ACD is supported by a grant from the National Institute of Health (R01-CA177875). LX is a fellow in the HHMI-funded translational medicine program at UNC Chapel Hill. JVM is supported by a T32 pre-doctoral fellowship from the Integrative Vascular Biology Program at UNC Chapel Hill. We thank Clayton Davis for assistance with confocal microscopy.

以下协议进行根据实验动物医学系在北卡罗莱纳大学教堂山分校制定的准则。 1.准备以下材料和试剂开始之前<ol><li>通过补充400毫升低葡萄糖制备乳油媒体（1克/升D-葡萄糖或LG）的Dulbecco氏改良Eagle氏用50ml热灭活的胎牛血清，将50ml的Nu-血清IV，加入5ml抗菌 - 抗霉菌培养基（DMEM），和hFGF，血管内皮生长因子，表皮生长因子，R3-IGF-1，和肝素部件从商业试剂盒。 </li><li>制成500毫升之FACS缓冲液（0.5％BSA和2mM EDTA的PBS）;过滤通过无菌0.22微米的过滤杯。 </li><li>消毒或消毒解剖板。 </li><li>消毒清扫针，手术剪，和解剖。 </li></ol> 2. EC隔离（第1天，约5小时） <ol><li>安乐死小鼠与二氧化碳或其它方法兼容机智小时机构动物护理和使用委员会（IACUC）政策。 注：在大小不同的多个乳腺肿瘤（5 - 直径15mm），在一个单一的转基因小鼠可能发展，如C3标签，一定要收获所有的人都对TEC隔离。如果使用的是肿瘤的原位移植的小鼠，池二点五十八1厘米3的肿瘤为一个单一的EC隔离。在这里，我们使用乳腺肿瘤作为示范，但协议可以被修改为其它类型的肿瘤。 </li><li>通过喷洒或擦拭鼠标腹侧与足量的75％V / V乙醇消毒鼠标。 </li><li>切除肿瘤的一对使用无菌技术在无菌罩或超净工作台剪刀和解剖的。 <ol><li>伸出和引脚小鼠的四肢在解剖板。让中线腹侧切开用剪刀无需打开腹膜。皮肤并朝向乳腺肿瘤，其中位于腹膜之间解剖横向。不要打开腹膜。 </li><li>消费税只能从乳腺肿瘤组织，离开了正常乳腺的利润。仔细修剪过的非肿瘤组织如皮肤和肌肉，然后将解剖的肿瘤在锥形管中，冰上静置30 ml的LG-DMEM中。 </li></ol></li><li>把肿瘤样本，以组织培养罩; 2次 - 用无菌LG-DMEM 1洗净组织。 </li><li>从锥形管到无菌组织培养培养皿转移的肿瘤，在盘中加2ml LG-DMEM中，并剁碎有一对无菌剪刀成片&lt;5毫米。 </li><li>添加5胶原酶毫升（股票= 2毫克/毫升在Hank氏平衡盐溶液，在下文HBSS）中，1毫升分散酶（股份=在HBSS 2.5 U / ml）和75微升脱氧核糖核酸酶（股份= 1mg / ml的在PBS中）放入培养皿。总成交量为现在〜10毫升</li><li>从培养皿转移胶原酶/组织混合于组织离解器管和在组织离解器60秒运行两次（预先设定的离解公关ogram的离解：1270总发每运行）。孵育在37℃下振荡光在振荡器上75分钟。 </li><li>过滤通过100微米的细胞过滤消化的组织在一个50ml锥形管中。冲洗过滤用5ml FACS缓冲液洗任何剩余的细胞。旋在280×g离心5分钟，小心地吸出上清液，而不干扰细胞沉淀。 </li><li>稀释1毫升库存RBC裂解缓冲液（10倍）的9ml无菌水。裂解红血细胞，用10ml的裂解缓冲液（1×），并立即自旋5分钟，在280×g下。注：此步骤可以，如果血少是可见的被跳过。 </li><li>重悬在10ml的FACS缓冲液。混合10微升细胞悬浮液与10微升台盼蓝的，并使用血球计数活细胞。 </li><li>重悬的细胞在约10 7个细胞/ 100微升。加10微升每100微升细胞悬浮液的FcR块，并在冰上孵育10分钟。 </li><li>根据添加大鼠抗小鼠PE标记的CD31抗体。 表 1冰上孵育10 - 15分钟，并轻弹管偶尔。 </li><li>加入10毫升FACS缓冲液到管和旋转，在280×g离心5分钟。小心地取出上清液，并用5毫升FACS缓冲液再清洗细胞沉淀。离心以沉淀细胞。吸上清而不会破坏细胞沉淀。 </li><li> 根据表2加入FACS缓冲液和抗PE微珠冰上孵育10 - 15分钟。弗里克管偶然。 </li><li>加入10 mL FACS缓冲液和自旋样品在280×g离心5分钟;用5ml FACS缓冲液和离心再次洗一次。吸上清而不干扰沉淀。 </li><li>把体积为300微升FACS缓冲液。通过35微米的细胞过滤皑皑管5分钟旋转280×g的。使用2管和更大的体积，如果需要的。 </li><li>设置磁性multistand和磁列在罩，一柱连接到分离器和平衡柱用2ml FACS缓冲液。 </li><li> AspiratE中的上清，重悬在0.5细胞沉淀 - 1毫升FACS缓冲液。 </li><li>通过平衡的磁列通过细胞悬液。 </li><li>用2ml FACS缓冲液洗涤柱3次，并在一个15毫升管（FT级分）收集流过（FT）。 </li><li>取柱关闭隔板和用2ml FACS缓冲液洗脱入另一个15ml试管（洗脱级分）。用柱塞，以确保所有单元都断列。重复洗脱两次用2ml FACS每次缓冲。 </li><li>旋洗脱液在280×g离心5分钟。 </li><li>去除上清，悬浮颗粒在10毫升EC媒体。 </li><li>等分洗脱液级分（〜6毫升）中成10厘米明胶包被的培养皿。对了三片1 立方厘米的肿瘤，板至少在四个板块洗脱细胞。替代地，板洗脱的细胞在不同浓度的多块板（例如，种子0.5毫升，1毫升，1.5毫升，和3ml洗脱液为四个板），以确保一t个最低一个板块稀疏接种洗脱细胞。检查附着的细胞的汇合是在〜1％，第二天（即，约1.0 - 2.0×10 5附着的细胞）。 注：细胞需要被镀稀疏以便乳油菌落可以形成而不被污染的其它细胞类型。 </li><li>平板FT分数1个10 cm培养皿，让细胞恢复O / N。检查板为80 ​​- 100％，第二天汇合。冻结向下细胞（-80℃）中在低温管250微升的细胞冷冻培养基，并将它们存储在液氮中的第二天。注意：FT部分可在以后的阶段和/或作为阴性对照为隔离EC克隆的乳油的基因表达分析可用于肿瘤细胞的分离。 </li><li>更换介质每2 - 3天。菌落开始形成后7 - 10天。小EC殖民地可以早一日3.标记上盘底部的细尖标记的菌落鉴定。 </li><li>刮去非特定的CELLS周围所识别的菌落用无菌200μl的pippet小费。 </li></ol> 3.殖民地选择使用克隆环<ol><li>更换介质每2 - 3天。 7天 - EC纯度可以通过低密度脂蛋白（DII-AC-LDL）增加约5进行检查。加入50微升的LDL每10 ml的EC平台，孵育3 - 荧光显微镜下检查细胞前4小时。 注：多个低密度脂蛋白+ EC克隆可以在〜7个一天观察。 </li><li>开始收获乳油菌落，当他们到达直径的3 - 5毫米大小。 （选择一个都挤满了小LDL +细胞最好的结果大殖民地。） </li><li>收获前乳油用克隆环，预涂几6孔板用0.5％明胶。抽吸明胶，加2ml EC培养基到每个孔中，并且直到需要保持所述板在培养箱中。 </li><li>刮去非欧共体对殖民地的边缘，以确保没有其他类型的细胞将在克隆环内被困。 </li><li>使用相差显微镜（4X或10X的目标），轮廓用细尖笔在培养皿底部的含EC的殖民地区。 </li><li>用10ml的PBS洗涤板和吸移当离开一个非常薄的层的PBS中的板。 （重要提示：少量（约0.5毫升）的PBS将保持细胞克隆环过程中存活;另外，组​​织粘合剂需要水粘合。） </li><li>选择适当大小的克隆环。用一双解剖镊子拿起一个环，并用10微升枪头均匀涂抹少量的组织粘合剂到克隆环。 注：组织粘合剂对克隆环只使用少量（〜0.2微升的小环），并确保组织粘合剂分散均匀地绕在底面，以保证良好的密封性。过多的组织粘合剂产生热量，并形成薄膜，可能会杀死细胞。 </li><li>放置克隆环比EC殖民地。轻轻按下克隆环粘上里纳克到板。确保殖民地粘合环之前不会干涸。 </li><li>立即吸管25微升酶细胞剥离溶液进入克隆环孵育约1分钟或直至细胞松散附着。 </li><li>吸管中的细胞克隆环滴加入6孔板含有预热的EC介质的一个孔中。 （重要提示：不要摇晃板分散细胞; EC喜欢在紧张的集群发展。）50洗净的克隆环 - 100微升EC媒体收集尽可能多的细胞成为可能，并且所有洗涤转移到相同的6孔。 </li><li>如果在10 cm培养皿一些群体过小的收获，加入10 mL新鲜的媒体，让菌落长出了几天，然后再次重复克隆环的过程。 </li><li>生长在6孔板收获菌落直到80 - 100％汇合，并转移到细胞3个孔一个6孔板，扩大它们在10厘米组织培养皿之前。刮去污染物细胞。重复一轮又一轮的克隆环过程（步骤3.5 - 3.10），如果有必要的。保持细胞相对汇合（〜60 - 70％）扩大时。欧盟可能会停止，如果镀过于稀疏增长。 </li><li>表征乳油通过FACS，染色，PCR等。注意，语-AC-LDL是荧光灯和与PE或其他荧光抗体用于FACS可能会干扰。 </li></ol>

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Res. 99 (8), 861-869 (2006).</li><li>Wylie-Sears, J., Aikawa, E., Levine, R. A., Yang, J. -. H., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitral+valve+endothelial+cells+with+osteogenic+differentiation+potential.">Mitral valve endothelial cells with osteogenic differentiation potential.</a> Arterioscler. Thromb. Vasc. Biol. 31 (3), 598-607 (2011).</li><li>Ginsberg, 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=Efficient+direct+reprogramming+of+mature+amniotic+cells+into+endothelial+cells+by+ETS+factors+and+TGF&#946;+suppression.">Efficient direct reprogramming of mature amniotic cells into endothelial cells by ETS factors and TGF&#946; suppression.</a> Cell. 151 (3), 559-575 (2012).</li><li>Sapino, A., 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=Expression+of+CD31+by+cells+of+extensive+ductal+in+situ+and+invasive+carcinomas+of+the+breast.">Expression of CD31 by cells of extensive ductal in situ and invasive carcinomas of the breast.</a> J. Path. 194 (2), 254-261 (2001).</li><li>Maddaluno, L., 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=EndMT+contributes+to+the+onset+and+progression+of+cerebral+cavernous+malformations.">EndMT contributes to the onset and progression of cerebral cavernous malformations.</a> Nature. 498 (7455), 492-496 (2013).</li><li>Cooley, B. C., 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=TGF-&#946;+signaling+mediates+endothelial-to-mesenchymal+transition+(EndMT)+during+vein+graft+remodeling.">TGF-&#946; signaling mediates endothelial-to-mesenchymal transition (EndMT) during vein graft remodeling.</a> Sci. Transl. Med. 6 (227), 227ra34-227ra34 (2014).</li><li>Garcia, 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=Tie1+deficiency+induces+endothelial-mesenchymal+transition.">Tie1 deficiency induces endothelial-mesenchymal transition.</a> EMBO Rep. 13 (5), 431-439 (2012).</li><li>Xiao, L., 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=Tumor+endothelial+cells+with+distinct+patterns+of+TGF&#946;-driven+endothelial-to-mesenchymal+transition.">Tumor endothelial cells with distinct patterns of TGF&#946;-driven endothelial-to-mesenchymal transition.</a> Cancer Res. 75 (7), 1244-1254 (2015).</li><li>Kusumbe, A. P., Ramasamy, S. K., Adams, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+angiogenesis+and+osteogenesis+by+a+specific+vessel+subtype+in+bone.">Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone.</a> Nature. 507 (7492), 323-328 (2014).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+clonally+expanding+haematopoietic+compartment+in+bone+marrow.">Identification of a clonally expanding haematopoietic compartment in bone marrow.</a> EMBO J. 32 (2), 219-230 (2012).</li><li>Sawamiphak, 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=Ephrin-B2+regulates+VEGFR2+function+in+developmental+and+tumour+angiogenesis.">Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis.</a> Nature. 465 (7297), 487-491 (2010).</li><li>Chi, J. -. T., 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+cell+diversity+revealed+by+global+expression+profiling.">Endothelial cell diversity revealed by global expression profiling.</a> Proc. Natl. Acad. Sci. 100 (19), 10623-10628 (2003).</li><li>Nolan, D. 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=Molecular+signatures+of+tissue-specific+microvascular+endothelial+cell+heterogeneity+in+organ+maintenance+and+regeneration.">Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.</a> Dev. Cell. 26 (2), 204-219 (2013).</li><li>Ingram, D. A., 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=Vessel+wall-derived+endothelial+cells+rapidly+proliferate+because+they+contain+a+complete+hierarchy+of+endothelial+progenitor+cells.">Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells.</a> Blood. 105 (7), 2783-2786 (2005).</li></ol>

The authors have nothing to disclose.

由于获 ​​得纯TEC初级培养物，许多体外研究替代TEC与市售的EC系或原乳油例如人脐静脉EC（HUVEC）13的困难。然而，从正常组织这些EC人群可能只能作为TEC从正常的同行明显不同的代理。例如，TEC表型上和功能异常在体内和一些这些异常可能是可透射体外 14-18。 TEC有异常生长，迁移和分化的能力，并与CD31 +肿瘤细胞可能聚结形成血管样结构16,19,20。...

内皮细胞（EC）是实体肿瘤的发展过程中是至关重要的。从休眠中的肿瘤血管生成开关来传播和远处转移的播种开始，欧共体形成提供血液，氧气管道，和营养，以维持肿瘤生长1。作为最近提出，EC也有灌注无关的功能，并形成一个支持癌症干细胞和其他肿瘤基质细胞2-5的生长利基。因此，高纯度的肿瘤特异性EC（TEC） 进行体外培养允许常规功能研究，将揭示介导肿瘤血管生成和肿瘤细胞串扰新型的分子机制的光。 EC是高度专业化的视原产6的组织。由于不同的肿瘤类型的异质性和肿瘤微环境，TEC也可以显示反映肿瘤特异性专业化ö独特的功能f显示血管。例如，有在TEC中的基因表达签名来自不同类型或肿瘤7,8-牌号分离引人注目变性。然而，频繁的共纯化非欧共体，特别是肿瘤相关的成纤维细胞和肿瘤细胞，与TEC的可混淆的全基因组表达分析。这些不需要的细胞类型是依赖于长期的 TEC培养体外扩增研究，特别是有问题的。 这里介绍的是，始终从生产肿瘤和其他组织的纯EC文化高保真方法。继欧盟分数和去除共纯化的非欧共体的免疫柱富集，一个额外的克隆环一步捕捉到纯粹的欧共体殖民地使用9。各菌落可以在培养物中多次传代而不会污染非EC中的出现进行扩展。这种方法也产生多重EC克隆从单一孤立的过程，这是理想的内切的研究thelial异质性。另外，它表明，CDH5 CRE：ZsGreen升/秒/升报告小鼠是一种有价值的工具，用于产生“命运映射”和擦掉标记EC其中保持ZsGreen荧光培养10。有了小幅调整方案，这种方法应该适应不同的肿瘤类型或正常组织。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s medium (1 g/L D-glucose) (LG-DMEM)</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 Bullet Kit&#160;</td>
			<td>Lonza</td>
			<td>CC4176</td>
			<td>Not all components used</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (Hyclone)</td>
			<td>Thermo Scientific</td>
			<td>SH30071.03</td>
			<td>Heat inactivated at 56&#176;C for 30 min</td>
		</tr>
		<tr>
			<td>Nu-Serum IV</td>
			<td>Corning</td>
			<td>CB-51004</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBBS)</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS buffer&#160;</td>
			<td>-</td>
			<td>-</td>
			<td>0.5 % BSA and 2 mM EDTA in PBS, filtered through a 0.22 &#956;m filter</td>
		</tr>
		<tr>
			<td>75% v/v ethanol for disinfection</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE microbeads&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) fraction V, 7.5%</td>
			<td>Gibco</td>
			<td>15260-37</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell freezing media (Bambanker)</td>
			<td>Wako Chemicals</td>
			<td>302-14681</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type II&#160;&#160;</td>
			<td>Worthington Biochemical</td>
			<td>LS004176</td>
			<td>Make stock concentration 2 mg/ml in HBSS</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I (DNase)</td>
			<td>Worthington Biochemical</td>
			<td>LS002004</td>
			<td>Make stock concentration 1 mg/mL in PBS</td>
		</tr>
		<tr>
			<td>Dil-Ac-LDL</td>
			<td>Biomedical Technologies</td>
			<td>BT-902</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5M, pH 8.0</td>
			<td>Cellgro</td>
			<td>46-034-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic cell detachment solution (Accutase)</td>
			<td>Sigma-Aldrich</td>
			<td>A6964-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin, 2 % in water, tissue culture grade</td>
			<td>Sigma-Aldrich</td>
			<td>G1393-100ML</td>
			<td>Dilute in PBS to make 0.5 % gelatin solution</td>
		</tr>
		<tr>
			<td>Mouse FcR Blocking Reagent&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral protease (Dispase)</td>
			<td>Worthington Biochemical</td>
			<td>LS02104</td>
			<td>Make stock concentration 2.5 U/mL in HBSS</td>
		</tr>
		<tr>
			<td>PE-rat anti-mouse CD31 antibody</td>
			<td>BD Pharmingen</td>
			<td>553373</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer (BD Pharm Lyse)</td>
			<td>BD Pharmingen</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4 %&#160;</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mm tissue culture dishes</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes (sterile)</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes (sterile)&#160;</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-dissociator tubes (gentleMACS) C tubes)&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Separator&#160; (MidiMACS)</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 100 &#956;m&#160;</td>
			<td>Corning</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Cloning rings (assorted sizes)</td>
			<td>Bel-Art Products</td>
			<td>378470000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotubes</td>
			<td>Thermo Scientific</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting board</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize or disinfect with 75% v/v ethanol before use&#160;</td>
		</tr>
		<tr>
			<td>Dissecting forceps and scissors</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize before use&#160;</td>
		</tr>
		<tr>
			<td>Dissecting pins 2&#34;</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize before use&#160;</td>
		</tr>
		<tr>
			<td>FACS tubes with 35 &#956;m filter cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter cup (Stericup, 0.22 &#956;m)</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-tip marker</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Multistand</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue adhesive (Vetbond)</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Or a centrifuge with similar capacity for 15 mL and 50 mL conical tube centrifugation</td>
		</tr>
		<tr>
			<td>Tissue culture hood</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue dissociator (gentleMACS)</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-235</td>
			<td>Preset program &#34;m_impTumor_01&#34; used for tissue dissociation&#160;</td>
		</tr>
		<tr>
			<td>Liquid nitrogen freezer</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate or rotary shaker</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast light microscope</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture expansion of tumor-specific endothelial cells

新鲜分离的肿瘤特异性内皮细胞（TEC）可以用来探索肿瘤血管生成的分子机制，并用作体外模型用于开发新的血管生成抑制剂用于癌症。然而，长期的鼠内皮细胞（EC）的体外扩增是具有挑战性，因为文化的表型漂移（内皮细胞间质转化），并污染非欧共体。这是为TEC其通过在培养共纯化的成纤维细胞或肿瘤细胞容易outcompeted尤其如此。在这里，高保真的隔离方法，它利用免疫磁珠富集加上菌落筛选和体外扩增的描述。这种方法产生纯乳油级分是完全不含污染基质或肿瘤细胞。它也表明，谱系追踪CDH5 CRE：ZsGreen升/秒/升报告小鼠，与该协议使用本文中所描述，是一种宝贵的工具来验证细胞纯度从这些小鼠孤立欧盟的菌落表现出持久的文化和灿烂的ZsGreen荧光。

统代表了总细胞群体中大多数成人组织11只有轻微的一小部分。因此重要的是充分消化收获组织成单细胞悬浮液，以确保从细胞外基质（ECM）和结缔组织的最大释放乳油。根据我们的经验，CD31介导的免疫选择仅提供丰富的，但不是纯统的部分;因此，另一个关键步骤是物理去除使用克隆环（图1）EC菌落共纯化非EC和选择/扩张。例如，当CD31富集EC铺板无需进一步菌落筛选，非欧?...

Watch this Scientific Journal Video about Isolation and Culture Expansion of Tumor-specific Endothelial Cells at JoVE.com

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

isolation-and-culture-expansion-of-tumor-specific-endothelial-cells

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11.1K 次观看.  University of North Carolina at Chapel Hill. We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models. 