7.4。以1毫升等分的该缓冲液中，添加：0.62毫克/毫升的木瓜蛋白酶，1.5毫克/毫升胶原酶和1.0毫克/毫升二硫赤藓醇。由于来自不同供应商的酶可以有不同的酶活性水平，包括在材料表中引用那些用于这项工作的产品编号。注：在协议双方分离缓冲液和分离缓冲液的酶是用来在不同的步骤。 </li><li>表面灌流液：用于superfusin的生理盐溶液（PSS）克分离的内皮管是由（以毫摩尔/升）：137氯化钠，氯化钾5，1的MgCl 2，10 HEPES，10葡萄糖和2的CaCl 2，pH为7.4。 </li><li>开始外科手术前准备缓冲区和解决方案。从4°C和等分加入50ml缓冲解剖取出溶液到200ml锥形瓶中并置于冰上。此外，分装50毫升解离缓冲无酶，并将其放置在台面上，以平衡至室温。从4℃取出灌流液，并使其平衡至室温在台面上。 </li></ol></li></ol> 2。动物的制备确保所有程序和协议，涉及动物是符合健康指南的国家研究院实验动物的护理和使用 。这里所描述的程序批准了大学的动物护理和使用委员会密苏里州。 <ol><li>使用雄性或雌性小鼠至少9周龄和独立研究每个。麻醉小鼠通过腹腔（IP）注射戊巴比妥钠（60毫克/千克）。提示：稀释的戊巴比妥至10毫克/毫升的无菌盐水注射前降低了过量的可能性。 </li><li>麻醉前注射戊巴比妥后立即危及温度调节，所以保持鼠标温暖。上加热板（校准到〜37°C）的顶部采用了金属载体（通风铝篮）的作品很好地保持鼠标的温度稳定。或者，使用烤灯，小心的从鼠标适当的距离来定位它。监视鼠标每次5-10分钟，直到麻醉的适当水平达到（缺乏撤出到脚趾或尾巴捏）。的补充剂量后15-20分钟是必需的。最好是循序渐进，注意避免过量，特别是与肥胖，年龄，或动物S的方式强调。 </li><li>通过小心地用小宠物电推剪剃它从腹侧取出头发。应特别注意注意避免外伤的动物。它有利于从一个地区跨越腋下到耻骨区中删除所有的头发。删除任何松散执着的头发用新鲜的酒精棉签。 </li><li>放置在它的后面鼠标，躺在既脱颖而出，后肢散开，露出尽可能多的腹壁越好。如有必要，使用实验室的胶带沾住腿在一条传播鹰的方式。 </li></ol> 3。在腹壁上动脉的分离<ol><li>通过皮肤只是丹田的区域的上方做一个小切口。确保只有皮肤，同时避免损坏底层腹部肌肉被切开;横向延伸在每个方向上的切口出到各自的后肢。继续切口吻侧沿腹侧中线到肋笼的顶部和n以每个方向横向延伸的切口，以各自的前肢。 </li><li>轻轻提起皮肤并用剪刀切断结缔组织保持皮肤下面的肌肉，从而露出腹部肌肉的整个表面上（ 见图3A）。灌溉外露肌肉用室温0.9％的生理盐水溶液。 </li><li>由于海水的体积小，放置动物体视显微镜作进一步的程序下方。抬起脂肪垫位于与钳胸骨的底部，并通过它使一个切口。继续沿着底部肋切口，确保不要剪得太深进入皮下组织。战略环评应该是可见的;小心，避免损坏。一旦切口完成后，灌溉用室温下0.9％的生理盐水溶液中的暴露组织。 </li><li>后骨骼肌的顶层已经缩回，大海和下面的肌肉很容易识别。提示：请注意长度隔离延长内皮管（沿其轴线在隔离的缩短），接近其原始长度前海在体内 。使用镊子和剪刀，小心地切除爪哇下方的薄肌肉层。 </li><li>使用倾斜的镊子，通过海下6-0丝线缝合的长度和结扎动脉及其邻近静脉，以保持它的加压和血液保留血管管腔内，以在后续的分离和清洗方便的可视化。 </li><li>重复的海结扎程序上的对侧。 </li><li>一旦双方的策略性已被结扎，使沿腹部肌肉分开各边的中线切口。 </li><li>横向延伸的切口在每一个方向上进行的皮肤，然后继续切口垂直沿外边缘完全从主体分离的腹部肌肉。避免损伤血管的爪哇美联储维持加压管腔。 </li><li> Ç用ut爪哇结扎以上，以保持密封，然后将分离的肌肉和血管中的50ml烧杯中含有10毫升4℃解剖缓冲区。 </li><li>重复上述步骤，腹部的另一侧，并培育组织的解剖缓冲10分钟。 </li><li>放置在冷藏（4℃）含夹层腔剥离缓冲液含有爪哇的腹肌（ 见图3B）。解剖腔由一个培养皿底部一层的Sylgard（聚二甲基硅氧烷[PDMS]）的。 </li><li>用昆虫针（0.15 mm）的，舒展隔离爪哇和腹部的肌肉出近似体内的长度（如上所述），并将其固定到的Sylgard。提示：定向肌肉，从而面临腹膜薄层在顶部，使爪哇随时可见使用透。 </li><li>利用精细显微切割工具及从结扎对上游现场工作下游终端，清除海从其配对静脉和周围组织，直到第一个主要分支站点（1-2厘米）。然后通过切割其正上方的分支站点和正下方的结扎切除大海。 </li><li>要清除血液血管管腔内保留，导管插入大海，与冰冷的解剖缓冲冲洗。采用了一块硅橡胶管，附上吸管插管的后端到5毫升注射器含冰冷的解剖缓冲和固定微量的显微操作的夹层腔内放置其尖端导管插入大海。 </li><li>一旦所有的红细胞被冲出，从插管吸管取出海，然后提起吸管出了夹层菜。 </li><li>切海成较小的段，每段1-3毫米的长度。这些短段促进内皮管的隔离。 </li></ol> 4。内皮管的隔离<ol><li>填补12毫米×75毫米的玻璃邪教URE管的中途与冰冷的解剖缓冲区。使用倾斜的镊子，转移动脉块放入培养管置于冰上。此时，结合消化酶与解离缓冲液1毫升中单独12毫米×75mm的培养管中的最终体积（见1.3.2解决方案）并预热该溶液至37℃加热块。 </li><li>而分离缓冲液和酶正在升温，小心翼翼地从培养管含留下的血管段小体积吸解剖缓冲区。 </li><li>轻轻地添加室温下解离缓冲无酶洗去剩余的剥离缓冲。提示：添加的解离缓冲缓慢，使得动脉件留在培养管的底部，以节省时间等待他们重新定居。提高缓冲温度超过从冰（4℃）多个步骤，以室温（〜24℃）至37℃，以减少震动。 </li><li>小心吸了dissoc从培养管iation缓冲液，再次是一定要留下含有血管区段小体积。 </li><li>取出预热解离缓冲液从加热块的酶，在加入1ml酶溶液转移至包含血管区段的培养管中，然后将培养管后面的加热块。在37°C为30分钟。 </li><li>在用酶温育，准备研磨移液管，与矿物油回填它和将其固定在其被安装在一个显微微量调节。然后缩回微量柱塞，以填补吸管2 NL的分离缓冲液和枪头过流室位置。 </li><li>在30分钟温育结束时，从加热块取出培养管中，小心地吸出缓冲如上。 </li><li>用4ml室温下解离缓冲液冲洗动脉段。注意：它不再是必要的，以保持容器SEGM已废除在培养试管底部;干扰动脉件实际上有助于确保剩余的酶有效地除去。 </li><li>转让一艘船段流室轻轻用1毫升微量吸它，并把它放入流室用1毫升分离缓冲液中。 </li><li>利用含有微量的显微操作器，定位在研磨移液管的附近的血管段的一端的一角。 </li><li>吸出血管区段（500 NL撤回）进入捣碎吸管慢慢（225升/秒），以便不引起机械应变的内皮细胞，然后将其弹出返回到腔室中。如果消化成功，平滑肌细胞和外膜将会从管内皮细胞解离。 </li><li>重复研碎，直至所有的平滑肌细胞解离。要知道，过度研磨（4倍以上）会损坏内皮细胞，使它们反应迟钝激动剂。提示：使用移液管研碎，将外膜远离内皮管尽快，以防止它缠结的管。注意：使用移液管研碎，分离的内皮管可吸出并转移到一个单独的腔室中。 </li><li>定位内皮管在中心的流动腔室的，沿流动方向对齐，并取出捣碎吸管。 </li><li>在显微操作安全的钉扎移液器安装在所述流动室的任一端，并在内皮细胞管的相应端部定位自己的技巧。 </li><li>降低一个钉扎吸管在管的一端连接到其按压在腔室的底部。位置以同样的方式，第二锁定吸管在所述管的另一端。注意：所述钉扎移液器位置是固定的管（它们放置在管的端部越远），并允许多个成像/实验区（将它们更接近该管的端部）之间的一个折衷。定位吨下摆〜从所述管的每个端部50μm的效果很好。正如钉扎吸管接触管，慢慢沿管的轴线缩回它，从而延长其近似体内长度，然后按下移液器针对该室底部，以确保所述管。 </li><li>开始的灌流液的流动，并允许内皮管到休息至少20分钟，以平衡并附着（参见图3C）。使用蠕动泵跨内皮管保持恒定的流体流量（灌流）。注意：钢钉移液器可以一次内皮管附着在流量腔室的底部（〜25分钟）或留在适当位置，以确保内皮管变得不脱落除去。 </li></ol> 5。染料加载和钙成像<ol><li>为了形象化细胞内钙离子反应，加载内皮管荧上午04时染料在室温（〜24℃）。停在洗澡流体流动，并添加荧光-4AM到10μM的终浓度在腔室中。等待10分钟，以使染料进入细胞，然后superfuse的内皮管的新鲜PSS 20分钟，以确保细胞外染料被除去，而细胞内的染料已完全脱酯化。 </li><li>进行钙成像。对于这项工作，直立显微镜（参见图2）装备有一个旋转的圆盘共焦成像系统被用来在环境室温。用491 nm激光激发荧光钙染料和使用增强的数码相机采集图像（在500-550 nm发射）。 </li></ol>

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J., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+electrical+conduction+along+endothelial+tubes+of+resistance+arteries+through+Ca2+-activated+K++channels.">Tuning electrical conduction along endothelial tubes of resistance arteries through Ca2+-activated K+ channels.</a> Circ. Res. 110, 1311-1321 (2012).</li><li>Bagher, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+intravascular+pressure+activates+endothelial+cell+TRPV4+channels,+local+Ca2++events,+and+IKCa+channels,+reducing+arteriolar+tone.">Low intravascular pressure activates endothelial cell TRPV4 channels, local Ca2+ events, and IKCa channels, reducing arteriolar tone.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 18174-18179 (2012).</li><li>Faraci, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+vascular+effects+of+angiotensin+II:+new+insights+from+genetic+models.">Cerebral vascular effects of angiotensin II: new insights from genetic models.</a> J. Cereb. Blood Flow Metab. 26, 449-455 (2006).</li><li>Emerson, G. G., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+coupling+between+endothelial+cells+and+smooth+muscle+cells+in+hamster+feed+arteries:+role+in+vasomotor+control.">Electrical coupling between endothelial cells and smooth muscle cells in hamster feed arteries: role in vasomotor control.</a> Circ. Res. 87, 474-479 (2000).</li><li>Ledoux, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+architecture+of+inositol+1,4,5-trisphosphate+signaling+in+restricted+spaces+of+myoendothelial+projections.">Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 9627-9632 (2008).</li><li>Tran, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Ca2++wavelets+and+the+induction+of+myoendothelial+feedback.">Endothelial Ca2+ wavelets and the induction of myoendothelial feedback.</a> Am. J. Physiol. Cell Physiol. 302, 1226-1242 (2012).</li><li>Nausch, L. W., 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=Sympathetic+nerve+stimulation+induces+local+endothelial+Ca2++signals+to+oppose+vasoconstriction+of+mouse+mesenteric+arteries.">Sympathetic nerve stimulation induces local endothelial Ca2+ signals to oppose vasoconstriction of mouse mesenteric arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 302, 594-602 (2012).</li><li>Duza, T., Sarelius, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+endothelial+cell+Ca2++in+response+to+mouse+cremaster+muscle+contraction.">Increase in endothelial cell Ca2+ in response to mouse cremaster muscle contraction.</a> J Physiol. 555, 459-469 (2004).</li><li>Colden-Stanfield, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bradykinin-induced+increases+in+cytosolic+calcium+and+ionic+currents+in+cultured+bovine+aortic+endothelial+cells.">Bradykinin-induced increases in cytosolic calcium and ionic currents in cultured bovine aortic endothelial cells.</a> Circ. Res. 61, 632-640 (1987).</li><li>Brunner, F., Kukovetz, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioligand+binding+to+muscarinic+receptors+of+bovine+aortic+endothelial+cells.">Radioligand binding to muscarinic receptors of bovine aortic endothelial cells.</a> Br. J. Pharmacol. 102, 373-380 (1991).</li><li>Tracey, W. R., Peach, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+muscarinic+receptor+mRNA+expression+by+freshly+isolated+and+cultured+bovine+aortic+endothelial+cells.">Differential muscarinic receptor mRNA expression by freshly isolated and cultured bovine aortic endothelial cells.</a> Circ. Res. 70, 234-240 (1992).</li><li>Adams, D. J., Hill, M. 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在这里，我们描述的内皮管从海上隔离和使用该制剂的形象化的Ca 2 +的构内皮细胞内信号事件。这个过程被改编从一个最初开发分离的内皮管从仓鼠提睾肌8的小动脉。利用这里提出的技术的微小变化，我们已经分离出了各种血管床内皮管，包括：仓鼠拉钩肌肉和颊的饲料袋动脉动脉10，鼠标腹壁上动脉6,7,9，小鼠肠系膜和脑动脉和微淋巴管（未发表意见;引用包括隔离肠系膜血管12和脑血管13）。隔离内皮管新血管床，可能需要修改原来的协议。如果分离是不成功的，最好是开始通过改变消化时间：增加的消化时间，如果内皮管是难以从平滑肌细胞和外膜分离和减少消化时间，如果内皮管不保持不变。如果改变消化时间是不成功的，调节消化酶的浓度或消解温度应该受到审判。另一种选择是降低研磨移液管，这增加了剪切力去除平滑肌细胞的内直径。增加液体喷射速度也可以尝试了同样的效果，但更可能是损坏的制备。应当认识到，也未必可以分离完整的内皮管从其中内皮细胞没有得到很好的相互连接通过连接蛋白血管。 最初执行使用手摇式移液器8平滑肌细胞以下酶消化分解。使用microsyr我们修订了研磨过程铰链系统，这使再内皮管一致的隔离（至3毫米），6。当使用这个系统时，移液管研碎回填与​​矿物油以提供所述活塞控制流体运动和含有血管区段的PSS之间的连续的流体柱。流体一起的微量活塞产生剪不断的为容器中的恒定的驱动力不可压缩是通过枪头用力。与此相反，手技术通常用于解离的细胞（ 如挤压橡胶球在巴斯德吸管的端部）涉及空气的压缩，它引入变异的驱动压力，并由此剪切在枪头作用。 有多种优点管子准备在微血管内皮学习功能。第一个是隔离工艺提供了内皮细胞的独特的同质细胞原生种群和流畅亩SCLE细胞。由于内皮细胞保持物理连接成管，它们是由个体平滑肌细胞，从而保持一个“C”的结构，如果它们保持研磨过程中放宽鲜明。因此，各类型的细胞很容易区分， 如获取样本，如实时PCR和免疫组织化学10分子技术的时候。因为多个管从单个容器中（或从作为完成的海上船只双边）分离，分子数据和功能的数据可以从一个给定的动物的同一容器中获得。因此，分子的表达特征可与微血管内皮的功能行为。此外，内和细胞间的信号传导事件固有的微血管内皮可以从血流动力学的力（压力，流量），在血流中（ 如激素）进行血管活性剂的影响来解决独立的，或者从周围的平滑肌细胞（ 如60;通过myoendothelial耦合14-16），神经17，或组织实质18。 重要的是，由于内皮细胞是新鲜从指定的微血管孤立的，没有改动的表型在其他方面与内皮细胞培养19-21关联。例如，培养的内皮细胞失去毒蕈碱受体的表达，从而改变他们的钙信号轮廓。此外，内皮细胞的电生理特性可以在文化22改变。因为个别内皮仍通过官能的间隙连接通道相连，内皮管呈现为研究细胞6,7之间的电与Ca 2 +信号传导的理想模型。还应当认识到，分离平滑肌细胞可容易地研究了膜片钳技术的互补数据相关的微血管功能23。 一个关键限制的内皮管米ODEL是准备随温度升高的不稳定。虽然我们从海准备已经证明在环境室温（〜24℃）和32℃数小时稳定，健康，形态和功能退化发生在不到一个小时，在37℃9。的内皮管的第二个重要的限制是myoendothelial路口和其固有的信令结构域是不可或缺的未受影响的血管壁14-16 EC功能的丧失。还应当认识到，虽然更长的管使细胞间信号传导来进行研究在相对 ​​大的距离6，制备管中是复杂的，因为它们变得更长，因为它完全解离周围的平滑肌细胞和外膜变得更加困难。我们已经发现，试管超过1-2毫米长，也更加难以定位，并在流动室固定。与此相反，而较短的管子（ 如 &lt;1毫米）ENA竹叶提取的Ca 2 +和电信号来进行研究8，它们是很难灌流在流动室中维持。最后，即使管的最优隔离两侧，没有足够的材料为使用Western印迹蛋白表达的传统的量化，尽管免疫标记提供了蛋白质表达和定位的指标。尽管有这些限制，内皮管代表提供新的洞察体内微血管内皮细胞功能的机制，新的制备。

在这个协议中，我们描述了从小鼠SEA内皮细胞管的隔离。策略性源于胸廓内动脉和供应含氧血液到前腹部肌肉。我们与大海为模型的工作，是由于它提供了相对较长，不分枝的微血管段，非常适合用于研究细胞间的信号事件的内皮细胞的治理和协调平滑肌细胞松弛作用相关。对于那些有兴趣的组织比骨骼肌等，我们发现这种技术可以很容易地适于获得内皮管脑，肠和淋巴系统的微血管。 这种方法的主要特点是微血管内皮完整长度（1-3毫米）是孤立的，抵押在流室的玻璃盖玻片中，他们灌流PSS，以及成像的Ca 2 +信号或7-9用于刺穿电信号6,8。内的流动室中，一个管的上半部分是暴露在表面灌流液，并响应实验的干预，而下半部分（与盖玻片接触）被保护并保持静态。除了 ​​研究内和细胞间的Ca 2 +信号的事件，内皮管可用于定量实时PCR来评估基因表达6,10，免疫组织化学研究蛋白质表达6,10，和电生理研究，以确定生物物理特性的电传导6,11。 有几个好处内皮管准备学习内皮功能。第一个问题是隔离工艺提供了两种细胞群之间的一个简单的区分：内皮细胞（其保持在管形成）和平滑肌细胞（单独分离;通常为“C”形，当放宽）。这允许选择性取样和各自的细胞的能力，血管功能的贡献相关的独特性能研究。第二，这种模式使信号事件固有的内皮，独立于血流的影响下，周围平滑肌，神经和实质的分辨率。第三，内皮细胞进行了研究，而新鲜分离的，从而避免了在基因或细胞培养相关蛋白表达的改变。

<table border="0" cellpadding="0" cellspacing="0" width="632">
	<colgroup>
		<col span="4" />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:159px;">Sodium Chloride</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:159px;">S642-212</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:159px;">Potassium Chloride</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:159px;">P9541</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:159px;">Magnesium Sulfate</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:159px;">M2643</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:159px;">Calcium Chloride</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:159px;">C1016</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:159px;">Sodium Nitroprusside</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:159px;">431451</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:159px;">Bovine Serum Albumin</td>
			<td style="width:159px;">USB Products</td>
			<td style="width:159px;">9048-46-8</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:159px;">Papain</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:159px;">P-4762</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:159px;">Collagenase</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:159px;">C-8051</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:159px;">Dithioerythritol</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:159px;">D-8255</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:159px;">Boroscilicate Glass (Cannulation and Pinning Pipettes)</td>
			<td style="width:159px;">Warner Instruments</td>
			<td style="width:159px;">G150-4</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:159px;">Boroscilicate Glass (Trituration Pipettes)</td>
			<td style="width:159px;">World Precision Instruments</td>
			<td style="width:159px;">4897</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:159px;">Nanoliter Injector Microsyringe</td>
			<td style="width:159px;">World Precision Instruments</td>
			<td style="width:159px;">B203MC4</td>
			<td style="width:159px;">Updated to Nanoliter 2010 from Nanoliter 2000</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:159px;">Micro4 Controller</td>
			<td style="width:159px;">World Precision Instruments</td>
			<td style="width:159px;">SYS-MICRO4</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:159px;">12 mm x 75 mm Culture Tubes</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:159px;">14-961-26</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:159px;">3-Axis Micromanipulator</td>
			<td style="width:159px;">Siskiyou Corp</td>
			<td style="width:159px;">DT3-100</td>
			<td style="width:159px;">Holding and positioning the pinning pipettes</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:159px;">Flow Chamber</td>
			<td style="width:159px;">Warner Instruments</td>
			<td style="width:159px;">RC-27N</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:159px;">100-1,000 &#181;l Pipette</td>
			<td style="width:159px;">Eppendorf</td>
			<td>3120000062</td>
			<td style="width:159px;">For transferring digested vessel segment to the flow chamber</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:159px;">1 ml Pipette Tips</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:159px;">02-707-405</td>
			<td style="width:159px;">For transferring digested vessel segment to the flow chamber</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:159px;">Upright Microscope</td>
			<td style="width:159px;">Olympus</td>
			<td style="width:159px;">BX51W1</td>
			<td style="width:159px;">The actual microscope is up to the user</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:159px;">Spinning Disc Confocal System</td>
			<td style="width:159px;">Yokogawa</td>
			<td style="width:159px;">CSU-X1</td>
			<td style="width:159px;">Confocal imaging is optional</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:159px;">XR/TURBO EX Camera</td>
			<td style="width:159px;">Stanford Photonics</td>
			<td style="width:159px;">XR/TURBO EX</td>
			<td style="width:159px;">Ideal for our needs; may vary with user</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:159px;">Piper Control Software</td>
			<td style="width:159px;">Stanford Photonics</td>
			<td style="width:159px;"></td>
			<td style="width:159px;">Imaging software for TURBO EX camera</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:159px;">Stereo Microscrope</td>
			<td style="width:159px;">Leica</td>
			<td style="width:159px;">MZ8</td>
			<td style="width:159px;">may vary with user</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:159px;">Sylgard</td>
			<td style="width:159px;">Dow Corning</td>
			<td style="width:159px;">184</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:159px;">Microdissection Scissors</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td style="width:159px;">15003-08</td>
			<td style="width:159px;"></td>
		</tr>
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isolation of microvascular endothelial tubes from mouse resistance arteries

血流的阻力血管的控制调节氧的供给量和营养物质伴随着副产物去除代谢的，其实例由运动的骨骼肌。内皮细胞（ECs）行中的所有阻力血管的内膜和服务控制直径的关键作用（ 如内皮依赖性血管舒张），并组织血流量，从而，大小和分布。由内皮细胞血管阻力的调节是通过细胞内Ca 2 +信号，从而导致产自体有效物质扩散（ 如氧化氮和花生四烯酸代谢产物）的影响1-3和超极化4,5是引起平滑肌细胞松弛。因此，了解内皮钙离子信号的动态是理解执政血流控制机制的关键一步。隔离内皮管消除了与基本法有关的混杂变量OOD在血管腔，并与周围的平滑肌细胞和血管周围的神经，否则影响EC的结构和功能。在这里，我们提出从腹壁上动脉（SEA）使用这个容器优化的协议内皮管的隔离。 为了隔离从麻醉小鼠内皮管的，海是连接在原地 ，保持血液的血管管腔内（便于清扫过程中可视化它），以及腹肌的整个工作表被切除。爪哇被解剖不受周围骨骼肌纤维和结缔组织，血液从腔冲洗，温和的酶消化进行到使除去外膜，用温和的研磨神经和平滑肌细胞。这些新鲜分离的完整内皮的制剂保留其天然形态，与个别内皮其余功能上彼此耦合，能够传送化学和电气SI信号无法intercellularly通过间隙连接6,7。除了提供新的洞察钙信号和膜生物物理学，这些准备工作使原生内微血管内皮细胞基因表达和蛋白定位的分子生物学研究。

钙反应可以使用乙酰胆碱新鲜分离的内皮管（ACH）来启动。在这部影片中，内皮管装有10微米荧光-4 AM的刺激与1微米乙酰胆碱（ 电影1）的灌流。 <a href="https://www.jove.com/files/ftp_upload/50759/SochaandSegal_movie1.avi" target="_blank">点击这里观看电影</a> 。染料被激发在491 nm，发射是从500-550 nm的使用增强电荷耦合器件摄像机记录。图像栈后天在120帧/秒的25秒期间内，然后可以进行平均（ 例如 ，以40帧/秒）。代表性的荧光图像随着时间的推移示于图4。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/50759/50759fig1.jpg" width="500px" /> 图1。流动腔和血管内皮管隔离期间使用移液器。 </stroNG&gt; A）一拉（ 左 ）和一把拉住，破碎，抛光和有角度（ 右 ）移液管插管的流动室装配有24毫米×50毫米的玻璃盖玻片为基数B）的例子。此吸移管被用于从动脉的管腔以下夹层冲洗红细胞。一个拉（ 左 ）和拉，破碎抛光（ 右 ）研磨吸管比例尺= 200微米。C）的例子。此移液管的内部直径是由动脉的外径来确定。一个拉（ 左 ）和拉，破碎抛光（ 右 ）钉扎吸管的比例尺= 200微米D）的例子。此吸移管的前端被四舍五入并完全密封，以确保内皮管中流动室。比例尺= 200微米。 <a href="https://www.jove.com/files/ftp_upload/50759/50759fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <p类=“jove_content”FO：保持together.within页=“总是”&gt; <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/50759/50759fig2.jpg" width="500px" /> 图2。旋转盘共聚焦显微镜的Ca 2 +信号的成像A）用于钙成像立式显微镜B）（A）与（二）增强型电荷耦合器件摄像机。℃）浸泡目标（ 一 ）63X（共焦旋转的光盘装置NA = 1）和（b）20X（NA = 0.5）用于成像。 <a href="https://www.jove.com/files/ftp_upload/50759/50759fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/50759/50759fig3.jpg" width="500px" /> 图3。腹部肌肉的分离和腹壁上动脉，一个 </st荣&gt;）麻醉小鼠腹部肌肉暴露和灌溉用室温0.9％生理盐水。红色箭头标记下脂肪垫的地点在哪个各自的战 ​​略环评进入每个腹直肌（ 即血管的地方，应结扎）。比例尺= 1厘米。 二）隔离腹肌（单边）和海中的解剖腔的显微解剖固定了。红色箭头表示海的第一个主要分歧点（ 即在该清洗容器的停止点）。从海比例尺= 1厘米。C）隔离内皮细胞管型。内皮管以下1小时在室温下孵育的微分干涉相衬图像。内皮管灌流灌流缓冲液以3毫升/分钟。比例尺= 50微米。 <a href="https://www.jove.com/files/ftp_upload/50759/50759fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 图4。钙荧光内皮细胞管型 。内皮管的响应与刺激1微米乙酰胆碱A）的刺激。前B荧光）内皮管的荧光在乙酰胆碱给药时代表荧光图像，T = 0秒。 三）内皮细胞荧光管在t = 5秒。 D）内皮管的荧光在t = 10秒E）内皮细胞荧光管在t = 15秒F）内皮细胞荧光管在t = 20秒。比例尺= 40微米。 <a href="https://www.jove.com/files/ftp_upload/50759/50759fig4highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries at JoVE.com

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

我们提出了在原生的，完整的微血管内皮细胞可视化和操纵钙信号的准备。内皮管新鲜小鼠抗动脉供应骨骼肌保留在体内的形态和动态信令内和之间的相邻细胞分离。内皮管可从其它组织和器官的微血管来制备。

从鼠标阻力动脉分离微血管内皮管

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

isolation-microvascular-endothelial-tubes-from-mouse-resistance

Research

JoVE Journal

Biology

15.5K 次观看.  University of Missouri. 我们提出了在原生的，完整的微血管内皮细胞可视化和操纵钙信号的准备。内皮管新鲜小鼠抗动脉供应骨骼肌保留在体内的形态和动态信令内和之间的相邻细胞分离。内皮管可从其它组织和器官的微血管来制备。 

视频: Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

瓣膜内皮细胞的分离

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

科研

生物学

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 ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

RNA-seq的凝血酶处理和控制人肺微血管内皮细胞的转录组分析

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

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 and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

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

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

一氧化氮和超氧化物歧化酶恩的人脸检测在完整的动脉内皮细胞层

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

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

鼠标冠状动脉内皮细胞的分离

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

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

微型猪原主主动脉内皮细胞的隔离与培养

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

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

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

小鼠原发性肝正弦内皮细胞的分离与表征

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

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

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

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

从小鼠颈动脉管腔中分离内皮细胞以进行单细胞多组学实验

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