Dr. K. Yuan was supported by an American Heart Association Scientist Development Grant (15SDG25710448) and the Pulmonary Hypertension Association Proof of Concept Award (SPO121940). Dr. V. de Jesus Perez was supported by a career development award from the Robert Wood Johnson Foundation, an NIH K08 HL105884-01 award, a Pulmonary Hypertension Association Award, a Biomedical Research Award from the American Lung Association and a Translational Research and Applied Medicine award from Stanford University.

伦理学声明:涉及动物主题程序已在医学院斯坦福大学医学院被批准的机构动物护理和使用委员会。 注意:动物是在用3％的蒸发器异氟醚和O 2气体的3％的电源麻醉。眼睛上的药膏兽医使用可能有助于防止干燥时的麻醉下。 1.细胞的制备<ol><li>培养人类内皮细胞和周细胞的细胞培养100 MM平板用10毫升适当的媒体。更换10毫升新鲜培养基每2 - 1天，直至细胞达到80％汇合。 <ol><li>培养的内皮细胞（EC）的补充有公司完整内皮细胞培养基（ECM）提供5％牛胎儿血清（FBS），10％青霉素/链霉素和10％的内皮细胞生长补充。 </li><li>在补充有公司完整周细胞介质（PM）的培养周细胞供给2％FBS，10％青霉素/链霉素和10％周细胞生长添加剂。 </li&gt; </ol></li><li>准备细胞混合物<ol><li>计算实验组和对照组的所需数量的细胞。 注:实验组每个插件都包含一个万美元（1×10 6）人内皮细胞和二十万（2×10 5）周细胞。对于对照组，每组插头包含120万只的人类内皮细胞。阴性对照组仅具有基质凝胶。每个组应该包括至少四塞，这将需要两只小鼠作为插头可注入的小鼠的腰部两侧。三个插头将作为一式三份进行实验和第四可能的情况下，一个发生故障被使用。准备25％的额外细胞相匹配凝胶的额外容量。 </li><li>收集和培养板细胞计数。 <ol><li>要取下电池，取出媒体和室温1X磷酸盐缓冲液（PBS）洗一次。除去PBS，加入1ml 0.25％胰蛋白酶/ 2.21毫摩尔EDTA，并孵育1分钟。加入2洗涤细胞断板毫升培养基，并收集在一个15毫升管。 </li><li>算使用根据制造商的说明血球细胞。 </li><li>离心细胞的适当数量的成丸，在400×g离心5分钟。为实验组，离心既内皮和周下来连成一片沉淀。 </li></ol></li></ol></li></ol> 2.基质凝胶制备<ol><li>解冻完全基质凝胶在4℃下4小时或过夜，得到均匀的溶液。没有混合是必需的。 </li><li>在1.5ml管分装基质凝胶和储存在4℃。 </li><li>预寒意无菌1.5毫升管和28克1在4℃毫升胰岛素注射器。注意:保持基质胶冰在任何时候。 </li><li>混合与bFGF的冷基质凝胶在1的体积比:100（bFGF的终浓度= 0.5微克/毫升; bFGF的储备溶液= 50微克/毫升）通过上下吹打数次混合矩阵凝胶溶液后，在冰上孵育30 - 60分钟b安伏与细胞在步骤3.1中混合。 注:混合前解冻股票的bFGF。计算所需要的注射插头的数量。例如，每个插头需要200微升基质凝胶。因此，准备25％的额外凝胶。 </li></ol> 3.混合矩阵凝胶细胞<ol><li>对于每个组，重悬细胞沉淀（含有适当的细胞数）与含有bFGF的母质凝胶的计算量。轻轻混匀，避免起泡，直到小鼠注射准备在15ml试管离开冰。 </li></ol> 4.鼠标准备<ol><li>在感应腔麻醉在3％异氟醚5分钟加3％O 2 1 SCID小鼠。集中在一个实验组，或2小鼠，一次。 </li><li>从感应腔在加热垫腹侧移开鼠标，地点向下并迅速经由非再呼吸系整个仁济供给麻醉附加一个吻喷嘴到鼠标的脸ction过程。切换从感应腔室到非再呼吸系统中的气体流量和降低气体压力，以1.5％的异氟醚和1.5％ 的 O 2。 </li><li>从两侧后区（大约直径1厘米），用剃刀或脱毛霜清除头发。 </li><li>擦拭用70％酒精垫皮肤区域。 </li><li>与第二只老鼠4.4 - 返回鼠标感应室，并重复步骤4.2。 </li></ol> 5.矩阵注射凝胶<ol><li>通过附着到非再呼吸系统和加热垫腹侧放置向下准备一只小鼠注射。 </li><li>加载基质凝胶成预冷的28克1毫升胰岛素注射器一次一个鼠标。 <ol><li>负载两个插头（每头200微升加25％的额外）到注射器矩阵凝胶的完整的500微升的体积。 </li><li>确保将细胞装载到注射器，以防止在CE的3分钟内注入基质凝胶到小鼠从沉降到注射器的侧面并防止基质凝胶从注射器的内部固化LLS。 </li></ol></li><li>抬起背部皮肤定位皮下空间，然后注入200微升基质凝胶的缓慢而均匀地进入左肋背面后的皮下空间。 </li><li>取出注射针慢慢地，小心翼翼地防止基质凝胶漏出注射部位。撞了会形成在注射的部位。擦拭注射部位用酒精垫。 </li><li>重复注射步骤5.2 - 5.5对小鼠的背部的另一侧，然后离开鼠标在它的后面，持续1分钟，以允许在热垫的基质的凝胶的凝胶化。 </li><li>介绍了使用永久性标记后14天内，以确定磕碰都颠簸。经过一番头发长回来，它会更容易找到肿块。 </li></ol> 6.基质凝胶插件隔离:注射后14天<ol><li>通过将动物安乐死人道的老鼠s到&gt; 5分钟吸入二氧化碳颈椎脱位，以确保死亡。 </li><li>卸下鼠标的背部基质胶栓。 <ol><li>轻轻地取出周围注射部位其插头重复步骤4.3重新长出位于毛。标记线表示原始凸点的皮肤下的大小。 </li><li>切除插头分别安装在插头的顶侧和底侧，周围皮肤和肌肉层沿。 <ol><li>使用精细手术剪，使上即垂直于皮肤插头的明显边界的切口，并通过切向插头下方的肌肉。然后，小心地切绕沿标记的边界的插头的外周。 </li><li>拔出插头，保持它夹在皮肤和肌肉层之间。在小烧杯用1X PBS立即冲洗洗去多余的血液。该插件现在可以是固定的（下一步，6.3）。 </li></ol></li></ol></li><li>修复PLU克4％的多聚甲醛。 <ol><li>将整个插头，包括皮肤和肌肉层，在50ml管中，用25ml 4％的多聚甲醛在室温下24小时而不摆动。第二天，转移插头成25个1ml 70％乙醇的供石蜡包埋之前另外24小时。 注:处理多聚甲醛慎用。戴手套。 </li></ol></li></ol> 7.石蜡过程中的基质凝胶插件<ol><li>准备石蜡包埋插头。 <ol><li>定向插头， 如图1a中 ;放置插头在一个组织盒，使得插头的侧面朝上，皮肤和肌肉层都是横跨组织块的表面，将第一个切片中被切割可见。 </li></ol></li><li>石蜡根据标准石蜡包埋协议13嵌入插头。 </li><li>削减8 - 10微米厚的部分离组织块，并安装到显微镜载玻片雅鼎标准石蜡切片协议13。 </li><li>贮存于室温阴凉干燥处的幻灯片，直到准备染色。 </li></ol> 8. HE染色（H＆E）染色第13 <ol><li>通过100％二甲苯传递载玻片两次去paraffinize组织切片，100％的乙醇，95％乙醇，70％的乙醇，最后1×PBS中，每一个用于在室温下5分钟。留在1×PBS中滑动，直到下一个步骤。 </li><li>吸取100微升H＆E解决方案的上段和孵育1 - 5分钟，或直到有作为在放大10倍的直立光学显微镜下观察细胞细胞核的蓝色和粉红色之间的细胞质鲜明对比。小心不要让H＆E解决方案触及目标。 </li><li>洗涤H＆E溶液关闭的扣篮在一个大烧杯中的自来水的滑动的滑动件，然后将滑动在烧杯齿条和与2分钟自来水冲洗。 </li><li>莫五个步骤9.8安装的幻灯片。 </li></ol> 9.染色其他幻灯片人类毛细血管和周<ol><li>通过100％二甲苯传递载玻片两次去paraffinize组织切片，100％的乙醇，95％乙醇，70％的乙醇，最后1×PBS中，每一个用于在室温下5分钟。留在1×PBS中滑动，直到下一个步骤。 </li><li>煮抗原检索解决方案的部分。 <ol><li>在一个2升的烧杯中，准备在95℃的加热块上沸腾抗原使用1倍的柠檬酸缓冲液（pH7.0）中检索溶液。 </li><li>一旦沸腾，将载玻片在金属机架上，并在20分钟的煮沸抗原修复溶液淹没。 </li><li>小心地取出从仍然浸没幻灯片加热块的烧杯中，并让溶液慢慢恢复到室温约30分钟。 </li><li>放置在1×PBS中滑动，直到下一个步骤。 </li></ol></li><li>方框在封闭缓冲液（章节1×PBS中，5％山羊血清，00.3％的Triton X-100）。 <ol><li>周围画有PAP笔每一部分的边缘疏水圆。 </li><li>放置在用蒸馏水湿度托盘的幻灯片在托盘的底部。 </li><li>吸移管将100μl封闭缓冲液到确保所有的组织的切片被流体覆盖（可能需要超过100微升）。 </li><li>孵育部分为1小时封闭液。 </li></ol></li><li>孵育初级抗体部分。 <ol><li>备具有两个初级抗体的一个解决方案探测CD31（内皮细胞），并在稀释缓冲液中稀释平滑肌肌动蛋白（周细胞）的每个部分（1×PBS，1％BSA，0.3％的Triton X-100）。 注意:在此，抗CD31浓度为1:50和抗平滑肌肌动蛋白浓度为1:300。 </li><li>孵育切片在4℃下在含有蒸馏水的湿度盘100微升第一抗体过夜。 </li></ol></li><li>洗幻灯片三次1X PBST（1X PBS + 0.1％TWEEN20），每次5分钟洗涤。 </li><li>孵育二级抗体部分。 <ol><li>准备用荧光团缀合的山羊抗兔第二抗体的溶液以识别在稀释缓冲液中稀释的抗CD31兔抗体。 <ol><li>注:平滑肌肌动蛋白的第一抗体已结合有CY3荧光团，所以不需要第二抗体染色。山羊抗兔次级抗体浓度为1:250。 </li></ol></li><li>孵育切片1小时，在含蒸馏水的湿度盘100微升次级抗体。 </li></ol></li><li>洗玻片三次在1×PBST，每次5分钟洗涤。 </li><li>装入幻灯片。 <ol><li>吸取2 - 3滴（〜40 - 60微升）与DAPI核染色防褪色安装解决方案，在每个部分。 </li><li>轻轻地放在一个没有。 1.5盖玻片在部分和安装的解决方案，从而允许流体在部分展开和向的边缘盖玻片。轻轻用手指压出气泡可能位于在截面。 </li><li>让幻灯片处理之前干燥至少1小时。 </li></ol></li></ol> 10.量化毛细血管密度和结构14 <ol><li>算在H＆E染色的切片的毛细血管的数目，表示为＃/毫米2。 <ol><li>获取下放大40倍一个堂堂正正的光学显微镜四个不同领域的照片。 注:毛细管被标识为管充满红血球。 </li><li>可替代地，代替手工计数毛细管，使用图像分析软件（ 例如，Wimasis图像分析）来量化各种血管生成的参数，如容器数，平均容器的长度和直径，和分支点。 </li></ol></li><li>免疫荧光染色切片分析毛细结构。 <ol><li>获得四个不同领域的图片在一个倒置荧光显微镜与FITC和TxRed过滤立方体。 <ol><li>在488纳米波长使用FITC立方体图像内皮通过激励和激发在594纳米波长使用TxRed立方体图像周细胞。 <style type="text/css">p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 8.0px Arial}
			</style><style type="text/css">p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 8.0px Arial}
span.s1 {letter-spacing: -0.1px}
			</style><style type="text/css">p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 8.0px Arial}
span.s1 {letter-spacing: -0.1px}
			</style><style type="text/css">p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 8.0px Arial}
span.s1 {letter-spacing: -0.1px}
			</style></li></ol></li></ol></li></ol>

<ol>
	<li>Risau, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+angiogenesis.">Mechanisms of angiogenesis.</a> Nature. 386 (6626), 671-674 (1997).</li><li>Ribatti, D., Nico, B., Crivellato, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+pericytes+in+angiogenesis.">The role of pericytes in angiogenesis.</a> Int J Dev Biol. 55 (3), 261-268 (2011).</li><li>Koh, W., Stratman, A. N., Sacharidou, A., Davis, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+three+dimensional+collagen+matrix+models+of+endothelial+lumen+formation+during+vasculogenesis+and+angiogenesis.">In vitro three dimensional collagen matrix models of endothelial lumen formation during vasculogenesis and angiogenesis.</a> Methods Enzymol. 443, 83-101 (2008).</li><li>Liu, Y., Senger, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-specific+activation+of+Src+and+Rho+initiates+capillary+morphogenesis+of+endothelial+cells.">Matrix-specific activation of Src and Rho initiates capillary morphogenesis of endothelial cells.</a> FASEB J. 18 (3), 457-468 (2004).</li><li>Grant, D. S., 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=Interaction+of+endothelial+cells+with+a+laminin+A+chain+peptide+(SIKVAV)+in+vitro+and+induction+of+angiogenic+behavior+in+vivo.">Interaction of endothelial cells with a laminin A chain peptide (SIKVAV) in vitro and induction of angiogenic behavior in vivo.</a> J Cell Physiol. 153 (3), 614-625 (1992).</li><li>Madri, J. A., Pratt, B. M., Tucker, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+modulation+of+endothelial+cells+by+transforming+growth+factor-beta+depends+upon+the+composition+and+organization+of+the+extracellular+matrix.">Phenotypic modulation of endothelial cells by transforming growth factor-beta depends upon the composition and organization of the extracellular matrix.</a> J Cell Biol. 106 (4), 1375-1384 (1988).</li><li>Brooks, P. C., Montgomery, A. M., Cheresh, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+10-day-old+chick+embryo+model+for+studying+angiogenesis.">Use of the 10-day-old chick embryo model for studying angiogenesis.</a> Methods Mol Biol. 129, 257-269 (1999).</li><li>Yuan, K., 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=Activation+of+the+Wnt/planar+cell+polarity+pathway+is+required+for+pericyte+recruitment+during+pulmonary+angiogenesis.">Activation of the Wnt/planar cell polarity pathway is required for pericyte recruitment during pulmonary angiogenesis.</a> Am J Pathol. 185 (1), 69-84 (2015).</li><li>Johns, A., Freay, A. D., Fraser, W., Korach, K. S., Rubanyi, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+estrogen+receptor+gene+prevents+17+beta+estradiol-induced+angiogenesis+in+transgenic+mice.">Disruption of estrogen receptor gene prevents 17 beta estradiol-induced angiogenesis in transgenic mice.</a> Endocrinology. 137 (10), 4511-4513 (1996).</li><li>Chen, C. 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=Human+pericytes+for+ischemic+heart+repair.">Human pericytes for ischemic heart repair.</a> Stem Cells. 31 (2), 305-316 (2013).</li><li>Richards, O. C., Raines, S. M., Attie, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+blood+vessels,+endothelial+cells,+and+vascular+pericytes+in+insulin+secretion+and+peripheral+insulin+action.">The role of blood vessels, endothelial cells, and vascular pericytes in insulin secretion and peripheral insulin action.</a> Endocr Rev. 31 (3), 343-363 (2010).</li><li>Ricard, N., 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=Increased+pericyte+coverage+mediated+by+endothelial-derived+fibroblast+growth+factor-2+and+interleukin-6+is+a+source+of+smooth+muscle-like+cells+in+pulmonary+hypertension.">Increased pericyte coverage mediated by endothelial-derived fibroblast growth factor-2 and interleukin-6 is a source of smooth muscle-like cells in pulmonary hypertension.</a> Circulation. 129 (15), 1586-1597 (2014).</li><li>Buchwalow, I. B., Bocker, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunohistochemistry: Basics and Methods. , 1-153 (2010).</li><li>Tata, D. A., Anderson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+investigation+of+capillary+structure.">A new method for the investigation of capillary structure.</a> J Neurosci Methods. 113 (2), 199-206 (2002).</li></ol>

No competing financial interests enclosed.

基质凝胶塞法已被证明是评估在血管发生， 在体内血管生成和抗血管生成的化合物基因调节，并以补充在体外试验方便和有效的方法。这里，我们详细描述人类血管生成的新颖基质凝胶塞测定该调查期间血管形成的内皮细胞和周细胞之间的相互作用。 还有在这个协议的几个新颖的关键步骤。在低通道（通道1 - 4）细胞是优选的，因为在14天的实验设置中年轻细?...

血管生成是由该新的血管从预先存在的血管网1在许多领域从正常发展到疾病形成和是正在进行的研究的焦点的过程。这种动态过程涉及的增殖和血管内皮细胞（ECS）和周构建血管管的朝向需要的氧气和营养输送2点定向招募的迁移。为了研究这个过程需要一个同样动态测定中，最重要的一个可以概括的管道形成三维性质。 体外 3D基质测定已经开发了满足这种需要，并已运作良好，使研究人员以限定在空间和时间上离散的步骤，其中的血管生成发生3，4，5，6。然而，这些体外 3D基质模型限于研究非灌注容器的ð因此缺乏相关的血管生成过程中的关键组件（ 例如，循环增长和抑制因子，非自然的紧张/势力横跨血管床），并不能模拟存在于活组织的复杂环境。为了解决此限制，几个体内血管生成测定法已开发7，包括基质凝胶塞测定法，其将是我们的报告8,9的焦点。 基质胶栓法是一个行之有效的体内血管生成实验，呼吁研究人员，因为它提供了一个强大的平台，以测试在不同的血管细胞和物质的作用。基质凝胶是由在37℃下固化成凝胶样物质的Engelbreth-Holm的-群（EHS）小鼠肉瘤细胞系分泌的市售基底膜溶液。基质凝胶可与细胞和/或物质，如生长因子，和麟蹄混合皮下反恐执行局到鼠标。主机内皮细胞会侵入插头超过14天，形成血管网，并成为与宿主的血液灌注。迄今为止，基质凝胶塞测定已专注于内皮细胞行为的血管生成过程中的研究，但是，据我们所知的任何努力尚未作出判断该测定是否可用于共培养的内皮细胞和周细胞研究这两种细胞类型的血管生成过程中如何相互作用。具体地，理解的EC和周之间的关系是用于研究疾病，其中血管损失是病理性的，包括微血管缺血和周围血管疾病10，11，12有价值。 在这里，我们描述了引入了源于人的周细胞与人类内皮和成纤维细胞生长因子（bFGF）沿着矩阵凝胶混合物的协议。该混合物然后可以皮下注射我ñSCID小鼠的背部，使形成功能齐全，周细胞涂，混合型血管。我们的协议描述了如何制备含有人内皮有或没有人的周细胞，放置到SCID小鼠以及如何分析关键血管生成端点组织切片基质凝胶塞。

<table border="0" cellpadding="0" cellspacing="0" width="806">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">PBS (Phosphate buffered saline)</td>
			<td style="width:115px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">0.25% Trypsin/0.53mM EDTA</td>
			<td style="width:115px;">Corning</td>
			<td style="width:119px;">25-053-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Endothelial Cell Media (ECM) kit</td>
			<td style="width:115px;">Sciencell</td>
			<td style="width:119px;">1001</td>
			<td>includes media, EC growth supplement, and penicillin/streptomycin, each supplied at the appropriate volume for easy mixing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Pericyte Media (PM) kit</td>
			<td style="width:115px;">Sciencell</td>
			<td style="width:119px;">1201</td>
			<td>includes media, Pericyte growth supplement, and penicillin/streptomycin, each supplied at the appropriate volume for easy mixing</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:405px;">primary human pulmonary microvascular endothelial cells</td>
			<td style="width:115px;">Pulmonary Hypertension Breakthrough Initiative</td>
			<td style="width:119px;"></td>
			<td>this cell type is also available commercially. Cells used at passage 1-4</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:405px;">primary human pulmonary pericytes</td>
			<td style="width:115px;">Pulmonary Hypertension Breakthrough Initiative</td>
			<td style="width:119px;"></td>
			<td>this cell type is not available commercially, but brain paricytes are.&#160; Cells used at passage 1-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">bFGF (basic Fibroblast Growth Factor)</td>
			<td style="width:115px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
			<td>stock solution is 50ug/ml in 0.1% BSA in PBS, aliquots at 50 uL and stored at -20 degrees</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Matrigel Basement Membrane Matrix</td>
			<td style="width:115px;">BD</td>
			<td style="width:119px;">356237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">28G 1cc Insluin Syringe</td>
			<td style="width:115px;">BD</td>
			<td style="width:119px;">329410</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">SCID (Severe Combined Immune Deficiency) mice</td>
			<td style="width:115px;">The Jackson Laboratory</td>
			<td>5557</td>
			<td>NOD.SCID IL2R gamma knockout strain is the best strain; 4-6 weeks of age</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">1.5mL microcentrifuge tubes, sterile</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">15 mL screw top tubes, sterile</td>
			<td style="width:115px;">BD Biosciences</td>
			<td style="width:119px;">352096</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">PAP pen</td>
			<td style="width:115px;">Life Technologies</td>
			<td style="width:119px;">8899</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">hemocytometer</td>
			<td style="width:115px;">ThermoFisher Scientific</td>
			<td style="width:119px;">02-671-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Nair hair removal cream</td>
			<td style="width:115px;">Walmart</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">anti-human CD31 primary antibody</td>
			<td style="width:115px;">LifeSpan Biosciences</td>
			<td>LS-B4737</td>
			<td>working solution is 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-human Smooth Muscle actin CY3 primary fluorescent antibody</td>
			<td style="width:115px;">Sigma</td>
			<td>C6198</td>
			<td>working solution is 1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">goat anti-rabbit secondary antibody; 488/green</td>
			<td style="width:115px;">ThermoFisher Scientific</td>
			<td style="width:119px;">A-11008</td>
			<td>working solution is 1:250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Prolong Gold DAPI solution</td>
			<td style="width:115px;">Cell Signaling</td>
			<td style="width:119px;">8961S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">microscope slides</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">48300-047</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">no. 1.5 cover slips</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12-544-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">citrate buffer 10x</td>
			<td style="width:115px;">Millipore</td>
			<td style="width:119px;">21545</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">extra fine surgical scissors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td style="width:119px;">14084-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">Formalin (paraformaldehyde)</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">245-685</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Tissue cassettes</td>
			<td style="width:115px;">Simport</td>
			<td>M492-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">goat serum</td>
			<td style="width:115px;">Dako</td>
			<td style="width:119px;">X0907</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo study of human endothelial-pericyte interaction using the matrix gel plug assay in mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

基质凝胶塞段的代表性H＆E和免疫荧光染色， 如图2所示。节从EC仅插头显示一些血管被大多不与血液（ 图2左上，黑色箭头），而含有插头灌注既内皮和周显示多个灌注血管直径较大和完整周细胞包裹，就证明了紧邻的CD31阳性内皮细胞阳性的SMA染色。这些结果表明，周细胞中的新生血管形成，而这动态过程可以概括并容易在体内</e...

Watch this Scientific Journal Video about In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse at JoVE.com

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

我们提出了一个协议使用基质凝胶塞血管生成测定的变化来研究小鼠的人内皮周细胞的相互作用。

在人类内皮周细胞的相互作用使用基质凝胶插件检测小鼠的体内研究

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

in-vivo-study-human-endothelial-pericyte-interaction-using-matrix-gel

Research

JoVE Journal

Biology

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

9.6K 次观看.  Stanford University. 我们提出了一个协议使用基质凝胶塞血管生成测定的变化来研究小鼠的人内皮周细胞的相互作用。 

视频: In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

优化的纤维蛋白凝胶珠检测血管生成的研究

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

In vivo and in vitro Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9.
Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue).
Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

In vivo and in vitro Studies of Adaptor-clathrin Interaction

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

适配器，网格蛋白相互作用在体内和体外研究

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

克隆形成含量:贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯:天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...