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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</ol>

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，...

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。

<table><tbody><tr><td>RPMI 1640 + glutamine</td><td>Invitrogen</td><td>6187-044</td><td></td></tr><tr><td>penicilin/streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>BD Falcon Round Bottom Tube</td><td>BD</td><td>352008</td><td></td></tr><tr><td>fetal calf serum</td><td>Invitrogen</td><td>10270-106</td><td>heat inactivated before use</td></tr><tr><td>T-flask</td><td>Greiner Bio-One</td><td>690195</td><td></td></tr><tr><td>K562 tumor cell line</td><td>DSMZ GmbH</td><td>ACC 10</td><td></td></tr><tr><td>ammonium-chloride-potassium (ACK) lysis buffer</td><td>made in house</td><td>/</td><td>components are listed in the text</td></tr><tr><td>Distilled water: Ampuwa Sp&amp;uuml;ll&amp;ouml;sung 1,000 ml Plastipur</td><td>Fresenius Kabi</td><td>1088813</td><td></td></tr><tr><td>Megafuge 40R Centrifuge</td><td>Heraeus</td><td>/</td><td></td></tr><tr><td>EDTA blood collector tubes</td><td>Sarstedt</td><td>386453</td><td>S-Monovette 7.5 ml, K&lt;sub&gt;3&lt;/sub&gt;EDTA</td></tr><tr><td>UltraPure 0.5 M EDTA, pH 8.0</td><td>Life Technologies</td><td>15575-020</td><td></td></tr><tr><td>Hematopoietic media (XVIVO)</td><td>Lonza Group Ltd</td><td>BE04-743Q</td><td></td></tr><tr><td>human serum</td><td>DRK Blutspendedienst, Frankfurt/M&amp;nbsp;</td><td>/</td><td>healthy donors with blood type AB; heat inactivated before use</td></tr><tr><td>Neubauer-improved counting chamber, bright line</td><td>Marienfeld superior/ LO-Laboroptik Ltd.</td><td>0640030/ 1110000</td><td></td></tr><tr><td>trypan blue solution (0.4%)</td><td>Invitrogen</td><td>15250-061</td><td></td></tr><tr><td>3% acetic acid with methylene blue</td><td>Stemcell Technologies&amp;nbsp;</td><td>07060</td><td></td></tr><tr><td>Corning 96 Well Clear V-Bottom TC-treated Microplates</td><td>Corning&amp;nbsp;</td><td>3894</td><td></td></tr><tr><td>Falcon 96 Well Round Bottom Not Treated Microplates</td><td>Corning&amp;nbsp;</td><td>351177</td><td></td></tr><tr><td>DPBS (Ca&lt;sup&gt;2+&lt;/sup&gt;- and Mg&lt;sup&gt;2+&lt;/sup&gt;-free)</td><td>Gibco Invitrogen</td><td>14190-169</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A2153-100G</td><td></td></tr><tr><td>NaN&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>08591-1ML-F</td><td></td></tr><tr><td>phorbol 12-myristate 13-acetate (PMA)&amp;nbsp;</td><td>Merck</td><td>524400-1MG</td><td></td></tr><tr><td>ionomycin&amp;nbsp;</td><td>PromoKine</td><td>PK-CA577-1566-5</td><td></td></tr><tr><td>interleukin 15 (IL-15)</td><td>PeproTech</td><td>200-15</td><td></td></tr><tr><td>Proleukin S (IL-2)&amp;nbsp;</td><td>Novartis Pharma</td><td>730523</td><td></td></tr><tr><td>Golgi Stop, Protein Transport Inhibitor (containing Monensin)</td><td>BD Biosciences</td><td>554724</td><td>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><td>Golgi Plug, Protein Transport Inhibitor (containing Brefeldin A)</td><td>BD Biosciences</td><td>555029</td><td></td></tr><tr><td>paraformaldehyde</td><td>AppliChem</td><td>UN2209</td><td></td></tr><tr><td>saponin</td><td>Sigma Aldrich</td><td>47036</td><td></td></tr><tr><td>flow cytometer: Canto10C</td><td>BD Biosciences</td><td>/</td><td></td></tr><tr><td>FlowJo</td><td>TreeStar Inc.</td><td>/</td><td></td></tr><tr><td>Graph Pad</td><td>Graph Pad Inc.</td><td>/</td><td></td></tr><tr><td>MACSxpress Separator</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-098-308</td><td></td></tr><tr><td>MACSxpress NK isolation kit</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-098-185</td><td></td></tr><tr><td>MACSxpress Erythrocyte Depletion Kit, human</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-098-196</td><td></td></tr><tr><td>MACSmix Tube Rotator&amp;nbsp;</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-090-753</td><td></td></tr><tr><td>anti-human CD3 APC</td><td>Biolegend</td><td>300412</td><td></td></tr><tr><td>anti-human CD3 V450</td><td>BD Biosciences</td><td>560366</td><td></td></tr><tr><td>anti-human CD14 PerCP</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-094-969</td><td></td></tr><tr><td>anti-human CD14 V450</td><td>BD Biosciences</td><td>560349</td><td></td></tr><tr><td>anti-human CD16 PE</td><td>Biolegend</td><td>302008</td><td></td></tr><tr><td>anti-human CD16 PerCP</td><td>Biolegend</td><td>302029</td><td></td></tr><tr><td>anti-human CD19 PE-Cy7</td><td>Biolegend</td><td>302216</td><td></td></tr><tr><td>anti-human CD19 V450</td><td>BD Biosciences</td><td>560353</td><td></td></tr><tr><td>anti-human CD45 BV510</td><td>BD Biosciences</td><td>563204</td><td></td></tr><tr><td>anti-human CD56 FITC</td><td>Biolegend</td><td>345811</td><td></td></tr><tr><td>anti-human CD107a PE</td><td>Biolegend</td><td>328608</td><td></td></tr><tr><td>anti-human IFN-&amp;gamma; AF-647</td><td>BD Biosciences</td><td>557729</td><td></td></tr><tr><td>anti-human MIP-1&amp;beta; APC-H7</td><td>BD Biosciences</td><td>561280</td><td></td></tr><tr><td>DAPI</td><td>Biolegend</td><td>422801</td><td></td></tr><tr><td>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...

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

Research

JoVE Journal

Immunology and Infection

21.0K 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

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.

We present two different staining protocols for NKG2D ligand (NKG2DL) detection in human primary acute myeloid leukemia (AML) samples. The first approach is based on a fusion protein, able to recognize all known and potentially yet unknown ligands, while the second protocol relies on the addition of multiple anti-NKG2DL antibodies.

NKG2D 利甘德表面检测的两种流细胞学方法，以区分干细胞与急性骨髓性白血病中的散装亚群

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

科研

癌症研究

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 ...

免疫与感染

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

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, ...

Assessment of Human Natural Killer Cell Events Driven by Fc&#947;RIIIa Engagement in the Presence of Therapeutic Antibodies

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by Fc&#947;RIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the Fc&#947;RIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.

A protocol for studying Fc&#947;RIIIa-driven events by therapeutic antibodies in human natural killer cells is described here. This artificial stimulation platform permits the interrogation of downstream effector functions such as degranulation, chemokine/cytokine production, and signaling pathways mediated by the Fc&#947;RIIIa and Fc portions of antibodies involved in binding.

在存在治疗性抗体的情况下，由FcγRIIIa参与驱动的人类自然杀伤细胞事件的评估

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and ...

Analysis of Human Natural Killer Cell Metabolism

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to promote survival, cellular proliferation, production of cytokines such as interferon gamma (IFN&#947;) and/or cytotoxicity programs. NK cell activation by cytokine stimulation requires a substantial remodeling of metabolic pathways to support their bioenergetic and biosynthetic requirements. There is a large body of evidence that suggests that impaired NK cell metabolism is associated with a number of chronic diseases including obesity and cancer, which highlights the clinical importance of the availability of a method to determine NK cell metabolism. Here we describe the use of an extracellular flux analyzer, a platform that allows real-time measurements of glycolysis and mitochondrial oxygen consumption, as a tool to monitor changes in the energy metabolism of human NK cells. The method described here also allows for the monitoring of metabolic changes after stimulation of NK cells with cytokines such as IL-15, a system that is currently being investigated in a wide range of clinical trials.

In this paper, we describe a method to measure glycolysis and mitochondrial respiration in primary human Natural Killer (NK) cells isolated from peripheral blood, at rest or following IL15-induced activation. The protocol described could be easily extended to primary human NK cells activated by other cytokines or soluble stimuli.

人类自然杀手细胞代谢分析

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to ...

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells is regulated by their expression of germline-encoded inhibitory and activating receptors. The engagement of these receptors by their cognate ligands on target cells determines whether the intercellular interaction will result in NK cell killing. This protocol details the design and optimization of two complementary mass cytometry (CyTOF) panels. One panel was designed to phenotype NK cells based on receptor expression. The other panel was designed to interrogate expression of known ligands for NK cell receptors on several immune cell subsets. Together, these two panels allow for the profiling of the human NK cell receptor-ligand repertoire. Furthermore, this protocol also details the process by which we stain samples for CyTOF. This process has been optimized for improved reproducibility and standardization. An advantage of CyTOF is its ability to measure over 40 markers in each panel, with minimal signal overlap, allowing researchers to capture the breadth of the NK cell receptor-ligand repertoire. Palladium barcoding also reduces inter-sample variation, as well as consumption of reagents, making it easier to stain samples with each panel in parallel. Limitations of this protocol include the relatively low throughput of CyTOF and the inability to recover cells after analysis. These panels were designed for the analysis of clinical samples from patients suffering from acute and chronic viral infections, including dengue virus, human immunodeficiency virus (HIV), and influenza. However, they can be utilized in any setting to investigate the human NK cell receptor-ligand repertoire. Importantly, these methods can be applied broadly to the design and execution of future CyTOF panels.

<meta charset="utf8"></head><body>人自然杀伤细胞受体-配体库分析

Here we design two complementary mass cytometry (CyTOF) panels and optimize a CyTOF staining protocol with the aim of profiling the natural killer cell receptor and ligand repertoire in the setting of viral infections.

人自然杀伤细胞受体-配体库分析

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells ...

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by clearing pathogen-infected, malignant, and stressed cells. The ability of NK cells to eradicate cancer cells makes them an important tool in the fight against cancer. Several new immune-based therapies are under investigation for cancer treatment which rely either on enhancing NK cell activity or increasing the sensitivity of cancer cells to NK cell-mediated eradication. However, to effectively develop these therapeutic approaches, cost-effective in vitro assays to monitor NK cell-mediated cytotoxicity and migration are also needed. Here, we present two in vitro protocols that can reliably and reproducibly monitor the effect of NK-cell cytotoxicity on cancer cells (or other target cells). These protocols are non-radioactivity-based, simple to set up, and can be scaled up for high-throughput screening. We also present a flow cytometry-based protocol to quantitatively monitor NK cell migration, which can also be scaled up for high-throughput screening. Collectively, these three protocols can be used to monitor key aspects of NK cell activity that are necessary for the cells&#39; ability to eradicate dysfunctional target cells.

Evasion of natural killer (NK) cell-mediated eradication by cancer cells is important for cancer initiation and progression. Here, we present two non-radioactivity-based protocols to evaluate NK cell-mediated cytotoxicity toward hepatic tumor cells. Additionally, a third protocol is presented to analyze NK cell migration.

肝肿瘤细胞背景下自然杀伤细胞介导细胞毒性和迁移的测量

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by ...

医学

Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level

The assessment of the cytotoxic potential of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell treatments, is instrumental in assessing their efficacy and is a prerequisite for clinical application. However, traditional cytotoxicity assays are conducted as bulk assays and do not provide detailed information about the functional heterogeneity of the CAR T cell population. In this study, we describe a double-emulsion droplet-based method that allows for large-scale co-encapsulation of single effector CAR T cells with single target cells while enabling dual quantification of both cytotoxic effector molecules from T cells and cell death of the target cell. The protocol outlines a method for the generation and purification of CD19-specific CAR T cells, followed by their co-encapsulation in droplets with the CD19+ cell line JeKo-1, along with reagents to visualize cytotoxic effector molecule secretion (Granzyme B) and cell death (using propidium iodide, PI). We demonstrate how to generate droplets containing single CAR T and target cells using a commercially available microfluidics device for generating double emulsion droplets. Additionally, we provide examples of how to assay the functional diversity of CAR T cells in droplets using standard flow cytometry equipment. Finally, we briefly describe the temporal kinetics and heterogeneity of CD19-specific CAR T-cell killing. While this method focuses on cell death following a CAR T cell attack, it is also adaptable for examining other types of T cells, cytotoxic immune cells, and effector cell functions, such as cytokine secretion.

Here, we describe a method for creating double-emulsion droplets encapsulating one T cell and a cancer target cell to examine cell killing at the single-cell level. This method allows for dual quantification of both cytotoxic molecules and target-cell apoptosis within a large population of T cells.

流式细胞仪为基础的检测方法的NK细胞功能的监控

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