这项工作是由卫生（HL115578到A牧野）全国学院的资助。

对小鼠研究批准了亚利桑那大学的机构动物护理和使用委员会（IACUC），并根据美国国立卫生研究院（NIH）的指导方针进行。 注:第1，第2和第3应的实验，安装前进行了一天。 1.准备解决方案<ol><li> CD31缓冲液（50ml）中<ol><li>加入50mL 1×磷酸盐缓冲盐水（PBS），以50毫升管中。权衡0.05克牛血清白蛋白（BSA）和0.037克乙二胺四乙酸（EDTA），并加入到50毫升管，并把管子转动约1小时，直至粉末溶解。调节pH至7.4，通过0.2微米的过滤器过滤到一个新的50ml管中，并在4℃直至使用存储。 </li></ol></li><li> Hank氏平衡盐溶液（HBSS）以HEPES（500毫升） <ol><li>添加1.1915克HEPES，使在HBSS溶液10毫米的HEPES拌匀。测量pH值调整至7.4。过滤器和储存在4C。 </li></ol></li><li> 1倍的Kreb氏溶液（1L） <ol><li>加入100毫升10×的Kreb氏溶液[ 表1]在1升烧瓶中以双蒸水。权衡2.1克碳酸氢钠和2克D-葡萄糖，并加入到烧瓶中。混合并调节总体积至1L </li><li>用95％ 的 O 2/5％CO 2的气泡溶液的罩内30分钟，并过滤溶液。存放在4℃和实验过程中保持在冰上。 </li></ol></li></ol> 2.准备磁珠<ol><li>加入1 ml的CD31缓冲器内每次1.5毫升管（每采样1管）。混合羊抗大鼠IgG珠和适量添加到每个样品[ 表1]。摇试管大力的30倍，并将其放置在磁板晃动1分钟。打开而上盘管和转储解决方案集成到一个桶里。 </li><li>加入1 ml的CD31缓冲到每个管中。添加2微升大鼠抗小鼠CD31抗体向每个管中并在4ºC旋转管O / N。 </li></ol> 3.高压灭菌<ol><li>高压灭菌所有的手术工具和枪头。切枪头的端部，以便有4种不同直径的提示，从最宽到最窄。的提示近似直径3毫米，2,5 mm开始，2毫米，并分别为1.5毫米。切割尖端，并在121°C的温度下具有杀菌30分钟，然后高压灭菌的一天。 注:第4 - 11应在实验当天进行。 </li></ol> 4.准备解决方案<ol><li>准备含2％铁强化小牛血清（IFCS）的M199洗涤液。对于成像，准备为30 mL /鼠标。 </li><li>制备的酶溶液（10毫升/样品）。称量0.6单位/ ml分散酶和1mg / ml的胶原酶II型的到50毫升管中。添加M199，并在4ºC旋转管15分钟。通过0.2微米的滤膜过滤到一个新的50ml管中insidË罩，并保持在4℃直至使用。 </li><li>准备HBSS / HEPES肝素。有10mM HEPES加入10 mL的HBSS在15毫升管中。加入100微升肝素到管，并保持在4℃直至使用。 </li></ol> 5.洗涤珠<ol><li>取出与来自旋转器珠粒的管，在4ºC并带至机罩。放置管在磁性板振摇1分钟。转储溶液和加入1 ml的CD31缓冲到每个管中。 </li><li>就拿管出来的磁板和用力振摇30倍。放管放回磁性板并重复总共3次洗涤。建成后，将管道回4C和保持旋转，直到使用。 </li></ol> 6.解剖<ol><li>用0.1ml肝素腹膜内注入小鼠，以防止血液凝固。等待10分钟，然后麻醉使用的0.01毫升戊巴比妥腹膜内注射小鼠。检查鼠标的感觉按捏脚的疼痛。 </li><li>广场上有头聚苯板鼠标并用针持有武器。使用镊子和强硬的切剪刀剪开在上胸区皮肤到喉咙。 </li><li>使在左肋只是隔膜上方的中心的切口。侧身向上切骨朝喉咙一起组成一个三角形暴露心脏和肺部。刚切膈上的主动脉。 </li><li>握住钳胸腺不拉并用剪刀小心切割任意的结缔组织去除肺部和心脏一起。在烧杯中的地方组织与1×克雷布斯的，握钳和转让〜6厘米培养皿1×克雷布斯的。 </li><li>删除使用剪刀和镊子的肺叶。插入一个20G的无菌导管进入主动脉。冲洗血液在用HBSS /肝素冠状循环系统。使心室一小截，摇心脏和转移到M199的样品管。置于冰上管。 </li></ol><p<html>类="jove_title"&gt; 7。组织消化<ol><li>的心脏转移到一个直径6cm培养皿填充用10ml酶溶液，并剁碎成用剪刀小块。放置在37ºC孵化的菜15分钟。通过与一切割端的前端上下吹打30次，以允许所述组织通过容易搅动消化的材料。 </li><li>把它放回在37ºC孵化了15分钟。重复搅拌三次，用枪头从最大开口每次最窄，每间15分钟孵化。 </li><li>过去的15分钟温育后，取样品并到罩。吸管溶液和使用吸管尖与最窄开口并通过40微米的细胞滤网到一个新的50ml管中下降了30倍。 </li><li>使用10毫升吸管洗净用10ml洗涤液的培养皿转移到细胞过滤。放置在离心机和自旋的50ml试管在400克在RT 10分钟。 </li></ol>书名"&gt; 8，洗涤和结合珠<ol><li>而不破坏沉淀除去上清液。洗用5毫升洗涤缓冲液细胞两次。 </li><li>获取先前一次中制备的1.5毫升管珠和工作在一个管。放一只1.5mL管中的磁板，摇3次，并打开它。 </li><li>最后离心后，取出尽可能多上清尽可能不中断沉淀。通过大力弹撇清的细胞团块。 </li><li>加入1ml洗涤缓冲液到50毫升管，将细胞用1毫升吸管混合。转储从1.5毫升管的溶液，并从50ml管中的珠加入1 ml的细胞悬浮液。振动小管的30倍，然后旋转，在4ºC30分钟。 </li></ol> 9.准备细胞培养/影像<ol><li>准备室电镀细胞。添加明胶溶液（5％重量/体积，300微升）到室以及涂覆表面，并在37ºC孵育30分钟。孵育后，REMOVE明胶和干燥的表面上。 </li><li>取出含珠/细胞的试管旋转后的30分钟，并把在发动机罩内的磁板。振摇2分钟，转储和加入1ml洗涤液。 </li><li>就拿出来管板，用力振摇30次，然后一抖30倍。将它们放回磁板，振摇1分钟。重复4次，总共5次洗涤的，但与上板1分钟进行摇动培养。 </li><li>最后一次洗涤后，取出一管并在每次打开一个管工作之一。摇30次，然后轻弹30倍。把它放在磁板，振摇1分钟。转储和加入1ml 20％乳油​​IFCS介质（37ºC预热）。 </li><li>剧烈摇晃30次，然后轻弹30倍。调整吸管500微升和吸管上下10次。转移500μl到每个室。 </li><li>把在37ºC孵化器，并留下O / N。第二天，洗涤细胞，用10％FBS的乳油介质。 </li></ol> 10.准备Western印迹（WB）样品<ol><li>德Ë出珠/细胞管旋转后的1小时。把它们放在磁板振摇2分钟。转储上清液，加入1毫升冷HBSS。剧烈摇晃30次，然后轻弹30倍。重复两次洗涤3次，但与上板1分钟进行摇动培养。 </li><li>最后一次洗涤后，把磁板管和振摇1分钟。使用移液管除去缓冲液和使用方法先前描述11,12裂解细胞为WB。收集裂解液中通常含有蛋白质约30微克。 </li></ol> 11.成像<ol><li>清洗细胞3次，用媒体，具有用于腔室的250微升最终体积。添加1.5微升语-acLDL的细胞中。从这一点来说，使细胞保持在黑暗中。 </li><li>在37℃孵育3.5小时。添加1.5微升Hoechst公司的，拌匀，并在37˚C.Wash细胞用PBS孵育30分钟，并用4％多聚甲醛（PFA）在室温20分钟固定。 </li><li>用PBS洗两次，并用5％的方框BSA-PBS进行30英里北，RT。用1％BSA-PBS中洗两次。稀释，用1％BSA-PBS BS-凝集素FITC至2微克/ ml的浓度，并添加300微升到每个室。 </li><li>放腔室上的轨道摇床在RT 30分钟。用PBS洗涤3次。 </li><li>可视化与FITC（凝集素染色，欧共体标志，激发/发射488纳米/ 519纳米），用于DAPI（赫斯特，核染色，激发/ 358 nm发射/ 30毫秒的200毫秒的曝光时间的荧光显微镜下观察细胞461纳米），并在557毫微米/ 576毫微米）300毫秒TRITC（acLDL染色，欧共体盯防，激发/发射。获得使用20X物镜图像。 </li></ol>

<ol><li>Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+cell+functions%5BTitle%5D)+AND+%22J+Cell+Physiol%22%5BJournal%5D)">Endothelial cell functions.</a> J Cell Physiol. 196, 430-443 (2003).</li>
<li>Sumpio, B. E., Riley, J. T., Dardik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cells+in+focus%3A+endothelial+cell%5BTitle%5D)+AND+%22Int+J+Biochem+Cell+Biol%22%5BJournal%5D)">Cells in focus: endothelial cell.</a> Int J Biochem Cell Biol. 34, 1508-1512 (2002).</li>
<li>Cines, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+cells+in+physiology+and+in+the+pathophysiology+of+vascular+disorders%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Endothelial cells in physiology and in the pathophysiology of vascular disorders.</a> Blood. 91, 3527-3561 (1998).</li>
<li>Deanfield, J. E., Halcox, J. P., Rabelink, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+function+and+dysfunction%3A+testing+and+clinical+relevance%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Endothelial function and dysfunction: testing and clinical relevance.</a> Circulation. 115, 1285-1295 (2007).</li>
<li>Jin, Y., Liu, Y., Antonyak, M., Peng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+characterization+of+vascular+endothelial+cells+from+murine+heart+and+lung%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Isolation and characterization of vascular endothelial cells from murine heart and lung.</a> Methods Mol Biol. 843, 147-154 (2012).</li>
<li>Makino, A., Platoshyn, O., Suarez, J., Yuan, J. X., Dillmann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Downregulation+of+connexin40+is+associated+with+coronary+endothelial+cell+dysfunction+in+streptozotocin-induced+diabetic+mice%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">Downregulation of connexin40 is associated with coronary endothelial cell dysfunction in streptozotocin-induced diabetic mice.</a> Am J Physiol Cell Physiol. 295, C221-C230 (2008).</li>
<li>Marelli-Berg, F. M., Peek, E., Lidington, E. A., Stauss, H. J., Lechler, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+endothelial+cells+from+murine+tissue%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Isolation of endothelial cells from murine tissue.</a> J Immunol Methods. 244, 205-215 (2000).</li>
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The authors have nothing to disclose.

在研究中最广泛使用的血管内皮细胞，因为它们方便和易于培养的人脐静脉内皮细胞（HUVECs）。然而，大量的研究，需要由内皮细胞从其它特定器官10的隔离达到生理模型的可用性。内皮细胞弥补心脏组织细胞的一个很小的群体;其分离和培养可以证明是非常困难的。 有此协议中的三个关键步骤。第一是冲洗血液良好。这是必要的，因为血液中的白细胞也表达CD31作为?...

各种心血管疾病和代谢性疾病的小鼠模型承受其类似于那些在患者中发现的生理和分子变化。此外，老鼠的基因的改变是一个强大的工具，使我们能够调查疾病发生和发展的特定基因的致病作用。时下，细胞类型特异性基因敲入或-out小鼠容易在特定的细胞类型许多实验室和mRNA的测定和蛋白水平产生在确定是否将小鼠真正遗传修饰的第一步。 内皮是内皮细胞（EC），该线的内血管壁的薄单层。内皮细胞在调节血管张力，血管通透性，及新血管形成1,2-关键作用。内皮功能障碍是许多病理学病症和血管内皮功能的改变的标志可导致各种汽车diovascular疾病3,4。它是这样显著研究生理和病理生理条件下的内皮细胞的功能。 有几种方法来分离内皮5-8和隔离方法具有这取决于组织和物种将被用来进行优化。这是因为来自不同组织的EC显示异质性的高水平的相对于它们的表面标记和蛋白质表达9。内皮细胞的成功分离往往是具有挑战性的，需要一定程度的培训和实践。一旦实现，在该协议提出隔离的方法证明是稳定的和有效的。 这种方法的总体目标是获得鼠标冠状动脉内皮细胞（MCECs）的高品质和数量。即使细胞从心脏采集的量小于从使用其他技术的其他组织中收集的细胞，该技术仍然提供赌注之三的品质。内皮细胞在所得人口的纯度为大于90％。因此，这种技术将是理想的，依赖于纯粹分离的内皮细胞，如细胞成像应用。

<table><tbody><tr><td>BSA</td><td>Sigma</td><td>A3311-10G</td><td></td></tr><tr><td>EDTA</td><td>Fisher</td><td>BP120-500</td><td></td></tr><tr><td>HBSS</td><td>Fisher</td><td>SH3026801</td><td></td></tr><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H4034-500G</td><td></td></tr><tr><td>Sodium Bicarbonate&amp;nbsp;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>D-(+)-Glucose&amp;nbsp;</td><td>Sigma</td><td>G7021-1KG</td><td></td></tr><tr><td>DynaBeads pan mouse IgG beads&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11035</td><td></td></tr><tr><td>Rat anti-mouse CD31 Antibody</td><td>BD Biosciences</td><td>550274</td><td></td></tr><tr><td>IFCS&amp;nbsp;</td><td>Fisher</td><td>SH30072.04</td><td></td></tr><tr><td>M199</td><td>Fisher</td><td>MT10060-CV</td><td></td></tr><tr><td>Dispase (Neutral Protease)</td><td>Worthington Biochemical Corp./Fisher</td><td>LS02109</td><td></td></tr><tr><td>Collagenase Type II&amp;nbsp;</td><td>Worthington Biochemical Corp./Fisher</td><td>LS004176</td><td></td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Fisher</td><td>BP381-1</td><td></td></tr><tr><td>Igepal</td><td>Sigma</td><td>I3021-50 ml</td><td></td></tr><tr><td>Phosphatase inhibitor cocktail</td><td>Sigma</td><td>P0044-1ml</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma</td><td>P8340-1 ml</td><td></td></tr><tr><td>Heparin&amp;nbsp;</td><td>Sigma</td><td>H3149-100KU</td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Fisher/Mediatech</td><td>MT35010CV</td><td></td></tr><tr><td>D-Valine</td><td>Sigma</td><td>V1255-5G</td><td></td></tr><tr><td>EGS</td><td>Corning</td><td>356006</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Fisher/Mediatech</td><td>MT-30-002-CI</td><td></td></tr></tbody></table>

isolation of mouse coronary endothelial cells

内皮细胞排列的血管的内壁，并在血管张力，血管通透性，及新血管形成的调节中起重要作用。内皮细胞功能障碍在许多心血管疾病，包括缺血性心脏疾病的发生和发展有关。审查功能和冠状内皮细胞的表征，细胞分离是第一步，它需要高纯度和数量进行随后的实验。这个协议描述了一种有效的方法，以分离成年鼠冠状内皮细胞。小鼠心脏解剖，切碎成小块。用分散酶和胶原酶II心脏的消化后，将细胞洗涤并与缀合有抗CD31抗体的磁珠孵育。与内皮细胞的珠洗涤几次，并准备在各种应用中，包括成像和分子生物学experime使用NTS。高效分离得到每一心脏大约10 4细胞有超过90％的纯度。

从小鼠心脏冠状内皮细胞的成功分离通常产生约10 4个细胞，纯度用鹅卵石形状90％以上。对内皮细胞的纯群体，应谨慎整个协议注意保证不受任何污染的无菌环境。细长细胞或群体内放大细胞的出现指示与其他细胞类型，平滑肌细胞和成纤维细胞的污染。内皮细胞的纯度试验是通过染色细胞与内皮细胞表面标记物，BS-凝集素和acLDL（ 图1）执行的。表?...

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Isolation of Mouse Coronary Endothelial Cells

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 ...

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Biology

11.2K 次观看.  University of Arizona, Tucson. 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.

视频: Isolation of Mouse Coronary Endothelial Cells

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

从鼠心心室自由壁分离内膜细胞和冠状内皮细胞

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

科研

发育生物学

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 ...

生物学

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

原代小鼠骨骼肌微血管内皮细胞的分离

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

神经科学

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

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 of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

小鼠原代肺内皮细胞的分离

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

免疫与感染

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 ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

鼠冠状动脉血管平滑肌细胞的分离

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

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 ...