我们承认在K·富塔默拉，谁也批判校订手稿的光谱仪的技术和理论贡献。我们也感谢C.艾特·曼苏尔，P.-H. Commere，A班代拉和P.佩雷拉批判地阅读手稿和无尽的技术咨询和支持。我们也感谢P·佩雷拉为防Vγ7-APC和Vδ4生物素标记的抗体的礼物。我们aknowledge从巴斯德研究所的中心D&#39;Enseignements的欢迎和支持拍摄物流。这项工作是由巴斯德研究所，研究所国家德拉桑特等德拉RECHERCHE MEDICALE（INSERM）的支持;瑞士国家科学基金会和巴斯德交易所鲁（SS）; Fundação第一个西恩西亚EA TECNOLOGIA - SFRH / BD /二千零十分之七万四千二百一十八（MV）;和巴黎大学狄德罗，法新社国立德拉RECHERCHE（ANR）项目Twothyme的ANR，并计划REVIVE（对于未来的投资）（AC）。

所有实验均根据巴斯德研究所伦理宪章和欧盟指南进行，并且法国农业部批准。 从成年小鼠器官1.细胞悬浮液的制备<ol><li> 脾细胞的分离 <ol><li>颈椎脱位安乐死的成年小鼠。请在腹部正中切口，打开用剪刀皮肤。嘉实用钳子脾。 </li><li>粉碎2玻璃显微镜之间的脾滑动以解离细胞，并稀释它们在5mL Hank平衡盐渍溶液（HBSS）含有1％胎牛血清（FCS）。 </li><li>离心机在120×g离心10分钟，4℃。弃去上清液。 </li><li>重悬在5ml HBSS 1％FCS的沉淀。计数与使用台盼蓝活力染色纽鲍尔室中的细胞。 注:80万个细胞应该从一个成人脾脏中获得。 </li></ol></li><li> Isolati对细胞从小肠<ol><li>颈椎脱位安乐死的成年小鼠。用剪刀，使皮肤切口在腹部正中线。抓住小肠用钳子，一手用剪刀剪下它的两端:第一，胃之后，然后在小肠的末端，只是大肠之前。使用2毫升的注射器用针冲洗小肠用1X Dulbecco氏磷酸盐缓冲盐水（DPBS）。 </li><li>放在纸巾小肠并小心地用一对锋利的剪刀的去除淋巴集结。 </li><li>用剪刀插入肠道内的剪刀的一个分支，以打开其长边肠道。切开肠成使用锋利的剪刀1厘米的片。 </li><li>孵育在37℃下持续搅拌下在30mL HBSS中有10％FCS 30分钟。 </li><li>放置与剩余的非解离的片段的细胞悬浮液在50mL塑料管，涡旋以高速5分钟。注:还有就是组织没有进一步的分解。 </li><li>孵育在冰上10分钟，以允许大的，非解离的片段以沉淀。 </li><li>收集上清液并通过离心，在120×g下7分钟集中它。 </li><li>弃上清，用1％FCS重悬在10mL HBSS的沉淀。计数与使用台盼蓝活力染色纽鲍尔室细胞。 注:〜6000万内上皮细胞（IELs）应该从一个成人的小肠中获得。 </li></ol></li><li> 面板设计策略 <ol><li>制备具有直接与荧光染料的抗体的面板。 注意:所有的抗体被预先滴定以获得细胞群的最佳清晰度（滴定可以与抗体批次而变化）。在19彩色面板用于染色的脾细胞的抗体列在表1中 。 </li><li>构建多科洛科洛[R面板的流式细胞仪。由于这是一项艰巨的任务，请遵循以下准则，以优化面板: <ol><li>检查频谱配置，并且可以在仪器上使用的荧光染料。 </li><li>使用由制造商提供的亮度指标/染色指数信息。 注:亮度指数/染色指数为荧光强度对背景对于每个荧光染料的相对指标。 </li><li>选择遵循以下规则抗体: <ol><li>选择对于那些弱表达的蛋白质中最亮的荧光染料和更多的高表达的蛋白质的最暗的荧光染料。 注意:例如，CD45，CD4，和CD8中高度表达，并且可以与暗淡的荧光染料使用。 </li><li>开始，首先选择具有最少的荧光染料的可能性抗体设计的面板。 </li><li>如果可能的话，应选择具有发射光谱f的最小重叠的荧光染料或标记上相同类型的细胞中表达。 注:双座（ 例如，PE-Cy5的，PE-Cy7的，和PerCP-Cy5.5的）应当谨慎使用，因为它们是通过光照射而降解更敏感。 </li></ol></li></ol></li></ol></li><li> 细胞仪分析的染色 <ol><li>制备一个5毫升管1×10 6脾细胞和一个5毫升管1×10 6个肠细胞并保持未染色用作阴性对照。 </li><li>准备并根据表1每个样品和每个面板标记一个5毫升管中。制备抗体的组合，根据表1，在HBSS 1％FCS。 </li><li>传送1×10 6个细胞-脾细胞要么或肠细胞-到每个5毫升管中。添加HBSS的2毫升1％FCS和离心机在4℃，5分钟和120×g下。弃上清，重悬沉淀的抗体混合物的50μL。 </li><li>标签的S全细胞分两步。首先，使用50μL含有PE-Cyanine 5，PerCP-Cyanine 5.5和PerCP（具有Fc受体阻断）标记的抗体的混合物在5mL管中。在4℃的黑暗中孵育20分钟后，加入50μL含有所有其他抗体的混合物至5 mL管中。 注意:这个策略限制空间位阻。 </li><li>在4℃的黑暗中孵育20分钟。 </li><li>加入2mL HBSS 1％FCS，并在4℃和120×g离心5分钟。弃去上清液，并将沉淀物重悬于200μL含有0.5μg/ mL碘化丙啶（PI）的HBSS 1％FCS中。 </li></ol></li></ol> 2.商业补偿珠微球（ 例如 UltraComp eBeads）上的每个荧光色素的单染色的制备，此后称为珠<ol><li>准备和标记与面板中使用的抗体数量相同的5 mL管。在每个管中放置1滴珠，并加入1＆＃181，L每管抗体。 注:这里使用的珠子（见材料表）包含染色（阳性）和未染色（阴性）珠。抗体被置于过量的小珠，以确保明亮的信号，从而明确的光谱标志对于每个染料。这是由软件需要能够做出精确的光谱分离。 </li><li>孵育在4℃下在黑暗中20分钟。 </li><li>加入2毫升HBSS 1％FCS和离心机的5分钟，在120×g下。弃上清，重悬沉淀在100μLHBSS 1％FCS的。 </li></ol>从胚胎小鼠心脏3.细胞悬浮液的制备<ol><li> 胚胎的解剖 <ol><li>打算通过在一天结束配合与一个阳（总是在男性的笼子）的女性2（17和19小时之间），以获得预先定时怀孕女性。第二天早上，阴道塞的女性被认为是0.5天后交配。 </li><li>安乐死E17.5孕雌性颈椎脱位ES。确保该动物是通过在小鼠爪（脚趾捏反射）用力按压响应。湿用70％乙醇的腹部消毒，防止老鼠皮毛的传播。 </li><li>使皮肤切口在腹部用剪刀中间，用手指拉开皮肤。 </li><li>通过朝向使用毛镊子和剪刀腹部的两侧从中心在腹部肌肉牵拉，使切口打开腹腔。 注:小心不要损坏小肠，这是立即腹腔肌肉下方。 </li><li>通过既在和卵巢的子宫体的水平切割收集子宫角（与胚胎）。浸泡其中在一个90×15毫米2培养皿用50mL DPBS的。 </li><li>由每个蜕膜，其次是子宫肌壁和用细镊子和剪刀的卵黄囊之间切割隔离每个胚胎。拉出胚胎INT法案。最后，剪断脐带。 </li><li>在开始解剖前切E17.5胚胎用剪刀（斩首）的头。 </li></ol></li><li> 心中的集合 <ol><li>浸泡断头胚在含有在培养皿底部的湿纸巾寝具和50mL HBSS的用1％FCS 90×15毫米2培养皿中。 </li><li>在立体显微镜下，用保持毛钳子每个胚使得腹侧朝上。 </li><li>小心地切割用剪刀胸部右侧，以避免对心脏的损伤打开胸腔电网。 </li><li>通过按住肋骨与毛钳暴露心脏。 </li><li>通过保持大血管（含有的入口和流出心脏的大血管）拉开心脏（与肺和胸腺仍然组装）并将它们放置在一个35×15毫米2培养皿用2mL的HBSS 1％有FCS。 </li><li>从周围器官隔离心和结缔组织用细镊子和手术刀。 </li><li>洗心在一个新的35×15毫米的培养皿用2mL HBSS 1％FCS的并保持在冰上20分钟，以允许心脏泵出腔室内部的血液。 </li></ol></li><li> 心脏细胞悬浮液的制备 <ol><li>预温热的HBSS + / +（以下简称为酶溶液）的200微克/毫升胶原酶至37℃。 注:稀释胶原酶在HBSS + / +（用Ca 2+和Mg 2+阳离子）最大化的酶活性;胶原酶活性是批次之间是稳定的。 </li><li>替换洗涤溶液中浸泡，用1mL预温酶溶液的心（HBSS 1％FCS）。 </li><li>在立体显微镜下，剁碎的心成1毫米使用细镊子和手术刀3件。添加另一个1毫升预热的酶溶液和切碎的组织有盖转移到5mL的试管中。 注:对于最佳的消化，放置最大的每2毫升的酶溶液5个E17.5心。如果有更多的心都在同一时间消化后，将它们分为不同的5个毫升管中。 </li><li>孵育剁碎心15分钟，在37℃。 注:放下在培养箱中5支毫升的管（正常关闭），以沿着所述溶液散布在组织。 <ol><li>在温育结束时，通过用移液管P1000上下吸液约20次均质化在相同的酶溶液的组织。把5毫升管在垂直位置，并让剩余的组织片段沉淀（大约3分钟）。 </li><li>收集上清液（含有在悬浮液中的消化的细胞）在50mL管中，加入2毫升HBSS的10％FCS（相同的体积的酶溶液的体积加入到该组织片段），并保持在冰上，同时继续消化过程。 注意:HBSS 10％FCS溶液和冰是重要的为不活动的酶的作用而剩余的组织是消化。 </li><li>加入2毫升预热的酶溶液于5 mL管与剩余的组织碎片和通过重复步骤3.3.4直到没有更多的组织被观察到继续消化。 注:消化的大约4个周期是足以完全解离的心脏组织（5个E17.5心在2mL酶溶液）。 </li></ol></li><li>离心50-mL管与细胞在悬浮液中，在300×g离心10分钟并弃去上清液。 </li><li>重悬细胞沉淀，加入1毫升的HBSS - / - 1％FCS，并过滤细胞悬浮液虽然70微米尼龙网。计数与使用台盼蓝活力染色纽鲍尔室中的细胞。 注:重悬细胞用HBSS - / - （不含Ca 2+和Mg 2+阳离子）以最小化细胞聚集。之间800,000 1,000,000个细胞从一个E17.5心脏获得。 </li></ol></li><li> 染色心肌细胞荧光抗体 注意:所有antibod先前滴定以获得细胞群体的最佳定义（滴定可以随抗体批次而变化）。心脏图谱中使用的抗体列于表1中。 <ol><li>通过96孔板（圆底）将心脏细胞分配在悬浮液中。将它们均匀分布在所有需要的孔中（每孔200μL），短距离旋转将96孔板以480xg离心1分钟，并弃去上清液。 注意:可以使用96孔板（圆底）的所有孔。 </li><li>重悬细胞沉淀，并在4℃黑暗中孵育心肌细胞20分钟，加入100μL抗体混合物（ 即 CD45-PE，TER119-PE，CD31-Alexa 647和Sca-1-PE-Cyanine5 ）稀释于HBSS - / - 1％FCS。 </li><li>通过加入200μLHBSS - / - 1％FCS，将细胞从过量的抗体中洗涤心脏细胞，并以480 x g将细胞短时间离心1分钟。 </li><li>弃上清并重复短旋转离心（步骤3.4.3）。 </li><li>将细胞重悬于HBSS的200μL - / - 1％FCS，并将它们传送到已经含有200μLHBSS的5mL的管 - / - 1％FCS。 </li><li>采集前，加入HBSS的400μL - / - 1％FCS用0.5微克/毫升PI和过滤细胞悬浮液虽然70微米尼龙网。 </li></ol></li></ol> 4.标记脾，肠，和心肌细胞与光谱流式细胞仪的采集<ol><li>采集数据之前，自动校准光谱仪按照制造商的说明。执行每日和每月的质量控制程序，实验的每一天。 </li><li>当细胞计数仪已准备就绪，开始含有标记与所有抗体的细胞悬浮液样品的预览。 注意:不要购买。 </li><li>确保所有激光器的要求上。调整FSC增益，使得所有细胞在各个可见地块。门控细胞群，并将该门应用于对应于488nm和638 / 405nm激光的两个光谱图。 注意:当荧光染料被638nm激光激发时（ 即，当638nm激光打开时），有一个屏蔽屏蔽617-662nm的光以防止638nm激光照射到PMT中。这导致光谱的一部分的损失，因此导致在这些波长中发射的近染料的较低分离。然而，如果不需要采集，可以通过关闭638nm激光器来收集整个信号。 </li><li>调整32通道光电倍增器（PMT）电压（％），使得所有荧光染料在显示488 nm和405/638的两个不同光谱图中显示在y轴（1-10 6 ）中的强度范围内nm激发。 注意:需要进行培训来识别所有使用的荧光染料的光谱。 </li><li>开始获取样品。点击 ";预览"以可视化的细胞在屏幕上，然后点击‘获取’，记录样品节省高达来自每个样品2×10 6个事件。 </li><li>获取染色样品后，取得与该活体染料，然后将补偿珠中使用的所有抗体单独染色染色的样品。点击"预览"可视化屏幕上的细胞，然后在"获取"，记录样品。 注意:保持对所有样品和对照（ 即，珠和未染色样品）相同的PMT电压; 5000个事件在很大程度上是足够的。 </li><li>最后，获得来自未染色的细胞悬浮液中的数据。点击"预览"可视化屏幕上的细胞，然后在"获取"，记录样品。 注:在实验结束时获取的未染色样品最小化荧光细胞的结转。 </li><li>清洁流式细胞仪根据制造商的指令的d合实验中采集的文件夹。 注意:只有在关闭实验后它会被保存，以便进行分析。 </li></ol>与光谱FCM软件进行数据的分析5 <ol><li>在"Analysis"标签的"调色板"的窗口，通过添加使用的每个荧光染料和相应的标记寄存器存在于面板的所有参数（从可用列表中选择或添加新的参数）。另外，包括自体荧光和生存能力参数;所有选定的参数将出现在"未混合的"窗口。 </li><li> "试管列表"这个实验中，"分析"选项卡上的下选择"控制"窗口中的第一单染色的样本（带补偿珠完成）。在工作表中，点击"多边形设计工具"，设计上在FSC / SSC图的珠子群门。在门的顶部双击打开频谱或点图以可视化的门控珠的女儿的情节。 <ol><li>设计，为积极的栅极和用于无论是在频谱上或散点图负珠一个门。检查对应于两个激光集对于每个正和负分数整个光谱和通过连续地选通和点击栅极验证女儿人口消除异常值。在"像元分解"窗口中输入的消极和积极的大门。 </li><li>重复步骤5.2每个单染色的样品。 注:请注意，正面和负面的门控策略对应于被分析的样品，虽然该计划允许它被放置在任何样本。代替确定用于每个单染色的样品的负级分，使用未染色样品珠设置一个通用负。 </li></ol></li><li>选择在"管表"设计门的自发荧光和非自发荧光细胞未染色的细胞样本。 </li> &lt;LI&gt;当所有的所有参数，包括自体荧光和生存力的负和正栅极，在"像元分解"的窗口被选择，点击"计算"。应用光谱分离到的样本进行分析。 </li><li> 通过设计门和创建双参数图分析数据。 <ol><li>首先，设计了一个门，用于对SSC FSC和然后通过排除标记有活力标记（PI与CD45）所有细胞去除死细胞。对细胞的其余部分，对于以下每个细胞悬浮液中描述的策略的所有感兴趣的种群设计栅极（ 图1-3）。 注意:如果需要，调整在补偿"参考光谱调节。"这是一个可以去混合后用来重新调整阳性群体的中位数为每一个荧光染料如果算法做不到完美的工具。 </li></ol></li></ol>

<ol>
	<li>Futamura, 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=Novel+full-spectral+flow+cytometry+with+multiple+spectrally-adjacent+fluorescent+proteins+and+fluorochromes+and+visualization+of+in+vivo+cellular+movement.">Novel full-spectral flow cytometry with multiple spectrally-adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement.</a> Cytometry A. 87 (9), 830-842 (2015).</li><li>Schmutz, S., Valente, M., Cumano, A., Novault, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+Cytometry+Has+Unique+Properties+Allowing+Multicolor+Analysis+of+Cell+Suspensions+Isolated+from+Solid+Tissues.">Spectral Cytometry Has Unique Properties Allowing Multicolor Analysis of Cell Suspensions Isolated from Solid Tissues.</a> PLoS One. 11 (8), e0159961 (2016).</li><li>Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparameter+FACS+analysis.">Multiparameter FACS analysis.</a> Curr Protoc Immunol. , (2002).</li><li>Perfetto, S. P., Chattopadhyay, P. K., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seventeen-colour+flow+cytometry:+unravelling+the+immune+system.">Seventeen-colour flow cytometry: unravelling the immune system.</a> Nat Rev Immunol. 4 (8), 648-655 (2004).</li><li>Grigoriadou, K., Boucontet, L., Pereira, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor-gamma+allele-specific+selection+of+V+gamma+1/V+delta+4+cells+in+the+intestinal+epithelium.">T cell receptor-gamma allele-specific selection of V gamma 1/V delta 4 cells in the intestinal epithelium.</a> J Immunol. 169 (7), 3736-3743 (2002).</li><li>Guy-Grand, D., 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=Origin,+trafficking,+and+intraepithelial+fate+of+gut-tropic+T+cells.">Origin, trafficking, and intraepithelial fate of gut-tropic T cells.</a> J Exp Med. 210 (9), 1839-1854 (2013).</li><li>Lavoinne, A., Cauliez, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Cardiac+troponin+I+and+T:+specific+biomarkers+of+cardiomyocyte].">[Cardiac troponin I and T: specific biomarkers of cardiomyocyte].</a> Rev Med Interne. 25 (2), 115-123 (2004).</li><li>Staudt, D. W., Liu, J., Thorn, K. S., Stuurman, N., Liebling, M., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+of+cardiomyocyte+behavior+reveals+two+distinct+steps+in+ventricular+trabeculation.">High-resolution imaging of cardiomyocyte behavior reveals two distinct steps in ventricular trabeculation.</a> Development. 141 (3), 585-593 (2014).</li><li>Beltrami, A. 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=Adult+cardiac+stem+cells+are+multipotent+and+support+myocardial+regeneration.">Adult cardiac stem cells are multipotent and support myocardial regeneration.</a> Cell. 114 (6), 763-776 (2003).</li><li>Keith, M. C., Bolli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="String+theory"+of+c-kit(pos)+cardiac+cells:+a+new+paradigm+regarding+the+nature+of+these+cells+that+may+reconcile+apparently+discrepant+results.">"String theory" of c-kit(pos) cardiac cells: a new paradigm regarding the nature of these cells that may reconcile apparently discrepant results.</a> Circ Res. 116 (7), 1216-1230 (2015).</li><li>Valente, M., Nascimento, D. S., Cumano, A., Pinto-do-&#211;, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sca-1++cardiac+progenitor+cells+and+heart-making:+a+critical+synopsis.">Sca-1+ cardiac progenitor cells and heart-making: a critical synopsis.</a> Stem Cells Dev. 23 (19), 2263-2273 (2014).</li></ol>

作者什么都没有透露。

常规FCM是基于检测的荧光探针的激励后发射的光子。在设计用于测量从另一荧光染料的信号的检测器检测到的一种荧光染料的荧光发射诱导物理重叠。发射光谱之间的这种溢出需要通过补偿进行校正。 光谱FCM和数据处理通过解混去卷积算法允许具有接近发射峰的荧光染料的无需额外补偿组合，条件是它们具有不同的光谱形状。用于分析的算法将自发荧光作为一个独立的参数?...

在过去的几十年中，流式细胞仪（FCM），成为细胞表型研究的一个必不可少的广泛使用的分析方法。出现了在可用的荧光染料的大幅增加，特别是荧光染料由紫色激光（405nm）的（ 例如，亮紫和新的Q点染料）激发。然而，现有的荧光染料的增长增加了重叠排放的风险和需要劳动密集型补偿矩阵。 FCM成为广泛用于分析从固体组织的细胞悬浮液，但自体荧光的细胞的存在限制特异性标记群的判别。 光谱FCM的基本原理被报告中详细二村等人。 1，2。简言之，此处所用的光谱FCM（见材料的表）配备有405，488，和638纳米的激光器。光谱捕获FCM所有散发氟luorescence如32通道线性阵列PMT（32CH PMT）500〜800nm的和，用于分别为420nm至44​​0nm和450nm至469纳米，2个独立的光电倍增管，其取代传统的带通滤波器的光谱。 488个六百三十八分之四百零五nm激光光斑在空间上分离，而405nm和638 nm的激光斑点是共线的。对于每一个单独的颗粒中，光谱FCM措施达到由405nm的激发的荧光数据的66个信道和488个纳米激光器。当细胞通过638 nm激光激发，光谱测量FCM 58个通道的荧光数据的，因为掩模被插入以防止638 nm激光从照入PMT。光谱与FCM基于加权最小二乘法（WLSM），使荧光光谱重叠的分离的算法分析所获取的全光谱数据。从单染色和未染色的样品衍生的光谱，确认为基本参考光谱。多染色的样品被数学嵌合的ND未混合，并用混合荧光标记的样品的光谱被分解成其组成的光谱的集合。解混，在光谱技术3,4，替换，可以消除除了所述一个测量给定染料的所有检测器的信号中的补偿。 在这项研究中，我们结合并在一个单一的，21参数分析其特征在于，所述小鼠脾脏中发现的主要的造血子集进行测试19点的荧光染料。此外，我们证明了光谱仪可管理的自体荧光信号，从而改善肠道上皮内淋巴细胞和胚胎心脏的表征。事实上，在这些组织中，自发荧光管理允许特定荧光的细胞的分配，将在从常规FCM分析中排除。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mice</td>
			<td>Janvier Labs</td>
			<td>CD45.2</td>
			<td>Source of cells</td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>VWR</td>
			<td>83801.36</td>
			<td>Sterilization</td>
		</tr>
		<tr>
			<td>DPBS (Ca2+, Mg2+)</td>
			<td>GIBCO ThermoFisher</td>
			<td>14040-174</td>
			<td>Embryos collection</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (+Ca2+ +Mg2+) (HBSS+/+)</td>
			<td>GIBCO Life ThermoFisher</td>
			<td>14025092</td>
			<td>Dissociation, digestion and staining solutions</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (-Ca2+, -Mg2+) (HBSS-/-)</td>
			<td>GIBCO Life ThermoFisher</td>
			<td>14175095</td>
			<td>Dissociation, digestion and staining solutions</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>EUROBIO</td>
			<td>CVFSVF00-0U</td>
			<td>Dissociation, digestion and staining solutions</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C2139</td>
			<td>Enzymatic solution</td>
		</tr>
		<tr>
			<td>90 x 15 mm, 
			plastic tissue culture Petri dishes.</td>
			<td rowspan="1">TPP</td>
			<td rowspan="1">T93100</td>
			<td rowspan="1">Embryos and hearts collection</td>
		</tr>
		<tr>
			<td>35 x 15 mm, 
			plastic tissue culture Petri dishes.</td>
			<td rowspan="1">TPP</td>
			<td rowspan="1">T9340</td>
			<td rowspan="1">Hearts collection</td>
		</tr>
		<tr>
			<td>Fine iris scissor</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
			<td>Dissection tools</td>
		</tr>
		<tr>
			<td>Gross forceps (narrow pattern forceps, curved 12 cm</td>
			<td>Fine Science Tools</td>
			<td>11003-12</td>
			<td>Dissection tools</td>
		</tr>
		<tr>
			<td>Fine forceps (Dumont no. 7 forceps,</td>
			<td>Fine Science Tools</td>
			<td>11272-30</td>
			<td>Dissection tools</td>
		</tr>
		<tr>
			<td>Scalpel&#160;</td>
			<td>VWR</td>
			<td>21909-668</td>
			<td>Dissection tools</td>
		</tr>
		<tr>
			<td>LEICA MZ6 Dissection microscope</td>
			<td>LEICA</td>
			<td>MZ6 10445111</td>
			<td>Sampling; Occular W-Pl10x/23</td>
		</tr>
		<tr>
			<td rowspan="1">Cold lamp source</td>
			<td rowspan="1">SCHOTT VWR</td>
			<td rowspan="1">KL1500 compact</td>
			<td>Sampling; 
			Two goose neck fibers adapted</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>VWR</td>
			<td>60819-310</td>
			<td>Digesting cells</td>
		</tr>
		<tr>
			<td>96 well plate (round bottom)</td>
			<td>VWR</td>
			<td>10861-564</td>
			<td>Staining cells</td>
		</tr>
		<tr>
			<td>Nylon mesh bolting cloth sterilized, 
			50/50 mm pieces.</td>
			<td rowspan="1">SEFAR NITEX</td>
			<td rowspan="1">SEFAR NITEX</td>
			<td rowspan="1">Cell filtration</td>
		</tr>
		<tr>
			<td>UltrasComp eBeads</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence labeled antibodies</td>
			<td>Biolegend</td>
			<td>Catalogue number see table below</td>
			<td>Staining cells</td>
		</tr>
	</tbody>
</table>

analysis of cell suspensions isolated from solid tissues by spectral flow cytometry

流式细胞仪已被用于在过去40年来定义和分析淋巴细胞和其他造血细胞的表型。最初仅限于少数荧光染料的分析，目前有几十种不同的荧光染料，以及多达14-18不同的染料可以在同一时间内合并。然而，一些限制仍然影响的分析能力。因为荧光探针的多重性的，数据分析已经变得越来越复杂，因为需要的大，多参数的补偿矩阵。此外，携带荧光蛋白，以检测和跟踪在不同的组织的特定细胞类型的突变体小鼠模型已经变得可用，因此需要的分析（通过流式细胞术）从实体器官得到自体荧光的细胞悬液。光谱流式细胞仪，其沿宽范围连续波长区分的发射光谱的形状，解决一些上述问题。分析数据w ^第i个的算法代替补偿矩阵和治疗自体荧光作为一个独立的参数。因此，光谱流式细胞仪应该能够以类似的发射峰鉴别荧光的，并且可以提供一个多参数分析，而无需补偿要求。 该方案描述的光谱流式细胞分析，从而允许一个21参数（19个荧光探针）的表征和自动荧光信号的管理，提供在微小的人口检测高分辨率。这里的结果表明，光谱流式细胞术呈现细胞群从组织难以在以往的流量表征流式细胞术，如心脏和肠的分析的优点。光谱流式细胞仪从而表明高性能，而不要求流式细胞术常规流动补偿，使自发荧光管理的多参数分析能力。

21-参数光谱FCM面板来分析脾细胞 图1示出了具有施加到包括识别T，B，NK，树突，和髓样细胞亚群的不同抗体的脾细胞中的19-荧光抗体面板所获得的结果，而CD45标记所有造血核细胞。该面板还包括一个生存力染料（PI），以及大小（FSC）和粒度（SSC）的参数。 图1A示出了参考荧光光谱。消除在CD45 +</sup...

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Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

本文介绍的光谱仪，在流式细胞术中使用的发射光谱的形状来区分的荧光染料的新方法。算法替代补偿，可以把自动荧光作为一个独立的参数。这种新方法允许从实体器官分离的细胞的正确分析。

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

analysis-cell-suspensions-isolated-from-solid-tissues-spectral-flow

Research

JoVE Journal

Biology

12.9K 次观看.  Institut Pasteur. 本文介绍的光谱仪，在流式细胞术中使用的发射光谱的形状来区分的荧光染料的新方法。算法替代补偿，可以把自动荧光作为一个独立的参数。这种新方法允许从实体器官分离的细胞的正确分析。 

视频: Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

从小鼠神经组织产生单细胞悬浮液

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

科研

生物学

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP - FCM:免疫沉淀，流式细胞仪检测

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

一个小鼠红细胞亚群富集去核活动由多光谱成像流式细胞仪鉴定

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

外层膜蛋白的分离线粒体的流式细胞仪免疫检测

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

对于外小泡的使用流式细胞仪分析技术

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

分离块茎原生质体流式细胞仪测量 Endoreduplication 的研究

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

流式细胞仪检测盘基柄柄Macropinocytosis 的高通量

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...