Name: analysis of cell suspensions isolated from solid tissues by spectral flow cytometry
Uploaded: 2017-05-05T15:00:00.000+00:00
Duration: 11 min 8 s
Description: Watch this Scientific Journal Video about Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry at JoVE.com

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>

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	<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><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 (Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>GIBCO ThermoFisher</td><td>14040-174</td><td>Embryos collection</td></tr><tr><td>Hanks&#039; Balanced Salt Solution (+Ca&lt;sup&gt;2+&lt;/sup&gt; +Mg&lt;sup&gt;2+&lt;/sup&gt;) (HBSS+/+)</td><td>GIBCO Life ThermoFisher</td><td>14025092</td><td>Dissociation, digestion and staining solutions</td></tr><tr><td>Hanks&#039; Balanced Salt Solution (-Ca&lt;sup&gt;2+&lt;/sup&gt;, -Mg&lt;sup&gt;2+&lt;/sup&gt;) (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,&lt;br/&gt; plastic tissue culture Petri dishes.</td><td>TPP</td><td>T93100</td><td>Embryos and hearts collection</td></tr><tr><td>35 x 15 mm,&lt;br/&gt; plastic tissue culture Petri dishes.</td><td>TPP</td><td>T9340</td><td>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&amp;nbsp;</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>Cold lamp source</td><td>SCHOTT VWR</td><td>KL1500 compact</td><td>Sampling;&lt;br/&gt; 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,&lt;br/&gt; 50/50 mm pieces.</td><td>SEFAR NITEX</td><td>SEFAR NITEX</td><td>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...

Watch this Scientific Journal Video about Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry at JoVE.com

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 ...

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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

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

加工的主要脑肿瘤组织的干细胞检测和流量排序

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

科研

医学

Characterizing Microbiome Dynamics &#8211; Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical applications driven by microorganisms. Next generation sequencing methods are widely utilized to resolve microbiomes, but they are generally resource and time intensive and deliver mostly qualitative information. Flow cytometric microbiome analysis does not suffer from those disadvantages and can provide relative subcommunity abundances and absolute cell numbers at-line. Although it does not deliver direct phylogenetic information, it can enhance the analysis depth and resolution of sequencing approaches. In sharp contrast to medical applications in both research and routine settings, flow cytometry is still not widely used for microbiome analysis. Missing information on sample preparation and data analysis pipelines may create an entry barrier for the researchers facing microbiome analysis challenges that would often be textbook flow cytometry applications. Here, we present three comprehensive workflows for pure cultures, complex communities in clear medium and complex communities in challenging matrices, respectively. We describe individual sampling and fixation procedures and optimized staining protocols for the respective sample sets. We elaborate the cytometric analysis with a complex research centered and an application focused bench top device, describe the cell sorting procedure and suggest data analysis packages. We furthermore propose important experimental controls and apply the presented workflows to the respective sample sets.

Flow cytometric analysis has proven valuable for investigating pure cultures and monitoring microbial community dynamics. We present three comprehensive workflows, from sampling to data analysis, for pure cultures and complex communities in clear medium as well as in challenging matrices, respectively.

微生物动力学的特征-基于流式细胞仪的工作流从纯文化到自然群落

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical ...

环境科学

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

神经细胞类型的流式细胞仪协议的表面和细胞内抗原分析

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

神经科学

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

应用流式细胞仪分析单侧坐骨神经和背根神经节的免疫细胞

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

流式细胞术测定小鼠胸腺、胰腺干淋巴结和脾脏的调节 t 细胞亚群

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

免疫与感染

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

高维流式细胞术用于解剖种植体组织的免疫功能分析

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

生物工程

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

简单和高效的睾丸细胞悬液的制备技术

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

生物学

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

通过不同的均质化方法从小鼠大脑/脊髓中检索到的多种细胞类型的同步流细胞测量特征

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Quality-Controlled Sputum Analysis by Flow Cytometry

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed using cytology-based methodologies. Its utility is limited because reading the slides is time-consuming and requires highly specialized personnel. Moreover, extensive debris and the presence of too many squamous epithelial cells (SECs), or cheek cells, often renders a sample inadequate for diagnosis. In contrast, flow cytometry allows for high-throughput phenotyping of cellular populations while simultaneously excluding debris and SECs.
The protocol presented here describes an efficient method to dissociate sputum into a single cell suspension, antibody stain and fix cellular populations, and acquire samples on a flow cytometric platform. A gating strategy that describes the exclusion of debris, dead cells (including SECs) and cell doublets is presented here. Further, this work also explains how to analyze viable, single sputum cells based on a cluster of differentiation (CD)45 positive and negative populations to characterize hematopoietic and epithelial lineage subsets. A quality control measure is also provided by identifying lung-specific macrophages as evidence that a sample is derived from the lung and is not saliva. Finally, it has been demonstrated that this method can be applied to different cytometric platforms by providing sputum profiles from the same patient analyzed on three flow cytometers; Navios EX, LSR II, and Lyric. Furthermore, this protocol can be modified to include additional cellular markers of interest. A method to analyze an entire sputum sample on a flow cytometric platform is presented here that makes sputum amenable for developing high-throughput diagnostics of lung disease.

This protocol describes an efficient method for dissociating sputum into a single cell suspension and the subsequent characterization of cellular subsets on standard flow cytometric platforms.

通过流式细胞术进行质量控制痰液分析

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed ...

Flow Cytometry and Fluorescence-Activated Cell Sorting (FACS): Isolation of Splenic B Lymphocytes

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
The overall function of the immune system is to defend the body against infectious organisms and other invaders. White blood cells, or leukocytes, are the key players of the immune system. Upon infection, they are activated and initiate an immune response. Leukocytes can be divided into various sub-populations (e.g., myeloid cells, lymphocytes, dendritic cells) based on different parameters that can be biological, physical, and/or functional (e.g., size, granularity, and secretion). One way to characterize leukocytes is through their surface proteins, which are mainly receptors. Each leukocyte population expresses a specific combination of receptors (e.g., cytotoxic, activating, migration receptors) that can define subsets among populations. As the immune system encompasses a wide range of cell populations, it is essential to characterize them to decipher their participation in the immune response.
Flow cytometry (FC or FCM) is a widely used method for analyzing the expression of cell surface and intracellular molecules, characterizing and defining different cell types in a heterogenous cell mixture. Flow cytometers are composed of three main sub-systems: fluidics, optics, and electronics. The fluidics system transports the cells in a stream such that they pass in front of a laser one by one. The optics system consists of light sources (lasers) to illuminate the particles, optical filters to direct the resulting light, and fluorescent signals to appropriate detectors. Finally, the electronics system converts the detected light signals into electronic signals that can be processed by the computer. As an individual cell passes in front of the laser beam, it scatters light. A detector in front of the beam measures forward scatter (FS) and several detectors to the side measure side scatter (SC). The FS correlates with cell size and SC is proportional to the granularity of the cells. In this manner, cell populations can often be distinguished based on differences in their size and granularity alone.
In addition to analyzing a cell&#39;s size, shape, and complexity, flow cytometry is widely used for detecting the expression of cell surface receptors (1). This is accomplished by using fluorochrome-labelled monoclonal antibodies which bind to known cell-specific receptors. Upon excitation, these bound fluorochromes emit a light of specific wavelength, called emission wavelength, which can be detected and scored. Fluorescence measurements provide quantitative and qualitative data about fluorochrome-labeled cell surface receptors. Hematologists were first to use FC for the therapeutic follow up of immune cell populations (2). Now, it is used for a wide range of applications such as immunophenotyping, cell viability, gene expression, cell counting, and GFP analysis.
FACS (Fluorescent Activated Cell Sorter) is a specialized type of flow cytometry, which sorts a population of cells into subpopulation using fluorescent labelling. Just like conventional flow cytometry, first FS, SC, and fluorescent data are collected. Then, the machine applies a charge (negative or positive) and an electrostatic deflection system (electromagnets) facilitates the collection of charged droplets containing cells into appropriate tubes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10494/10494fig1.jpg" /> 
Figure 1: Schematic representation of FACS. Sample (1) is aspirated in the FACS (2) and passed in front of the laser (3). Cell fluorescence is sensed by fluorescence detectors (4). Finally, cells are incorporated in droplets and the cells of interest are deflected by deflection plates (5) and collected in a collection tube (6). The remaining cells go into the trash (7). <a href="https://www.jove.com/files/ftp_upload/10494/10494fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The sorting aspect of the FACS presents many advantages. Many tests can help understand the role of specific cells in immune system, such as analyses of gene expression like RT-qPCR, cell cycle, or cytokine secretion. However, cells should be purified upstream to obtain clear and specific results. Here, FACS comes in useful and the desired cells can be sorted with great purity, yielding highly reliable and reproducible results. FACS can also be used to sort cells based on nuclear or other intracellular staining and according to the presence, absence, and density of surface receptors. FACS is now a standard technique for the purification of subpopulations of cells and has the ability sort up to four populations simultaneously.
This lab exercise demonstrates how to isolate splenic leukocytes and then how to specifically sort B lymphoid cells from the splenic leukocyte cell mixture using FACS.