Authors were supported by the German Cancer Aid (Max Eder Nachwuchsgruppe, Deutsche Krebshilfe; EU), the LOEWE Center for Cell and Gene Therapy Frankfurt (EU, ST) funded by the Hessian Ministry of Higher Education, Research and the Arts, Germany (III L 4- 518/17.004) and by the "Alfred- und Angelika Gutermuth-Stiftung", Frankfurt, Germany (EU). BJ was funded by a Mildred Scheel postdoctoral scholarship from the Dr. Mildred Scheel Foundation for Cancer Research. ST was founded by a GO-IN postdoctoral fellowship (PCOFUND-GA-2011-291776). The authors thank Becton Dickinson (BD) for providing the FACSCanto II and Canto10c Flow Cytometry Analyzers used in this study.

这项研究是根据法兰克福大学的当地伦理委员会的建议进行的。 1，K562细胞的培养<ol><li>培养的K562细胞在R10培养基（RPMI1640谷氨酰胺培养基，1％青霉素/链霉素，10％胎牛血清）中以每毫升0.5-1×10 6细胞在细胞培养瓶中的密度，在37℃和5％的CO 2。 </li><li>嘉实K562细胞24小时的新实验开始前。 <ol><li>删除含有从孵化器中的K562细胞的细胞培养瓶。通过上下轻轻吹打起来，再暂停培养基内的K562细胞。 </li><li>传送含有K562细胞培养基到15或50毫升管和沉淀细胞，在400×g离心8分钟。弃上清，重新悬浮在5毫升R10的媒体细胞拌匀。 </li><li>转移20微升细胞溶液到96-U，孔板的孔中的。加入20微升台盼蓝和通过上下吹打至少5次拌匀。吸移管将10μl该溶液到细胞计数室和计数细胞。 </li><li>沉淀细胞，在400×g离心8分钟。重悬在R10培养基中的细胞，并调整K562细胞浓度为每毫升0.5-1×10 6个细胞。 </li><li>孵育在细胞培养烧瓶中的K562细胞在37℃和5％的CO 2，直到使用。 </li></ol></li></ol> 2，NK细胞的分离<ol><li>据当地伦理委员会的批准和指导方针，6-10无论是EDTA或肝素化外周血毫升采集之前获得健康供者或患者的书面知情同意书。 </li><li>在4℃下的NK细胞隔离，直至实验开始商店试剂，然后在无菌条件下于室温（RT）下使用它们。使用磁微珠根据吨细胞分离特定磁体分离自外周血NK细胞他制造商的说明。 <ol><li>吹打7.5毫升提供的缓冲区A（包括NK细胞分离试剂盒中）进入小瓶每瓶冻干NK细胞阴性分离抗体混合物一小瓶。通过上下吹打3-4次轻轻拌匀。 注意:确保，即悬挂在每次使用前均匀。 </li><li>通过准备步骤2.2.1和缓冲液B（包括NK细胞分离试剂盒中）混合重组颗粒适当体积的最终解决方案的鸡尾酒。为了确定这些卷，以ml定义中的全血样品的体积为体积= 1.0。使用0.25体积重构的粒料（来自步骤2.2.1）和0.25体积的缓冲液B的处理1体积的全血。 </li><li>将混合物转移到含有全血样品的适当管。不吸管上下，以避免血液的吸移管内的损失。相反，关闭管和移动小心翼翼地把它向上和向下，直到suspen锡安是均匀的。不要旋涡。 </li><li>使用试管旋转器在RT 5分钟进一步均质化样品。 </li><li>在无菌条件下，取下盖和管放入15分钟的磁分离器所推荐的制造商。确保所述管的标签朝向磁体的背侧，以允许隔离线的不受干扰可视性。分离过程中不要移动磁铁，因为这个过程会被扰乱。 </li><li>小心将上清转移到新的15ml试管并用完全培养基填满（CM;造血细胞培养基，1％青霉素/链霉素，5％的人血清）。沉淀细胞，在400×g离心8分钟。 <ol><li>可选:如果颗粒是红色的，重新悬浮细胞在红细胞裂解缓冲液（1毫升例如 ，将1ml氯化铵-钾（ACK）裂解缓冲液:155毫氯化铵 ; 0.1毫摩尔EDTA; 10mM的KHCO 3在1升蒸馏水（DW））和在室温下孵育8分钟。另外，使用ERythrocyte枯竭套件。 注意:请记住，即使用ACK缓冲可能NK细胞功能14造成负面影响。 </li><li>通过加入至少1毫升的CM 400 XG停止裂解和离心8分钟以沉淀细胞迅速稀释该ACK缓冲器。 </li></ol></li><li>重悬在1ml的CM细胞沉淀并与计数继续进行。转移20微升的NK细胞溶液到96-U，孔板中。加入20μl亚甲蓝向每个孔并通过上下抽吸至少5倍拌匀。吸移管将10μl该溶液到细胞计数室和计数细胞。另外，使用30微升未稀释的细胞悬液与市售细胞计数器计数。 </li><li>调节细胞以每100微升的CM至少2×10 4个 NK细胞的浓度。 </li><li>执行分离的NK细胞群的表面抗体染色使用流式细胞仪检查纯度。 <ol><li>镨由指定抗体的体积混合按表1 epare一个主混合物溶液。 </li><li>重悬在88.5微升洗涤缓冲液将细胞（WB; 0.5％牛血清白蛋白（BSA），0.1％NaN 3的PBS中），并添加11.5微升主混合物溶液。孵育20分钟，将细胞在4℃在黑暗中。 注意:NaN的3有毒，诱变剂。采取相应措施，以相应的材料安全数据表（MSDS）。 </li><li>在400 XG加入1ml WB和沉淀细胞4分钟。 </li><li>重悬在300μl的染色缓冲液加DAPI细胞。获得使用流式细胞仪的细胞。 注意:只有当NK细胞纯度是所有活的淋巴细胞中的至少80％的实验进一步继续。 </li></ol></li></ol></li></ol> 3. K562细胞的NK细胞刺激的收获<ol><li>除去含有K562细胞（来自步骤1.2）从第细胞培养烧瓶<html> Ë孵化器。通过上下轻轻吹打起来，再暂停培养基内的K562细胞。 </li><li>传送整个培养基成15或50毫升管和沉淀细胞，在400×g离心8分钟。弃去上清液并重新悬浮在5ml磷酸盐缓冲盐水（PBS）将细胞并充分混合。 </li><li>转移20微升细胞溶液到96-U，孔板的孔中的。加入20微升台盼蓝和上下吹打至少5次拌匀。吸移管将10μl该溶液到细胞计数室和计数细胞。可替代地使用30微升未稀释的细胞悬浮液与市售的细胞计数器计数。 </li><li>沉淀细胞，在400×g离心8分钟。重悬在CM中的细胞以每100微升媒体至少2×10 4个 K562细胞的浓度。 注:使用相同的K562和NK细胞浓度，以便在效应孵化两个:目标:1（E T）:1的比例以后。 </li></ol>"&gt; 4。NK细胞与肿瘤细胞系K562及细胞因子的刺激<ol><li>轻轻吹打他们上下至少5倍混合细胞。分发100μl/孔的NK细胞溶液到96-V的孔板的所需要的孔中。 <ol><li>使用至少两个井对于每个供体，一种带有一个没有刺激（ 例如 ，K562肿瘤细胞和细胞因子）。只要有可能，至少一式两份进行实验，以添加阳性对照（ 例如，佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）和离子霉素，终浓度为50nM PMA，1μM的离子霉素每孔）。制备用于实验的更新日期流式细胞补偿。 注:滴定抗体事先使用（见讨论）。 </li></ol></li><li>关掉光线并添加抗CD107a抗体（2微升，最终浓度:1:100），以每孔NK细胞上的96-V的孔板中。 </li><li>轻轻吹打并上下在混合K562细胞至少5倍。 <ol><li>从含有NK细胞/抗CD107a抗体溶液的井，确定计划与K562细胞刺激的人。加入100微升/ K562的细胞溶液进入这些井的井。至少5倍仔细混匀通过上下抽吸。 </li><li>添加白介素-2（IL-2;终浓度100U / ml）和白细胞介素-15（IL-15;终浓度10纳克/毫升），以含有K562细胞和NK细胞/抗CD107a抗体溶液的孔中。 <ol><li>可选:测试或K562细胞或细胞因子的单独的分布式NK细胞的影响，以破译肿瘤诱导与细胞因子诱导NK细胞的功能。 </li></ol></li><li>确定的96-V孔板井规划为阴性对照无刺激。添加100μl/孔CM以仅含有NK细胞/抗CD107a抗体溶液，这些孔中。至少5倍仔细混匀通过上下抽吸。 <ol><li>可选:对于阳性对照，加入100μ含有PMA和离子霉素1立方厘米（最终浓度:50nM的PMA，1μM的离子霉素每孔）以一个很好地与NK细胞/抗CD107a抗体溶液。 </li></ol></li></ol></li><li>孵育细胞在37℃下在黑暗中3小时，5％ 的 CO 2。 </li><li>制备蛋白质转运抑制剂溶液（例如布雷菲德菌素A和/或莫能菌素）在CM中。孵育1小时后，添加溶液到96-V的孔板与所述光的每个孔关闭（最终浓度:0.5-1μM布雷菲德菌素A和2-3微米莫能）。至少5倍仔细混匀通过上下抽吸。继续对剩余2小时孵化。 </li></ol> 5.表面和细胞内染色<ol><li>在3小时温育期后，至少5倍混合96-V的孔板的仔细的孔中的细胞通过上下吹打，并将它们传送到流式细胞仪的管。加入1毫升/ WB管。沉淀细胞，在400×g离心4分钟。 <ol><li>可选的:前离心冲洗各孔用100μl/孔PBS中，为了将细胞转移到相应的FACS管之前，以优化细胞回收，通过上下吹打至少5次。 </li></ol></li><li>弃去上清液，并与表面染色继续。 </li><li>包括胺反应性荧光染料是不可穿透活细胞具有发射最大值的423纳米，可固定死细胞标记物（DCM）中。之前（见下文），或与这取决于DCM的类型的抗体表面染色用于并行地执行染色。 <ol><li>重悬的细胞在99微升/管PBS中，加入1微升/可定影的DCM（最终浓度:1:100）的管中。混合使用涡旋孔的细胞，并培育它们在室温在黑暗中15分钟。 </li><li>准备由表2中列出的"表面"抗体混合在一起（见染色步骤柱以蓝色突出显示）一个主混合物溶液。使用中dicated卷/管和流量的数量乘以他们仪来进行染色管。 </li><li>孵化后采取流式细胞仪与细胞管中流动，加入1毫升/ WB管。沉淀细胞，在400×g离心4分钟。 </li><li>弃去上清液，再暂停WB的84微升/管细胞，加入16微升/主混合溶液中管。使用一个旋涡混匀细胞。孵育细胞20分钟，在4℃下在黑暗中。 </li><li> 20分钟后加入1毫升/管WB和颗粒细胞在400 XG 4分钟。 </li></ol></li><li>弃上清，再暂停100微升/冷固定溶液（如 2％（最终）多聚甲醛或甲醛）的管细胞。使用一个旋涡混匀细胞。孵育细胞10分钟，在4℃下在黑暗中。 <ol><li>染色细胞内细胞因子/趋化因子的细胞此步骤之后，或它们在WB 4℃过夜储存。 注:过夜温育后，可能会发生低级细胞恢复。 </ol></li><li>在400 xg离心洗涤细胞在1ml /管WB 4分钟，并与透步骤进行。 </li><li>制备透化缓冲液（PB）（ 例如，0.2％皂苷，1％BSA的PBS溶液）。 </li><li>洗细胞两次PB中1毫升/管中，在400×g离心4分钟。与细胞内染色继续。 注意:皂素是潜在的刺激。采取相应措施，以相应的材料安全数据表（MSDS）。 </li><li>使用指定的"内"抗体表2册（绿色高亮）准备一个主结构的解决方案。乘这些卷用管的数目被染色。 <ol><li>重悬在90微升/ PB管细胞，并应用10微升/抗体预混液管。拌匀使用涡旋和孵化单元3在4℃在黑暗中0分钟。 </li></ol></li><li>在400 XG在PB 1毫升/管两次洗涤细胞4分钟。 </li><li>弃上清，再暂停400微升/管PB细胞。使用一个旋涡混匀细胞。放置在冰上的细胞在黑暗中，直至测量。 </li></ol> 6.流式细胞仪补偿和收购<ol><li>选择用于不同荧光通道（ 见表2）中的参数设置的文件夹流式细胞采集软件的流动范围内。此外，选择FSC-A的渠道和-H以及为SSC-A和-H。 </li><li>加载所选参数到收购文件中创建一个补偿矩阵。 <ol><li>创建由每个使用荧光进行单颜色污点用分离的NK细胞从步骤2.2.8一个补偿矩阵（ 见表2）。使用步骤5.1-5.4中描述的表面染色协议。包括未染色的对照（不加任何抗体），以及同种型对照和/或荧光减去一个对照（FMO）。 注意:作为一种替代的基于细胞的补偿，使用IgG结合珠补偿控制。 </li><li>将每个管为单染色样品和无抗体进入样品注射端口（SIP）的样品。点击在流式细胞仪采集软件的"记录"按钮，并获得每个样品至少为5×10 3的NK细胞的细胞数。 </li><li>使用流式细胞仪采集软件的补偿计算应用程序创建一个补偿矩阵。 </li></ol></li><li>用于识别NK细胞人口创建散点图。 <ol><li>确定采用FSC-A / FSC-H和SSC-A / SSC-H散点图单个细胞。点击"多边形门"按钮。周围绘制的细胞，其具有在两个点图的线性分布图案的栅极。在门双击打开一个新的点图。 </li><li>选择一个FSC-A / SSC-A散点图识别淋巴细胞/ NK细胞群（ 见图1）。绘制多边形门周围，并在门上双击。 </li></ol></li><li> NK细胞刺激实验的每个样品管放入SIP。点击"记录"按钮，并获得每个样品至少为5×10 3的NK细胞的细胞数。 </li></ol> 7.分析和统计<ol><li>开术分析软件程序的流程。点击装载样本按钮打开未受刺激的对照样品。 </li><li>对于不同的NK细胞亚群（ 见图1）创建浇注系统。 <ol><li>点击散点图按钮创建一个FSC-A / SSC-A地块。点击矩形门按钮，并绘制了所有事件的矩形门具有FSC-A值&gt; 5×10 4，排除杂物。通过在门双击打开门。 </li><li>再次选择新的散点图中的FSC-A / SSC-A参数，点击多边形门按钮。 ð生绕淋巴细胞群体的多边形栅极（ 见图1）。打开门。 </li><li>选择新散点图的SSC-A / SSC-H的参数和周围绘制多边形门的所有单个细胞。单细胞表现出横跨FSC-A / FSC-H和SSC-A / SSC-H参数的线性分布（ 见图1）。通过双击它打开门。 </li><li>重复步骤7.2.3使用FSC-A / FSC-H参数这段时间。 </li><li>使用参数CD45和转储通道（CD3，CD14，CD19; DCM）排除死细胞。画周围的一切CD45阳性的矩形门和转储通道阴性细胞。通过在门双击打开门。 </li><li>通过使用CD56识别整个的NK细胞群和转储通道参数。创建周围CD56阳性的矩形门和转储通道阴性细胞。通过双击它打开门。 </li><li>绘制一个矩形，门周围CD56 / CD16散点图中的所有三个NK细胞亚群。井ntify不同子集作为CD56 + CD16 - ，CD56 + CD16 +和CD56 + CD16 + NK细胞亚群。 </li></ol></li><li>分析每个NK细胞亚群NK细胞功能。 <ol><li>点击这三个NK细胞亚群大门打开之一。创建用于CD56 / CD107a，CD56 / IFN-γ和CD56 / MIP-1β三个不同的点图。 </li><li>绘制为CD56阳性细胞，这分别是正，以及对于CD107a，IFN-γ或MIP-1β的矩形栅（ 见图1）。 </li><li>重复步骤7.3.1-7.3.2其余每个NK细胞亚群。 </li></ol></li><li>重复步骤7.2和7.3都刺激了样品。通过点击分析软件内的统计出口按钮保存各个样品的所有统计值。 </li><li>打开包含个别样本的统计值来分析NK细胞的功能的文件。选择个别NK细胞值子集（ 例如 ，CD56 + CD16 - NK细胞），通过将其复制到另一个文件。 <ol><li>减去CD107a阳性NK细胞，其没有被用刺激治疗，从CD107a阳性NK细胞的百分比，已用一个刺激治疗的百分比。 注意:使用的结果证明在响应于刺激这个特定NK细胞子集的脱粒能力。 </li><li>对于IFN-γ和MIP-1β阳性NK细胞，重复步骤7.5.1证明响应于刺激的细胞因子和趋化因子生产。 </li><li>其余每个NK细胞亚群重复步骤7.5.1-7.5.2，以评估在刺激他们的脱粒能力和细胞因子/趋化因子的生产。 </li></ol></li></ol>

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G., 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=IDEC-C2B8+(Rituximab)+anti-CD20+monoclonal+antibody+therapy+in+patients+with+relapsed+low-grade+non-Hodgkin's+lymphoma.">IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma.</a> Blood. 90 (6), 2188-2195 (1997).</li><li>Salles, G., 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=Phase+1+study+results+of+the+type+II+glycoengineered+humanized+anti-CD20+monoclonal+antibody+obinutuzumab+(GA101)+in+B-cell+lymphoma+patients.">Phase 1 study results of the type II glycoengineered humanized anti-CD20 monoclonal antibody obinutuzumab (GA101) in B-cell lymphoma patients.</a> Blood. 119 (22), 5126-5132 (2012).</li><li>Terszowski, G., Klein, C., Stern, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KIR/HLA+interactions+negatively+affect+rituximab-+but+not+GA101+(obinutuzumab)-induced+antibody-dependent+cellular+cytotoxicity.">KIR/HLA interactions negatively affect rituximab- but not GA101 (obinutuzumab)-induced antibody-dependent cellular cytotoxicity.</a> J Immunol. 192 (12), 5618-5624 (2014).</li><li>Hsu, A. 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=The+immunostimulatory+effect+of+lenalidomide+on+NK-cell+function+is+profoundly+inhibited+by+concurrent+dexamethasone+therapy.">The immunostimulatory effect of lenalidomide on NK-cell function is profoundly inhibited by concurrent dexamethasone therapy.</a> Blood. 117 (5), 1605-1613 (2011).</li><li>Salih, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BCR/ABL-inhibitors+imatinib,+nilotinib+and+dasatinib+differentially+affect+NK+cell+reactivity.">The BCR/ABL-inhibitors imatinib, nilotinib and dasatinib differentially affect NK cell reactivity.</a> Int J Cancer. 127 (9), 2119-2128 (2010).</li><li>Benson, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+1+trial+of+the+anti-KIR+antibody+IPH2101+in+patients+with+relapsed/refractory+multiple+myeloma.">A phase 1 trial of the anti-KIR antibody IPH2101 in patients with relapsed/refractory multiple myeloma.</a> Blood. 120 (22), 4324-4333 (2012).</li><li>Vey, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+1+trial+of+the+anti-inhibitory+KIR+mAb+IPH2101+for+AML+in+complete+remission.">A phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission.</a> Blood. 120 (22), 4317-4323 (2012).</li><li>Terme, M., Ullrich, E., Delahaye, N. 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The authors have nothing to disclose.

所描述的方法是一种简单，快速和可靠的方法来研究健康人或病人全血标本NK细胞功能。这种方法提供了很大的优势，以直接净化从全血NK细胞，避免了费时密度梯度离心，这是强制性的许多其他纯化方法15。此外，它需要比较"经典的"NK细胞分离/富集方法较小的样本大小，这使得它对于儿科和/或免疫缺陷患者的样品的合适的替代。该协议可以被用来获得NK细胞功能的离体的基础值，但它也允许进一步NK细胞培养和扩大，以这种方式与其它方法互补先前描述8之中。然而某些关键步骤，必须考虑到。由于该测定是基于细胞内外抗体染色，关键的是要确定最佳的禾第一rking浓度为每抗体。特别是对于细胞内染色，强烈建议所用的抗体仔细评估。一个好的方法是测试系列稀释所使用的抗体（ 例如，1:12.5:100到1）在有或没有PMA /离子霉素刺激的细胞悬浮液。随后刺激和未刺激的样品之间的积极事件的比例计算并绘制。抗体稀释具有最高正倍数变化率（最佳信号-背景比）应当用于进一步的实验16。此外，使用像同种型和FMO控制控件可以证明是对于细胞内染色尤其基本以栅极上正确的阳性细胞群。 但是，也有所描述的方法的某些重要的限制。 CD107a表达是一个脱颗粒标记，因此只能间接地表明NK细胞的细胞毒性。 NK细胞细胞毒性一次脱粒容量可以是不同的。上调在分泌颗粒酶B的降解肿瘤细胞的结果自噬途径，并减少对NK细胞的相互作用17细胞死亡。因此一个附加的DCM（ 例如，裂解的caspase 3）可能被加入到染色面板以监测靶细胞18内的细胞死亡的事件。此外，NK细胞细胞毒性是由它们的颗粒内细胞毒性蛋白质（ 例如 ，穿孔素，粒酶B）的量，其可以通过细胞内染色19,20来量化影响。 此外，也有不同的可能性来修改协议。代替分离的NK细胞，外周血单核细胞（PBMCs）都可以使用。虽然一个人必须知道，PBMC群体内的绝对NK细胞数不同供体之间，甚至从在不同时间点的相同供体的样品之间是不同的。 NK细胞degranul通货膨胀是高度依赖于E:T比值。为了比较在不同时间点不同供体之间或NK细胞的功能，所述PBMC群体内的绝对NK细胞数量应该被用来建立一个常数E:在所有实验T比值。如果NK细胞功能的相同供体中在不同时间点监测的PBMC可冷冻和分析可以减少帧间实验变化11在一次对所有不同的时间点进行。 由于具有高达18的颜色是可能的染色板，的NK细胞功能分析可以扩展到一个更大的细节。使用额外的表面标志物包括CD57，NKG2A和杀伤细胞免疫球蛋白样受体（的KIRs），进一步NK细胞亚群可以被识别。表达至少一种自我KIR受过教育的NK细胞与K562细胞比未受过NK细胞相互作用后脱粒更强。相比之下，更多的差异化的CD57 + CD56 + </SUP&gt; NK细胞产生时相比较少分化的CD57，IL-12和IL-15的刺激少的IFN-γ - CD56 + NK细胞21。此外，NK细胞的功能的其他标记物可以解决。 LFA-1是NK细胞粘附的重要分子，并在后NK细胞活化其开放构象中被表达。这个所谓的"由内向外的信号"是早期NK细胞活化标志22。 最后，用于测试的NK细胞功能的刺激也可被修改。根据我们的经验，如果不加入细胞因子对K562细胞相互作用新鲜分离的NK细胞只脱颗粒甚少。用新鲜分离的PBMC不经进一步的细胞因子除了绕过这个问题，因为旁观者细胞的影响。此外，细胞因子的产生，特别是IFN-γ和TNF-α可以在IL-12和IL-18的刺激23启动。 NK细胞亚群内的IFN-γ产量可能会有所不同，取决于不g。在使用的刺激（细胞因子对目标诱发的刺激）24。此外，代替使用K562细胞作为靶细胞，从肿瘤患者来源的原发肿瘤细胞可以以在使用之前或期间免疫调节疗法（ 如 ，单克隆抗体治疗或免疫调节药物，以测试针对自体肿瘤细胞的NK细胞功能（各种IMiDs））。 在过去的几年中，免疫治疗的领域得到了迅速与免疫检查点抑制剂（ 如，ipilumumab，nivolumumab，pembrolizumab）的临床批准演变25和成功的试验中使用-expressing T细胞以及转基因的嵌合抗原受体（CAR）的CAR-NK细胞（在参考26中综述），为血液肿瘤患者27的治疗。因此，NK细胞为基础的免疫疗法越来越越来越成为关注的焦点。抗CD20抗体已在treatme取得了巨大成功过去二十年来28中的恶性淋巴瘤新台币。绝大多数NK细胞表达的低亲和性Fc受体CD16使细胞识别并杀死抗体 - 标记的靶细胞（抗体依赖性细胞的细胞毒性 - ADCC）。抗体的触发NK细胞功能的能力可以在体外使用所描述的协议29进行测试。 Terszowski 等分析NK细胞"对使用不同的临床批准的抗CD20抗体一个B类淋巴母细胞系的ADCC。而ADCC在利妥昔单抗治疗诱导的（第一临床批准的抗CD20抗体30）由KIR / HLA相互作用负面影响，使用obinutuzumab（一种新颖的糖基化改造的II型抗CD20单克隆抗体）31,32时这种效果，没有观察到。此外，NK细胞的功能是由不同的新的抗肿瘤药物像IMID来那度胺33和不同酪氨酸kinas影响Ë抑制剂（TKI）34。目前NK细胞特异性关卡抑制剂在临床试验中进行测试。实例是使用抗KIR 35,36或抗-NKG2A抗体（NCT02331875）以及诸如抗CD137抗体活化激动剂（NCT01775631; NCT02110082）的。 重要的是，过继NK细胞传送已经在各种不同的恶性疾病的有希望的临床效果37的执行。其目的选择最佳供体，对病人的肿瘤的NK细胞功能可能在体外使用的血液样品从潜在的NK细胞供体测试。 总之，所描述的协议被设计成分析来自健康供体或患者多样NK细胞的功能。这些功能可以在选定的时间点后，各种刺激多样化NK细胞亚群进行监测。

作为先天性免疫系统天然杀伤的一部分（NK）细胞有助于针对病毒感染或恶性转化的细胞防御的第一道防线。抑制和激活受体的一种系统使他们没有相反的T细胞之前，抗原引发健康和转化的细胞之间进行区分。当靶细胞相遇NK细胞释放它们的细胞毒性颗粒（ 如穿孔素，颗粒酶）的含量为免疫突触杀死他们的目标。此外，NK细胞产生和分泌不同种类的细胞因子（ 例如，干扰素γ:干扰素γ;肿瘤坏死因子α:TNF-α）和趋化因子（ 例如，巨噬细胞炎性蛋白1β:MIP-1β）在靶细胞相互作用或细胞因子刺激1。 足够的NK细胞功能，如细胞毒性，趋化因子和细胞因子的产生有不同的存款保险计划的命运产生重要影响简化。白血病患者显示增加的复发率，如果他们在诊断显示出有缺陷的NK细胞轮廓由减少的IFN-γ生产和活化NK细胞受体2的表达减少。 NK细胞数量和功能，包括细胞因子的产生对靶细胞相互作用的早期恢复与接收异基因造血干细胞移植3例患者减少复发和改善存活率有关。此外，当在C型肝炎病毒感染的病人的干扰素治疗开始外周血NK细胞的脱粒能力是在早期应答比在非应答者4更强。 NK细胞数（&gt; 80 /微升）在第15天在从淋巴瘤或多发性骨髓瘤的患者的自体干细胞移植（autoSCT）之后是预测性的改进的无进展生存率和总生存率5。在黑素瘤患者的T细胞中的表达immunoglobulin-和粘蛋白结构域containin克分子-3（TIM-3），NK细胞上的免疫调节蛋白，与疾病分期和预后6相关。 科学家们在整个过去几十年监测NK细胞的功能。抗肿瘤细胞NK细胞细胞毒性的不预先起动最初的分析是使用51 Cr-释放测定法7解决。最近，科学家开发了一种非放射性方法来评估膨胀NK细胞8的细胞毒性。细胞因子和趋化因子生产已使用酶联免疫吸附测定（ELISA）技术9,10-频繁评估。在过去几十年来，这些方法已通过流式细胞术基于测定补充。与常规的表面染色方案结合使用蛋白转运抑制剂（ 如，布雷菲德菌素A和莫能菌素）和细胞透方法已使科学家研究在不同的特定淋巴趋化因子和细胞因子产生ocyte子集（ 例如 ，T，B或NK细胞）11。此外，不同的流量基于流式细胞术的测定法已被开发并监控T和NK细胞的细胞毒性。在2004年阿尔特等人在目标小区遭遇所述NK细胞的溶酶体相关蛋白CD107a（灯1）的表面上表达作为细胞毒性颗粒12的脱粒标记。自宽范围的不同的荧光染料和多通道的流式细胞仪的是在我们的日子可用，它已成为可能同时监测在不同NK细胞亚群多样NK细胞功能（细胞毒性，细胞因子和趋化因子生产）。这成为在的情况下的样本大小是有限的， 例如 ，特别重要的是，在活检或从白细胞减少患者的血液样本。 为了测试全局NK细胞的功能，不同的流基于流式细胞仪的测定法可以有效地结合起来。 Theorell 等从HEA刺激NK细胞lthy捐助者与肿瘤细胞株K562和分析通过流式细胞仪13 NK细胞脱颗粒，由内向外信号和趋化因子的生产。最近NK细胞亚群，表型和肿瘤患者的功能autoSCT期间使用流式细胞仪为基础的试验进行了分析。据表明，NK细胞能够脱粒和autoSCT 11后在非常早的时间点产生在肿瘤细胞识别细胞因子/趋化因子。 这里一个协议描述用一个流式细胞术为基础的测定法，使得它能够同时监视在不同子集的NK细胞功能的肿瘤细胞，包括脱粒能力，趋化因子和细胞因子的产生来评估相互作用后NK细胞的功能。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 + glutamine</td>
			<td style="width:135px;">Invitrogen</td>
			<td style="width:119px;">6187-044</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">penicilin/streptomycin</td>
			<td style="width:135px;">Invitrogen</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD Falcon Round Bottom Tube</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">352008</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">fetal calf serum</td>
			<td style="width:135px;">Invitrogen</td>
			<td style="width:119px;">10270-106</td>
			<td style="width:167px;">heat inactivated before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T-flask</td>
			<td style="width:135px;">Greiner Bio-One</td>
			<td style="width:119px;">690195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">K562 tumor cell line</td>
			<td style="width:135px;">DSMZ GmbH</td>
			<td style="width:119px;">ACC 10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ammonium-chloride-potassium (ACK) lysis buffer</td>
			<td style="width:135px;">made in house</td>
			<td style="width:119px;">/</td>
			<td style="width:167px;">components are listed in the text</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Distilled water: Ampuwa Sp&#252;ll&#246;sung 1000ml Plastipur</td>
			<td style="width:135px;">Fresenius Kabi</td>
			<td style="width:119px;">1088813</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Megafuge 40R Centrifuge</td>
			<td>Heraeus</td>
			<td style="width:119px;">/</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EDTA blood collector tubes</td>
			<td style="width:135px;">Sarstedt</td>
			<td style="width:119px;">386453</td>
			<td style="width:167px;">S-Monovette 7,5 ml, K3 EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UltraPure 0.5M EDTA, pH 8.0</td>
			<td>Life Technologies</td>
			<td style="width:119px;">15575-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hematopoietic media (XVIVO)</td>
			<td>Lonza Group Ltd</td>
			<td style="width:119px;">BE04-743Q</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">human serum</td>
			<td style="width:135px;">DRK Blutspendedienst, Frankfurt/M&#160;</td>
			<td style="width:119px;">/</td>
			<td style="width:167px;">healthy donors with blood type AB; heat inactivated before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Neubauer-improved counting chamber, bright line</td>
			<td style="width:135px;">Marienfeld superior/ LO-Laboroptik Ltd.</td>
			<td style="width:119px;">0640030/ 1110000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">trypan blue solution (0,4%)</td>
			<td>Invitrogen</td>
			<td style="width:119px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3% acetic acid with methylene blue</td>
			<td style="width:135px;">Stemcell Technologies&#160;</td>
			<td style="width:119px;">07060</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning 96 Well Clear V-Bottom TC-treated Microplates</td>
			<td style="width:135px;">Corning&#160;</td>
			<td style="width:119px;">3894</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Falcon 96 Well Round Bottom Not Treated Microplates</td>
			<td style="width:135px;">Corning&#160;</td>
			<td style="width:119px;">351177</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">DPBS (Ca2+- and Mg2+-free)</td>
			<td style="width:135px;">Gibco Invitrogen</td>
			<td style="width:119px;">14190-169</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BSA</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">A2153-100G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NaN3</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">08591-1ML-F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">phorbol 12-myristate 13-acetate (PMA)&#160;</td>
			<td style="width:135px;">Merck</td>
			<td style="width:119px;">524400-1MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ionomycin&#160;</td>
			<td style="width:135px;">PromoKine</td>
			<td style="width:119px;">PK-CA577-1566-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">interleukin 15 (IL-15)</td>
			<td>PeproTech</td>
			<td>200-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proleukin S (IL-2)&#160;</td>
			<td style="width:135px;">Novartis Pharma</td>
			<td style="width:119px;">730523</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Golgi Stop, Protein Transport Inhibitor (containing Monensin)</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:119px;">554724</td>
			<td style="width:167px;">This product can be used in combination or instead of Golgi Plug. The best combination for the wished experimental setting has to be tested.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Golgi Plug, Protein Transport Inhibitor (containing Brefeldin A)</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:119px;">555029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paraformaldehyde</td>
			<td style="width:135px;">AppliChem</td>
			<td style="width:119px;">UN2209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">saponin</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">47036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">flow cytometer: Canto10C</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:119px;">/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FlowJo</td>
			<td style="width:135px;">TreeStar Inc.</td>
			<td style="width:119px;">/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Graph Pad</td>
			<td style="width:135px;">Graph Pad Inc.</td>
			<td style="width:119px;">/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACSxpress Separator</td>
			<td style="width:135px;">Miltenyi Biotec&#160;</td>
			<td style="width:119px;">130-098-308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACSxpress NK isolation kit</td>
			<td style="width:135px;">Miltenyi Biotec&#160;</td>
			<td style="width:119px;">130-098-185</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MACSxpress Erythrocyte Depletion Kit, human</td>
			<td style="width:135px;">Miltenyi Biotec&#160;</td>
			<td style="width:119px;">130-098-196</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACSmix Tube Rotator&#160;</td>
			<td style="width:135px;">Miltenyi Biotec&#160;</td>
			<td style="width:119px;">130-090-753</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD3 APC</td>
			<td style="width:135px;">Biolegend</td>
			<td>300412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD3 V450</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>560366</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD14 PerCP</td>
			<td style="width:135px;">Miltenyi Biotec&#160;</td>
			<td>130-094-969</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD14 V450</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>560349</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD16 PE</td>
			<td style="width:135px;">Biolegend</td>
			<td>302008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD16 PerCP</td>
			<td style="width:135px;">Biolegend</td>
			<td>302029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD19 PE-Cy7</td>
			<td style="width:135px;">Biolegend</td>
			<td>302216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD19 V450</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>560353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD45 BV510</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>563204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD56 FITC</td>
			<td style="width:135px;">Biolegend</td>
			<td>345811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD107a PE</td>
			<td style="width:135px;">Biolegend</td>
			<td>328608</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human IFN-&#947; AF-647</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>557729</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human MIP-1&#946; APC-H7</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>561280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DAPI</td>
			<td style="width:135px;">Biolegend</td>
			<td>422801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zombie Violet Fixable Viability Kit</td>
			<td>Biolegend</td>
			<td>423113</td>
			<td>fixable dead cell marker</td>
		</tr>
	</tbody>
</table>

flow cytometry-based assay for the monitoring of nk cell functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

<html>用于分析脱粒，细胞因子和趋化因子生产整个NK细胞种群和三种不同的NK细胞亚群的门控策略在图1中示出。 一种健康的供体的代表性结果显示于图2所示的NK细胞无任何刺激产生既不IFN-γ也不MIP-1β和不表达CD107a在其表面上（ 图2A）。相反，用肿瘤细胞和细胞因子刺激的NK细胞产生显著量胞内IFN-γ和MIP-1β。此外，其中的20％以上后，可以通过表面（ 图2C）上表达CD107a标明肿瘤细胞相互作用脱颗粒。 PMA和离子霉素刺激被用作对照（ 图2B）。 Ë1"SRC ="/文件/ ftp_upload / 54615 / 54615fig1.jpg"/&gt; 图1:门控策略后续栅极被作图。首先，碎片被排除在FSC-A / SSC-A地块。然后淋巴细胞识别和双峰是使用两个地块与SSC-A / SSC-H和FSC-A / FSC-H排除在外。此后，包括所有CD45 +细胞和不计都死了，CD3 +，CD14 +和CD19 +细胞栅极通过绘制CD45 /转储通道设置。接着，NK细胞被门控CD56 +细胞识别。与CD56 / CD16的作图可以用于确定主要NK细胞亚群。最后，整个的NK细胞群中，功能性标记由与CD107a，分别IFN-γ和MIP-1β，绘图CD56鉴定。这是可以做到，以及对各种不同类型的NK细胞亚群。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54615/54615fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图2:代表性的结果从一个健康的NK细胞供体使用图1中所描述的选通策略，像CD107a（用于脱粒）NK细胞功能的标记物，IFN-γ和MIP-1β可以识别。左栏（A）表示使用NK细胞在没有刺激的结果。中间列（B）中说明了在阳性对照PMA /离子霉素的存在的结果。右列（C）提出了与肿瘤细胞株K562刺激NK细胞，并在细胞因子的存在IL-2（100U / ml）和IL-15（10毫微克/毫升）。结果<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54615/54615fig2large.jpg" target="_blank">，请点击这里查看一个更大的版本这个数字。</a> <i毫克ALT ="表1"SRC ="/文件/ ftp_upload / 54615 / 54615table1.jpg"/&gt; 表 1: 纯度检查抗体。 <img alt="表2" src="/files/ftp_upload/54615/54615table2.jpg" /> 表2:细胞内外染色的抗体。

Watch this Scientific Journal Video about Flow Cytometry-based Assay for the Monitoring of NK Cell Functions at JoVE.com

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

一种简单和可靠的方法是此处描述的分析的一组NK细胞的功能，如不同的NK细胞亚群内脱粒，细胞因子和趋化因子生产。

流式细胞仪为基础的检测方法的NK细胞功能的监控

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

flow-cytometry-based-assay-for-the-monitoring-of-nk-cell-functions

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

20.7K 次观看.  Johann Wolfgang Goethe-University. 一种简单和可靠的方法是此处描述的分析的一组NK细胞的功能，如不同的NK细胞亚群内脱粒，细胞因子和趋化因子生产。 

视频: Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

科研

免疫与感染

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

人外周血 NK 细胞活性评估流基于流式细胞仪检测细胞毒性检测

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

基于流式细胞仪的 Zika 病毒疗养血清测定登革热病毒的体外抗体依赖性

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...