Name: isolation of human lymphatic endothelial cells by multi-parameter fluorescence-activated cell sorting
Uploaded: 2015-05-01T15:00:00.000+00:00
Duration: 7 min 36 s
Description: Watch this Scientific Journal Video about Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting at JoVE.com

作者要感谢贝克基金会和皇家儿童少年基金会"女科学家奖学金"支持Zerina Lokmic的。安德鲁G. Elefanty和爱德华G.士丹利NHMRC资深研究员的研究。在他们的实验室工作是由NHMRC和干细胞澳大利亚支持。

伦理声明:伦理学批准征收的淋巴管畸形及包皮组织从人类研究伦理委员会获得皇家儿童医院，墨尔本，澳大利亚。签名同意从患者的父母接受手术前。组织样本从确诊患者与LMS进行外科手术作为临床管理的一部分，并择期包皮环切收集。所有实验均符合国家卫生和医学研究理事会，澳大利亚的指引执行。 从包皮和淋巴管畸形组织1.准备细胞悬液<ol><li>缓冲器和媒体准备。 <ol><li>使用市售的EGM-2中压子弹套件温热的EGM-2培养基，轻轻解冻试剂盒组分在37℃水浴中制备完整的内皮细胞的介质。在II级生物安全柜，无菌每个组件添加到EGM-2介质。一旦所有的组件添加到媒体，请参阅此媒体称为"完整的内皮细胞培养基"。使用无菌50毫升吸管混合使用前的内容。 注 :所有涉及的细胞培养步骤是在II类生物危害柜进行。所有解决方案都存储在4℃按制造商的说明，并加热至在使用前室温。完整的内皮细胞培养基，细胞培养缓冲液和酶溶液加热至37℃，使用前20分钟。 </li><li>补充完整内皮细胞培养基用50ng / ml的VEGF-C。分装补充内皮细胞培养基到50ml无菌管中并储存于4℃直至使用。储存在4℃下，当媒体是稳定至少4周。 </li><li>通过溶解8.752克氯化钠，1.416克的Na 2 HPO制备钙和镁的磷酸盐缓冲盐水（PBS）4 .2H 2 O和0.395克KH 2 PO 4在1,000ml水中。调节pH至7.4。使用0.22微米的过滤器，并储存在4℃过滤消毒PBS。在使用之前，添加抗生素/抗真菌溶液（在1中使用:100）。 </li><li>为了制备人纤连蛋白，溶解在10毫升无菌水中1毫克纤维连接蛋白。商店重新溶解在100μl的等分溶液在-20℃下。上使用的当天无菌PBS加入10 mL至100μl纤连蛋白等分试样（以得到最终工作的10微克/毫升浓度）。 </li><li>用于酶介质，制备含有0.04％分散酶II，0.25％胶原酶II和0.01％DNA酶I在无菌PBS中的溶液。首先，称量分散酶和胶原酶，放置在无菌的50ml管中，加入的PBS所需的体积和30分钟孵育振荡在37℃下使其溶解。一旦溶解，在生物安全罩过滤消毒（0.22微米过滤器）分散酶/胶原酶溶液。无菌在37℃下加入DNA酶I.商店酶溶液直到使用。 注 :抗酶是一种商业细胞培养级试剂可作为无菌冻干粉。 </li></ol></li></ol>从包皮和淋巴管畸形2.准备细胞悬液<ol><li>称量50ml管含有10毫升的DMEM / 2％抗生素 - 抗真菌溶液。该管将用于组织采集。 </li><li>下列手术切除淋巴管畸形和包皮的组织，在无菌条件下在冰上添加组织标本到管并转移到细胞培养实验室。 </li><li>称量装有组织的管，并计算组织重量。使用该权重来计算的消化所述组织所需的酶溶液的体积。 </li><li>在II级生物安全柜，用无菌镊子来组织和媒体转移到100毫米组织培养皿。用无菌剪刀精细剁碎组织成约1 立方毫米件。 </li><li>转移组织糜混合媒体我NTO 50ml管含有额外的10毫升无菌DMEM / 2％抗生素 - 抗真菌溶液。通过颠倒混合到重悬浮组织。离心样品在300×g离心5分钟。 </li><li>去除上清。基于组织重量，加入1 ml预热（37℃）胶原酶II每100mg组织糜的/ DNA酶I /分散酶II消化溶液。通常，使用将10ml酶溶液对约1g组织糜的。 </li><li>孵育在37℃培养箱的组织为20-90分钟，恒定200rpm振荡。在这一时期结束时，确保几乎所有的组织的被消化成细碎片。 注 :在隔离的从主组织影响细胞存活的时间曝光的细胞胶原酶和分散酶的。我们已经发现根据经验，多数新生儿包皮样品最优地需要消化20-30分钟，而纤维化淋巴管畸形样品可能需要消化60-90分钟。 LM LEC产量S被纤维化的存在量的影响，更纤维组织产生较少的细胞，这可能是由于长期暴露的有害影响，胶原酶II和分散酶II。 </li><li>消化后，将管转移到生物安全柜，并通过一个70微米的过滤器放入一个无菌50ml管中通过消化的组织方案。使用3个毫升注射器活塞与柱塞橡胶，磨下来的剩余组织，直到细胞外基质的只有很小的痕迹观察。 </li><li>与卷的内皮细胞培养基相当于解离酶培养基以回收细胞粘到筛和使酶失活的体积洗细胞过滤网。例如，对于2毫升解离酶介质消化的组织糜的，内皮细胞培养基中加入2毫升至使酶失活。 </li><li>离心在300×g离心5分钟将细胞溶液和吸出上清液。 R&gt; E-暂停在10毫升PBS的细胞。离心细胞在300 XG5分钟后，除去上清液，然后重复细胞洗涤两次。 &gt;重悬细胞沉淀在20ml内皮细胞培养基中。使用台盼蓝然后种子计数细胞2×10&gt; 6个细胞在150厘米&gt; 2烧瓶中20ml的内皮细胞培养基的终体积预涂覆有纤连蛋白。培养在37℃，在5％CO&gt; 2的湿润空气培养箱中。&gt; 注 :预计有核细胞的75％-90％的存活组织消化估算由锥虫蓝排除。初始细胞计数反映所有有核细胞中的细胞悬浮液（ 即角质形成细胞，白细胞，巨噬细胞，结缔组织细胞和血管细胞）。在5-7天的细胞计数反映已附加，存活和细胞培养过程中增殖的细胞。涂层组织培养瓶中用纤连蛋白也提高了随后的LEC和LM LEC生存。 </li><li>过夜孵育后，在洗涤三次以P洗去未结合的细胞BS / 1％的抗生素，抗真菌的解决方案，并添加新鲜的内皮细胞培养基。进行三次洗涤，有效地除去未结合的细胞和红血细胞存在于烧瓶中。改变媒体每隔一天。细胞将是​​〜80％汇合后5-7天。 </li></ol>细胞淋巴管内皮细胞表面标志物流式细胞仪的3抗体染色<ol><li>后的细胞达到80％汇合时，吸出介质并漂洗细胞，用PBS。 </li><li>通过孵育细胞用7ml的细胞脱附溶液分离贴壁细胞如的Accutase每150cm 2烧瓶中5-7分钟，在37℃。 </li><li>收获分离的细胞，通过加入3倍体积的内皮细胞培养基的灭活细胞脱离溶液和细胞悬液转移到一个新的无菌试管中。 </li><li>离心细胞，在300×g离心5分钟。 </li><li>吸去上清液并重新悬浮在5ml PBS中的细胞。离心细胞，在300×g离心5分钟，除去的S然后重复upernatant洗细胞。重新悬浮在5ml PBS中的细胞。 </li><li>计数活细胞的数量。混合10微升细胞悬浮液与90微升0.4％台盼蓝。转移10微升此悬浮液至血细胞计数计数。 注 :最终细胞产率将取决于处理的样本大小。每150cm 2烧瓶范围通常的细胞产量从7×10 5〜1.2的活细胞×10 6。 </li><li>制备用于细胞染色的抗体溶液。 注意 :下面的稀释液进行滴定染色1×10 6中的100微升的染色体积。 <ol><li>稀PE偶联的小鼠抗人VEGFR-3（1:50）; PE-Cy7的缀合的小鼠抗人缀合的CD34（1:200）; APC缀合的小鼠抗人CD31（1:100）和Alexa 488缀合的鼠抗人PODOPLANIN（1:200）中的100微升每组织样品无菌5％FBS / PBS溶液总体积。下面的准备抗体溶液，保持在冰上直到使用。同样准备稀释的同型对照抗体的混合物，以方便流式细胞仪门的设置。 </li></ol></li><li>为了减少非特异性抗体结合，离心细胞，在300×g离心2分钟，除去上清液，然后悬浮细胞在100μl灭菌的5％FBS /每个样品PBS溶液进行染色。孵育细胞在冰上20分钟，在4℃。 </li><li>离心细胞在300 XG 5分钟，弃上清然后重新暂停细胞中的共轭抗体的鸡尾酒。还染色在100μl同种型对照抗体混合物的细胞（1×10 5）的更小的等分试样。孵育细胞在冰上进行20分钟。 </li><li>以除去未结合的抗体，加入2毫升的2％FBS / PBS溶液和离心机将细胞在300×g离心5分钟。吸出上清液，重复清洗。 </li><li>重悬细胞沉淀于同种型对照和抗体染色样品中加入300微升的0.5毫克/毫升丙锭io的要排序啶/ 2％FBS / PBS溶液。放在冰管，直到排序。 </li></ol> 4.细胞分选<ol><li>使用以下材料来设置仪器;对准珠如市售的荧光颗粒，磷酸盐缓冲的基于盐水鞘流体和下降延迟珠，如Accudrop荧光珠。 </li><li>隔离在一个多参数荧光激活细胞分选仪纯化的人淋巴管内皮细胞。设置仪器和对齐根据制造商的建议。此外，运行单染补偿控制与每个排序以生成补偿矩阵，并通过荧光的发射，以从而消除实质泄放如PE进入PE-CY通道。 注 :FACS分选的细胞比例较高生存的隔离，如果一个100微米的喷嘴被使用，细胞分类成内皮细胞培养基中。 </li></ol> 5.细胞培养后FACS排序ING <ol><li>以下排序，离心细胞，在300×g离心5分钟。吸出上清液并将细胞重新悬浮在5-10毫升的培养基（取决于所使用的种子的细胞在烧瓶）。如果低于50,000元分类中，细胞是在纤连蛋白涂覆为25cm 2烧瓶中培养。 </li><li>否则培养在5％的CO 2，空气增湿培养箱将细胞在75cm 2的纤连蛋白涂覆的烧瓶在37℃。术后第2天更换介质。 注 :细胞培养基改变每隔一天，因为延长介质变化的时间似乎有利于非内皮细胞的存活。 PROX-1，VEGFR-3，Podoplanin，CD34和CD31，当细胞是第一分割为进一步扩大15:我们使用免疫组织化学标记的检测验证的淋巴管内皮细胞的表型。 </li></ol>

<ol>
	<li>Baluk, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally+specialized+junctions+between+endothelial+cells+of+lymphatic+vessels.">Functionally specialized junctions between endothelial cells of lymphatic vessels.</a> J Exp Med. 204 (10), 2349-2362 (2007).</li><li>Yao, L. C., Baluk, P., Srinivasan, R. S., Oliver, G., McDonald, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+button-like+junctions+in+the+endothelium+of+airway+lymphatics+in+development+and+inflammation.">Plasticity of button-like junctions in the endothelium of airway lymphatics in development and inflammation.</a> Am J Pathol. 180 (6), 2561-2575 (2012).</li><li>Zawieja, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+physiology+of+lymphatics.">Contractile physiology of lymphatics.</a> Lymphat Res Biol. 7 (2), 87-96 (2009).</li><li>Witte, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+function+relationships+in+the+lymphatic+system+and+implications+for+cancer+biology.">Structure function relationships in the lymphatic system and implications for cancer biology.</a> Cancer Metastasis Rev. 25 (2), 159-184 (2006).</li><li>Andrade, M., Jacomo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+human+lymphatic+system.">Anatomy of the human lymphatic system.</a> Cancer Treat Res. 135, 55-77 (2007).</li><li>Kumar, V., Abbas, A. K., Aster, J. C., Ch Fausto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cotran Pathologic Basis of Disease. 43-78. 2, (2009).</li><li>Shayan, R., Achen, M. G., Stacker, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+vessels+in+cancer+metastasis:+bridging+the+gaps.">Lymphatic vessels in cancer metastasis: bridging the gaps.</a> Carcinogenesis. 27 (9), 1729-1738 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mulliken &amp; Young's Vascular Anomalies Hemangiomas and. , 1059-1076 (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mulliken &amp; Young's Vascular Anomalies Hemangiomas. 14, 562-594 (2013).</li><li>Bellini, C., Hennekam, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+disorders+of+primary+malfunctioning+of+the+lymphatic+system.">Clinical disorders of primary malfunctioning of the lymphatic system.</a> Adv Anat Embryol Cell Biol. 214, 187-204 (2014).</li><li>Schook, C. 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=Primary+lymphedema:+clinical+features+and+management+in+138+pediatric+patients.">Primary lymphedema: clinical features and management in 138 pediatric patients.</a> Plast Reconstr Surg. 127 (6), 2419-2431 (2011).</li><li>Brouillard, P., Boon, L., Vikkula, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+lymphatic+anomalies.">Genetics of lymphatic anomalies.</a> J Clin Invest. 124 (3), 898-904 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=for+the+Study+of+Vascular+Anomalies+(ISSVA)+classification.">for the Study of Vascular Anomalies (ISSVA) classification.</a> Available from: http://www.issva.org/classification.com. , (2014).</li><li>Croteau, S. E., 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=Kaposiform+lymphangiomatosis:+a+distinct+aggressive+lymphatic+anomaly.">Kaposiform lymphangiomatosis: a distinct aggressive lymphatic anomaly.</a> J Pediatr. 164 (2), 383-388 (2014).</li><li>Lokmic, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+lymphatic+malformation+endothelial+cells,+their+in+vitro+characterization+and+in+vivo+survival+in+a+mouse+xenograft+model.">Isolation of human lymphatic malformation endothelial cells, their in vitro characterization and in vivo survival in a mouse xenograft model.</a> Angiogenesis. 17 (1), 1-15 (2014).</li><li>Smith, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+malformations.">Lymphatic malformations.</a> Lymphat Res Biol. 2 (1), 25-31 (2004).</li><li>Chen, E. Y., 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=Similar+histologic+features+and+immunohistochemical+staining+in+microcystic+and+macrocystic+lymphatic+malformations.">Similar histologic features and immunohistochemical staining in microcystic and macrocystic lymphatic malformations.</a> Lymphat Res Biol. 7 (2), 75-80 (2009).</li><li>Norgall, 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=Elevated+expression+of+VEGFR-3+in+lymphatic+endothelial+cells+from+lymphangiomas.">Elevated expression of VEGFR-3 in lymphatic endothelial cells from lymphangiomas.</a> BMC cancer. 7 (2), 75-80 (2007).</li><li>Garzon, M. C., Huang, J. T., Enjolras, O., Frieden, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+malformations.">Vascular malformations.</a> Part I. J Am Acad Dermatol. 56 (3), 353-370 (2007).</li><li>Osborn, A., Dickie, P., Adams, D., Dickie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 20th International workshop on Vascular Anomalies. ISSVA. , (2014).</li><li>Baluk, P., McDonald, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+for+microscopic+imaging+of+lymphangiogenesis+and+angiogenesis.">Markers for microscopic imaging of lymphangiogenesis and angiogenesis.</a> Ann N Y Acad Sci. 1131, 1-12 (2008).</li><li>Lokmic, Z., Mitchell, 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=Engineering+the+microcirculation.+Tissue+engineering.">Engineering the microcirculation. Tissue engineering.</a> Part B, Reviews. 14 (1), 87-103 (2008).</li><li>Lokmic, Z., Mitchell, 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=Visualisation+and+stereological+assessment+of+blood+and+lymphatic+vessels.">Visualisation and stereological assessment of blood and lymphatic vessels.</a> Histol Histopathol. 26 (6), 781-796 (2011).</li><li>Hagerling, R., 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=A+novel+multistep+mechanism+for+initial+lymphangiogenesis+in+mouse+embryos+based+on+ultramicroscopy.">A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy.</a> EMBO J. 32 (5), 629-644 (2013).</li><li>Pollmann, C., Hagerling, R., Kiefer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+lymphatic+vessel+development,+growth,+and+function.">Visualization of lymphatic vessel development, growth, and function.</a> Adv Anat Embryol Cell Biol. 214, 167-186 (2014).</li><li>Breiteneder-Geleff, 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=Angiosarcomas+express+mixed+endothelial+phenotypes+of+blood+and+lymphatic+capillaries:+podoplanin+as+a+specific+marker+for+lymphatic+endothelium.">Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium.</a> Am J Pathol. 154 (2), 385-394 (1999).</li><li>Kaipainen, 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+the+fms-like+tyrosine+kinase+4+gene+becomes+restricted+to+lymphatic+endothelium+during+development.">Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development.</a> Proc Natl Acad Sci U S A. 92 (8), 3566-3570 (1995).</li><li>Schacht, V., 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=T1alpha/podoplanin+deficiency+disrupts+normal+lymphatic+vasculature+formation+and+causes+lymphedema.">T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema.</a> EMBO J. 22 (14), 3546-3556 (2003).</li><li>Kriehuber, E., 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=Isolation+and+characterization+of+dermal+lymphatic+and+blood+endothelial+cells+reveal+stable+and+functionally+specialized+cell+lineages.">Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages.</a> J Exp Med. 194 (6), 797-808 (2001).</li><li>Florez-Vargas, 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=Comparative+analysis+of+D2-40+and+LYVE-1+immunostaiing+in+lymphatic+malformations.">Comparative analysis of D2-40 and LYVE-1 immunostaiing in lymphatic malformations.</a> Lymphology. 41 (3), 103-110 (2008).</li><li>Makinen, 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=PDZ+interaction+site+in+ephrinB2+is+required+for+the+remodeling+of+lymphatic+vasculature.">PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature.</a> Genes Dev. 19 (3), 397-410 (2005).</li><li>Connell, F. 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=Analysis+of+coding+regions+of+VEGFR3+and+VEGFRC+in+Milroy+disese+and+other+primary+lymphoedemas.">Analysis of coding regions of VEGFR3 and VEGFRC in Milroy disese and other primary lymphoedemas.</a> Hum Genet. 124 (6), 625-631 (2009).</li></ol>

The authors have nothing to disclose.

淋巴管内皮细胞在维持流体稳态，对外来抗原的吸收和某些营养素转运免疫应答中起重要作用。 LEC动态平衡可受疾病过程，如细菌感染和肿瘤转移，但淋巴管内皮细胞还可以开发导致形成的不正常的淋巴管和显著发病为受影响的患者的体细胞突变。要通过植入体内和体外实验获得的淋巴管畸形的病因更多的理解，并发现新的治疗选择，我们首先开发了LEC分离方法的基础上使用CD34 CD31 负片波什选择策略15磁珠选择策略。然而，所得到的培养物常常含有细胞比内皮细胞是难以除去其他。随后，该方法是精制用流式细胞仪进行排序，表达CD34 低 CD31 波什 VEGR3 P晶状体上皮细胞OS PODOPLANIN 名次表型 ​​。这种做法造成了对后整理检查包含&lt;非CD34 低 CD31 波什 VEGR3 波什 PODOPLANIN 波什细胞的0.5％相对同质LEC文化。在实践中，通过FACS分选的细胞的优点是&lt;0.5％到减少的非淋巴管内皮细胞的存在。培养淋巴管内皮和晶状体上皮细胞LM有一个典型的鹅卵石形态，成为各地通行13衰老。 在这项研究中，我们已经描述了一种流式细胞仪基于流的协议的分离和的高度富集的淋巴管内皮细胞培养从基于其一套细胞表面标记（CD34 低 CD31 方位 VEGR3 方位 PODOPLANIN 方位 ）的表达正常和异常组织。细胞从主组织以下5-7天的体外扩增的，一个步骤，导致LEC大量富集相比，其在组织中，在隔离的时间频率进行排序。在Surgery我们获得组织从几微克到几十的重量克。这种组织可以大大于它的组合物相对于淋巴管畸形血管目前，组织结疤和淋巴管畸形囊肿血栓存在量会有所不同。所有这些组件将影响多少细胞可以从病变组织中分离出来。因此，初始细胞数的范围可以从几千细胞几百万个细胞。同样，这将影响到细胞悬浮液的组合物和起始意味着排序细胞的数量也将有所不同，从一个样品到另一个细胞的数量。 淋巴管内皮细胞的未分选的包皮样品在以下5-7天在培养的频率范围为0.52％至4.7％，而LM淋巴管内皮细胞的范围从0.8％至12.43％。 LEC的文化由&gt; 99％CD34 低 CD31 波什 VEGR3 波什 PODOPLANIN 波什细胞后排序重新分析并保持一个典型cobbleston后ê形态的文化，直至衰老的通道〜13没有过度生长由非内皮元素。 有必要扩大细胞体外以下初始组织消化和排序，因为无论包皮或淋巴管畸形细胞的产率通常很低。因此，淋巴管内皮细胞的前扩张I N 体外允许种子鼠标室所需的〜2×10 6个细胞的产生。这需要5-7天的体外扩增阶段。虽然我们接受细胞培养物可诱导的原代细胞的表型变化，我们已经表明，这些培养的细胞保持其形成淋巴管畸形状结构在小鼠异种移植模型15的容量。 有在协议中的几个关键步骤，通过FACS分选的细胞时即确定成功的程度。最关键的步骤是将细胞暴露的时间胶原酶并如步骤2.7中所述从主组织隔离期间分散酶。这不仅通过量和组织存在，但也通过分批使用的酶的类型的影响。此外，用于向所述细胞群的特征的荧光标记的抗体需要滴定，优选单克隆抗体。我们测试了几种不同的细胞分离解决方案，如胰蛋白酶/ EDTA，EDTA的TrypLE选择和细胞分离液。我们发现，EDTA不得不被用于在延长的时间周期，以分离细胞和残留的细胞团块往往残留。与此相反，3-5分钟孵育在胰蛋白酶/ EDTA，细胞脱离溶液并的TrypLE选择内被产生单细胞悬浮液。我们发现在CD31，CD34，Podoplanin和VEGFR-3的表达下列任何的酶溶液的治疗没有显着差异。单个染色的样品也应与每排序实验做是为了确保荧光发射光谱不重叠并且因此得出不正确的读数。这些关键步骤也构成流式细胞仪的地方分拣流程中可能需要或故障排除修改的细胞协议的步骤。 时相比，我们的先前公布的磁珠的LEC 15的隔离，隔离淋巴管内皮细胞通过FACS的优点包括以下方面:细胞具有高纯度和鉴定离散细胞亚群和稀有种群异构样品内的更迅速的隔离。流式细胞仪为基础的LEC和LM LEC隔离的主要限制是围绕着以下的限制细胞分选结果可用于验证细胞数。此外，虽然每一尝试我们的测定法和仪器设置规范，这些相同的参数不一定是可再现的其他实验室。因此，可能出现在结果中的一些可变性。此外，该淋巴生物学家面临的问题之一是缺乏的细胞特异性标记将最初的毛细管LEC标志和收集毛细管LEC标记区分。在这个阶段，我们还没有确定的LEC的标记，将健康的淋巴管内皮细胞和LM淋巴管内皮细胞之间进行区分。由于淋巴管畸形组织中还含有正常外观的淋巴管（ 图1A），将所得的CD34 低 CD31 方位 VEGR3 方位 PODOPLANIN 方位将包含正常淋巴管内皮细胞的比例，以及那些患病表型。未来的研究考察LEC和LM LEC基因表达和蛋白质可以是能够直接我们走向更具体的LM LEC标记，可以区分的LM淋巴管内皮细胞从淋巴管内皮细胞在相同的组织中。 在未来，我们希望利用流式细胞仪和这些新LEC标记在试图找出无论是在包皮和淋巴管畸形LEC人群罕见的子集。这将允许我们隔离这些细胞群，并研究在lymp发展的作用hatic血管系统和淋巴管畸形。 基于CD34 低 CD31 波什 VEGR3 波什 PODOPLANIN 波什隔离的LEC和LM的LEC显著降低非内皮细胞污染。此外，流式细胞仪技术使我们能够晶状体上皮细胞分成基于CD31，PODOPLANIN和VEGFR-3细胞表面标志物的表达亚型和理解这种细胞谱系发展的背景下。对于患病淋巴管内皮细胞，这允许细胞特异性的研究，将阐明基因引起的LEC疾病和LEC能力在体外和动物异种移植物模型响应各种细胞刺激另外的光。

淋巴血管系统的一个主要功能是吸收淋巴液，含有脂质，蛋白质和细胞成分的过量的组织液，并进行到血液静脉系统。毛细淋巴管的一个网络指示淋巴在那里它筛选外源抗原，在免疫监视和部署的白血细胞中和外来抗原的重要过程的存在的淋巴结。 单向淋巴系统开始在组织中与初始毛细淋巴管，一个独特的结构，薄壁扁平的内皮细胞的一个连续单层与专门的细胞连接，允许淋巴条目1,2。这些毛细管经由锚定纤丝以防止在增加间质的压力3存在容器塌陷附连到相邻的结缔组织基质。最初的毛细淋巴管直接排入收集毛细淋巴管这凝聚成较大的淋巴管静脉或。在比较初始淋巴毛细血管，收集淋巴管具有较厚的血管壁，配对淋巴阀和由不连续的基底膜，其中几个平滑肌细胞包埋4被装入。平滑肌细胞协调开口和淋巴阀门关闭和收缩有助于流淋巴结3。在人体中，从身体的各个区域淋巴静脉连接形成淋巴中继线其合并以形成两个淋巴管:胸导管和右淋巴管。胸导管排出淋巴液从身体的左侧和从右侧胸部下方而从右侧臂的头，颈和胸部的和右侧的右淋巴管下水道淋巴。两管道在颈部5进行淋巴进入锁骨下静脉。 淋巴系统疾病，大致分为获取的第二先天性（ 表1）。收购条件的例子是淋巴管炎和继发性淋巴水肿。淋巴管炎是一种淋巴管的炎症因细菌感染。受影响的淋巴管扩张并充满渗出液含有多形核细胞。在皮肤中，这些淋巴管是作为红，痛苦皮下条纹常伴相关引流淋巴结（淋巴结）6的放大可见。继发性淋巴水肿出现损坏或阻塞的淋巴管或淋巴结阻塞的后果。这导致慢性进行性肿大，由于淋巴结远端积累的损坏或阻塞。在发达国家中，继发性淋巴水肿是最常见的恶性肿瘤，其中转移性肿瘤妨碍淋巴管或区域淋巴结，或作为抗癌治疗的结果在手术切除的淋巴结，辐照后纤维化和炎症后血栓的相关bosis和疤痕7。在世界的其他地方，继发性淋巴水肿可继发于引起寄生蠕虫淋巴阻塞如班氏丝虫 6。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td colspan="4"> 淋巴血管系统疾病 </td></tr><tr><td> 后天 </td><td colspan="3"> 先天性 </td></tr><tr><td rowspan="3">淋巴结炎 继发性淋巴水肿</td><td> 原发性淋巴水肿10 </td><td> 零星的淋巴管畸形13 </td><td> 淋巴管畸形伴有证13 </td></tr><tr><td rowspan="2"> 例如: 米尔罗伊综合征 美格综合征</td><td> 简单 : 淋巴管畸形</td><td rowspan="2"> 例如: 畸形骨Tranaunay综合征 公园Weber综合征 斯特奇 - 韦伯综合征</td></tr><tr><td> 结合 : 毛细淋巴管畸形 毛细淋巴管静脉畸形 毛细淋巴管，动静脉畸形 毛细淋巴管静脉动静脉畸形</td></tr></tbody></table> 表1概述淋巴血管系统的疾病。 淋巴系统的先天性病症包括初级（特发性）淋巴水肿被认为是由基因突变，淋巴管扩张和淋巴系统8,9的异常所引起的。初级淋巴水肿可能是零星的推测是通过从头突变引起，或继承。淋巴病症也可以是分离的或包括一个更一般化综合征10的一部分。在儿科人群中，97淋巴结％水肿是零星的，在淋巴管结构异常的影响区域淋巴结引流11。米尔罗伊疾病是由突变的VEGFR-3的基因明显在出生时或不久后12初级淋巴水肿的一个例子。虽然大多家族条件下，米尔罗伊疾病，也可在婴儿识别未经米尔罗伊疾病32的家族史。任何淋巴水肿的严重程度是依赖于淋巴生产和运输淋巴回静脉循环6能力的量。 根据临床表现和原位内皮细胞增殖，淋巴系统的异常被分类为淋巴肿瘤或淋巴管畸形13。 Kaposiform淋巴血管瘤病是一个LEC肿瘤14的一个例子。淋巴管畸形被认为是胚胎发育过程中产生和成长的比例给孩子15,16。他们很少倒退，但可以remaiñ无症状直到外伤或感染沉淀的快速增长，导致临床并发症。淋巴管网和淋巴传导的有序结构，从组织到上述静脉循环受到干扰的淋巴管畸形其中包括充满淋巴液不正常的囊性结构的局部集合。虽然没有临床或实验证据表明，这些囊状容器被连接​​到淋巴循环或它们包含官能淋巴阀，其淋巴同一性是通过淋巴细胞标记物如PODOPLANIN，CD31，淋巴管内皮受体1范围表达证实（LYVE-1），普洛斯彼罗同源异型盒蛋白1（PROX-1）和VEGFR-3 15,17,18。这些囊性结构可以是小的（微囊）或大（macrocystic），但大多数淋巴管畸形同时包含微囊和macrocystic组件（ 图1）16。手术后，注射液ñ硬化和/或射频消融治疗的淋巴管畸形往往重演。 <img alt="图1" src="/files/ftp_upload/52691/52691fig1highres.jpg" width="700" /> 图1.形态人体淋巴血管和淋巴管畸形。正常人体淋巴（A）和淋巴管畸形血管（B和C）标记抗体PODOPLANIN（棕色标签，箭头）。人类淋巴管畸形血管的特征是显着的扩张和管腔大小相当大的变化。这些局部异常囊性结构可以是小的（微囊，*）（B）或大（macrocystic，＃）（℃）。多数淋巴管畸形同时含有微囊和macrocystic组件。 <a href="/files/ftp_upload/52691/52691fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> &lt;/ P&gt; 一些研究者已经提出，淋巴管畸形代表淋巴脉管系统的发育障碍，其中所述淋巴管内皮细胞不具有异常生长潜力，而是未能连接到正常循环19。然而，我们已经发现，在LM淋巴管内皮细胞增殖速度更快，对细胞凋亡比包皮的LEC 15提示有在LM淋巴管内皮细胞的主缺陷更有抵抗力。当LM晶状体上皮细胞植入小鼠异种移植模型中，他们形成的结构让人想起淋巴管畸形15。这支持一个假说，即淋巴管畸形可能由胎儿发育过程中的LM淋巴管内皮细胞产生的一种或多种体细胞突变引起的。事实上，最近的报告已经确定这样的一个突变在磷酸肌醇-3-激酶（PIK3CA）基因20的p110α催化亚基。 鉴于DNA测序技术，相关的突变c中的进步乌尔德在孤立的LM晶状体上皮细胞更容易识别，引导这些条件进一步研究。可行的淋巴管内皮细胞的分离将促进试验，例如迁移，增殖，管形成能力和存活异常和正常淋巴管内皮细胞之间的比较响应于降低的养分可用性或促凋亡剂15。孤立的LEC将进一步使我们能够进行细胞特异性基因表达和蛋白质组研究中，描绘新的LEC亚群，并发现适合淋巴管畸形的临床管理新的药理学试剂。 之前我们已经发布了基于晶状体上皮细胞从新生儿包皮和淋巴管畸形15磁珠分离LEC隔离法。我们报道从基于缺乏CD34的表达的，接着进行的CD34 负片细胞级分，以正选择为CD31血管内皮细胞的分离正常和患病的淋巴管内皮细胞的一个策略。然而，这种方法受到阻碍残留非内皮细胞的存在。这是独立前的后续结缔组织消化去除表皮。这些污染物通常增殖得更快，从而最终茂密生长尽管后续尝试重复LEC隔离的内皮细胞培养物。的确，非内皮细胞低至2％至5％的初始污染足以压倒LEC群体15。这促使我们探索荧光激活细胞分选法作为一种选择，以改善LEC细胞产率和纯度。此外，我们使用了多参数排序以增强LEC群体的特异性，增加VEGFR-3和PODOPLANIN到选择标记，以确定CD34 低 CD31 方位 VEGFR-3的方位 PODOPLANIN 方位淋巴管内皮细胞。 选择这些标记的基本原理是根据报告，虽然晶状体上皮细胞和血液血管内皮CELLS有许多共同的细胞表面标志物，例如CD31，淋巴管内皮细胞表现出在它们的CD34，PODOPLANIN和VEGFR-3的细胞表面标志物的表达表型变异时相比，血液血管内皮细胞21-23。 CD31是一种130 kDa的跨膜糖蛋白也被称为血小板内皮细胞粘附分子-1（PECAM-1）。它被认为是一个泛内皮细胞标记物，因为它表达于所有类型的血液和淋巴管21,24,25。 CD34是存在于大多数造血祖110-kDa的跨膜糖蛋白和干细胞，血管内皮细胞和某些淋巴管26。 VEGFR-3，受体对血管内皮细胞生长因子C和D，是最初存在于在小鼠胚胎显影静脉，但以下淋巴规范由转录调控因子SRY相关的HMG-盒（SOX）-18 角 hicken Øvalbuminüpstrea米p romoter 牛逼ranscription˚F演员2（COUPTF-II）和PROX-1，VEGFR-3静脉表达丢失，它成为限制胚胎的LEC 25,27。 PODOPLANIN，一个38 kDa的膜粘蛋白，首先注意对小鼠胚胎发育28淋巴管在胚胎大约11天（〜E11.0），并同时强烈微血管淋巴管，PODOPLANIN表达在淋巴管畸形macrocystic淋巴管内皮细胞表达更变15。流式细胞术实验表明，至少一些的CD34 高 CD31 方位内皮细胞表达淋巴标记PODOPLANIN 29。虽然LYVE-1和PODOPLANIN染色在人淋巴管畸形系统的评估表明，两者都是在染色淋巴管畸形内皮30有效，在正常组织中，LYVE-1据报道，强烈存在于初始淋巴毛细血管内皮但降低，即使在收集淋巴管内皮31不存在。因为我们的目标是隔离两个我们已选择的初始和收集淋巴管内皮细胞不使用LYVE-1作为我们的小区选择策略的一部分。最后，聘请这些标记的决定也是基于抗体的诊断被用于为显微成像标记淋巴管的可用性，这将允许流式细胞仪及免疫研究之间相关性的特征。 本文将描述该组织消化法，细胞染色和FACS设置所需的CD34 低 CD31 波什VEGFR-3 POS PODOPLANIN 波什晶状体上皮细胞的成功分离，以及来自包皮和淋巴管畸形高 ​​CD34 CD31 波什VEGFR-3 POS PODOPLANIN 波什内皮细胞组织。

<table><tbody><tr><td>DMEM</td><td>Life Technologies (Gibco)</td><td>11965-092</td><td>Used to collect tissue samples from the operating theatre.</td></tr><tr><td>EGM-2 MV Bullet Kit</td><td>Lonza</td><td>CC-3202</td><td>EGM-2 MV Bullet Kit contains 500 ml of EBM-2 media and human EGF, hydrocortisone, gentamycin (GA-1000), fetal bovine serum (FBS), VEGF, human FGF-b, R&lt;sup&gt;3&lt;/sup&gt;-IGF-1 and ascorbic acid.</td></tr><tr><td>VEGF-C</td><td>R&amp;amp;D Systems</td><td>2179-VC-025</td><td>Complete endothelial cell media is supplemented with 50 ng/ml VEGF-C</td></tr><tr><td>Antibiotic-antimycotic solution (100x)</td><td>Life Technologies (Gibco)</td><td>15240-062</td><td>This solution contains 10,000&amp;nbsp;U/ml of penicillin, 10,000&amp;nbsp;mg/ml of streptomycin and 25&amp;nbsp;mg/ml of Fungizone antimycotic. Use at 1:100 dilution.</td></tr><tr><td>Fibronectin from human plasma</td><td>Sigma-Aldrich</td><td>F2006</td><td>Used at 10 mg/ml concentrations. It can be re-used for up to 1 month if kept at 4&amp;nbsp;&amp;#176;C and maintained sterile. Use 7&amp;nbsp;ml to coat 150 cm&lt;sup&gt;2&lt;/sup&gt; flask.</td></tr><tr><td>StemPro Accutase Cell dissociation reagent</td><td>Life Technologies (Gibco)</td><td>A11105-01</td><td>StemPro&amp;reg;Accutase&amp;reg; is used at 0.05 ml per cm&lt;sup&gt;2&lt;/sup&gt; to cover the entire surface area of the flask. Using lesser volumes may result in incomplete cell detachment.</td></tr><tr><td>0.4% Trypan Blue dye</td><td>Life Technologies (Gibco)</td><td>15250-061</td><td>Used as a viability stain.</td></tr><tr><td>Dispase II</td><td>Roche Applied Biosciences</td><td>4942078001</td><td>Used at 0.04%</td></tr><tr><td>Collagenase II</td><td>Worthington Lab</td><td>4176</td><td>Used at 0.25%</td></tr><tr><td>DNase I</td><td>Roche Applied Biosciences</td><td>11284932001</td><td>Used at 0.01%</td></tr><tr><td>70 mm nylon cell strainers</td><td>BD Falcon</td><td>352350</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td>PE-conjugated VEGFR-3 clone 9D9F9, clone WM59</td><td>BioLegend</td><td>356204</td><td>Used at 1:50 dilution</td></tr><tr><td>PE-Cy7-conjugated CD34, clone 581</td><td>BioLegend</td><td>343516</td><td>Used at 1:200 dilution</td></tr><tr><td>APC-conjugated mouse anti human CD31, clone WM59</td><td>BioLegend</td><td>303116,</td><td>Used at 1:100 dilution</td></tr><tr><td>Alexa 488-conjugated rat anti human Podoplanin, clone NC-80.</td><td>BioLegend</td><td>337006</td><td>Used at 1:200 dilution</td></tr><tr><td>PE-conjugated mouse IgG1, k isotype, clone MOPC-21</td><td>BioLegend</td><td>400112</td><td>Used at 1:50 dilution</td></tr><tr><td>PE-Cy7-conjugated mouse IgG1, k isotype, clone MOPC-21</td><td>BioLegend</td><td>400126</td><td>Used at 1:200 dilution</td></tr><tr><td>APC-conjugated mouse IgG1, k isotype, clone MOPC-21</td><td>BioLegend</td><td>400120</td><td>Used at 1:100 dilution</td></tr><tr><td>Alexa 488-conjugated mouse IgG2a, k isotype, clone RATK2758</td><td>BioLegend</td><td>400525</td><td>Used at 1:200 dilution</td></tr></tbody></table>

isolation of human lymphatic endothelial cells by multi-parameter fluorescence-activated cell sorting

淋巴系统疾病如原发性淋巴水肿，淋巴管畸形和淋巴管瘤是导致发病率显著但鲜为人知的是，他们的生物学罕见的情况。隔离患病和健康组织高纯度的人淋巴管内皮细胞（晶状体上皮细胞）将促进淋巴管内皮细胞在基因，分子和细胞水平的研究。可以预料，这些调查可能揭示目标为新的疗法可能改变这些条件的临床管理。描写人包皮淋巴管内皮细胞和淋巴管畸形淋巴管内皮细胞（LM的LEC）的隔离协议提出。以获得单细胞悬液组织切碎并用分散酶II和胶原酶II酶处理。将所得的单细胞悬浮液，然后标记抗体的分化（CD）标志物CD34，CD31，血管内皮生长因子-3（VEGFR-3）和PODOPLANIN群集。 STAI奈德的活细胞进行分选上的荧光激活细胞分选（FACS）对CD34 低 CD31 方位 VEGFR-3的方位 PODOPLANIN 方位的LM LEC人口从其他内皮细胞和非内皮细胞中分离出来。排序LM淋巴管内皮细胞培养和扩增的纤维连接蛋白包瓶中进一步的实验中使用。

以下初始组织消化，在24小时中未分离的样品培养之后，不同的内皮细胞集落可以观察到（ 图2A）一起成纤维细胞样细胞和平滑肌细胞。以下排序和24小时细胞培养物后，将CD34 低 CD31 方位 VEGFR-3的方位 Podoplanin 方位细胞附着并显示典型鹅卵石形态（ 图2B和C）。使用上述流式细胞仪的方法，我们能够从CD34 高 CD31 方位 VEGFR-3的方位 Podoplanin 方位内皮细胞纯化细胞高达99.8％纯度和区分它们。门控策略和排序图3给出的代表性结果。这已经解决的问题用磁珠分离方法时，经验丰富。到目前为止，我们已经代这些细胞多达通道13这段话后，LM晶状体上皮细胞和包皮LEC先从衰老，伴随着形态学改变，降低细胞分裂。 <img alt="图2" src="/files/ftp_upload/52691/52691fig2.jpg" /> 图2.包皮淋巴管内皮细胞和LM-LEC。24小时酶消化后，未分选的细胞中含有两个内皮（箭头）和非内皮细胞（A）中 。继CD34 低 CD31 波什VEGFR-3 POS Podoplanin 波什流式细胞仪细胞分离，包皮的LEC（B）和LM-的LEC（C）是缺乏非内皮细胞和维护鹅卵石形态。 <a href="/files/ftp_upload/52691/52691fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。 。</a> <img alt="图3" src="/files/ftp_upload/52691/52691fig3.jpg" /> 图3.荧光激活细胞对血管的细胞分选和淋巴管内皮细胞分选门控策略第（A） 的在线细胞首先分别门上的CD34和CD31的表达，随后VEGFR-3和PODOPLANIN选通来排序血管（B）和淋巴管内皮（C）的细胞。 （D）重新分析排序CD34 高 CD31 波什VEGFR-3 POS PODOPLANIN 波什细胞显示&gt; 98％，血管内皮细胞的表型。 （ 五）LEC细胞分选上CD34 低 CD31 波什VEGFR-3 POS PODOPLANIN 名次表型 ​​也&gt; 98％的纯度。 <a href="/files/ftp_upload/52691/52691fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting at JoVE.com

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

这个协议的目标是隔离淋巴管内皮细胞衬里使用荧光激活细胞分选（FACS）人淋巴管畸形囊肿状容器和包皮。随后的细胞培养和扩大这些细胞的实验允许为复杂遗传，蛋白组学，功能和细胞分化研究的一个新的水平。

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

isolation-human-lymphatic-endothelial-cells-multi-parameter

Universidad San Sebastian

Research

JoVE Journal

Medicine

14.3K 次观看.  The Royal Children&#146;s Hospital. 这个协议的目标是隔离淋巴管内皮细胞衬里使用荧光激活细胞分选（FACS）人淋巴管畸形囊肿状容器和包皮。随后的细胞培养和扩大这些细胞的实验允许为复杂遗传，蛋白组学，功能和细胞分化研究的一个新的水平。 

视频: Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

鼠淋巴结基质细胞的分离

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

科研

免疫与感染

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

小鼠视网膜内皮细胞分离用于二代测序

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

发育生物学

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

小鼠小肠免疫系统淋巴细胞的分离

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

分离子宫先天淋巴细胞，用于流式细胞术分析

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and T lymphocytes. Studies of zebrafish lymphopoiesis are difficult because antibodies recognizing D. rerio cell surface markers are generally not available, complicating isolation and characterization of different lymphocyte populations, including B-lineage cells. Transgenic lines with lineage-specific fluorophore expression are often used to circumvent this challenge. The transgenic lck:eGFP line has been used to study D. rerio T cell development, and has also been utilized to model T cell development and acute lymphoblastic leukemia (T-ALL). Although lck:eGFP fish have been widely used to analyze the T-lineage, they have not been used to study B cells. Recently, we discovered that many zebrafish B cells also express lck, albeit at lower levels. Consequently, lck:eGFP B cells likewise express low levels of GFP. Based on this finding, we developed a protocol to purify B-lineage cells from lck:eGFP zebrafish, which we report here. Our method describes how to utilize a fluorescent-activated cell sorter (FACS) to purify B cells from lck:eGFP fish or related lines, such as double-transgenic rag2:hMYC; lck:eGFP fish. In these lines, B cells, particularly immature B cells, express GFP at low but detectable levels, allowing them to be distinguished from T cells, which express GFP highly. B cells can be isolated from marrow, thymus, spleen, blood, or other tissues. This protocol provides a new method to purify D. rerio B cells, enabling studies focused on topics like B cell development and B lymphocyte malignancies.

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Transgenic lck:eGFP zebrafish express GFP highly in T lymphocytes, and have been used to study T cell development and acute lymphoblastic leukemia. This line can be used to study B cells, which express lck at lower levels. This protocol describes purification of malignant and non-malignant B cells from lck:eGFP zebrafish.

从 lck 中分离恶性和非恶性 b 细胞: egfp斑马鱼

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and ...

医学

使用磁性细胞选择建立体外 LLEC 培养物

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro.
This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

作者聚焦：揭示软脑膜淋巴内皮细胞的细胞功能和潜在临床意义 

Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.

软脑膜淋巴内皮细胞的收获和原代培养

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...

人淋巴管内皮细胞中的多参数荧光激活细胞分选分离

摘要

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此视频中的章节

鼠淋巴结基质细胞的分离

从表型分析和人体皮肤淋巴细胞分离

小鼠视网膜内皮细胞分离用于二代测序

小鼠小肠免疫系统淋巴细胞的分离

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

分离子宫先天淋巴细胞，用于流式细胞术分析

从 lck 中分离恶性和非恶性 b 细胞: egfp斑马鱼

软脑膜淋巴内皮细胞的收获和原代培养

软脑膜淋巴内皮细胞的收获和原代培养

软脑膜淋巴内皮细胞的收获和原代培养

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

摘要

探索更多视频

此视频中的章节

鼠淋巴结基质细胞的分离

从表型分析和人体皮肤淋巴细胞分离

小鼠视网膜内皮细胞分离用于二代测序

小鼠小肠免疫系统淋巴细胞的分离

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

分离子宫先天淋巴细胞，用于流式细胞术分析

从 lck 中分离恶性和非恶性 b 细胞: egfp斑马鱼

软脑膜淋巴内皮细胞的收获和原代培养

软脑膜淋巴内皮细胞的收获和原代培养

软脑膜淋巴内皮细胞的收获和原代培养

人淋巴管内皮细胞中的多参数荧光激活细胞分选分离