作者想感谢他们的工作，在创建初始K562 AAPC和mIL21融合向量劳伦斯库珀，Harjeet辛格和LENKA Hurton。 为这项工作提供了资金的UT MD安德森医师科学家计划，圣Baldrick的基金会，Friendswood的传奇。

1。从Buffy大衣的外周血单个核细胞的分离外周血单个核细胞（PBMC）的聚蔗糖 - 帕确来自健康捐赠者巴菲外套leukapheresis派生样本的浮力密度离心获得。 <ol><li>聚蔗糖帕确离心作为生产商的协议稍作修改。 </li><li> PBS将正常的捐助者的血液银行捉鬼外套到终体积为140毫升（典型的棕黄色大衣量为40-70毫升）。 </li><li>层35毫升15毫升（4管）聚蔗糖 - 帕确捉鬼外套样品。 </li><li>在400克离心20分钟不带刹车。 </li><li>恢复从聚蔗糖 - 帕确外周血:等离子接口，不放弃的聚蔗糖 - 帕确底部的红细胞。 </li><li>洗净外周血单个核细胞用PBS洗3次，每次10分钟，离心于400克。 </li><li>在这个阶段的NK细胞膨胀或NK细胞，外周血单个核细胞可以直接使用可以通过RosetteSep隔离（第4） </li><li>剩余的外周血单核细胞可以被冻结在FBS的液态氮中含10％DMSO。 </li><li>吸聚蔗糖和收集到两个50 mL离心管中，从第5步中的红细胞，用PBS（添加50毫升大关）洗三次，每次吸出上清液略读红细胞层的顶部删除粒。 </li><li> RosetteSepNK细胞的纯化（参见第4节）的红细胞，可立即使用，或存储在同等体积的Alsever的解决方案，在4 ° C，为以后使用的红细胞（可存储最多为4周）。 </li></ol> 2。 NK细胞扩张使用外周血或纯化的NK细胞，NK细胞可以启动扩大。在结束了一个为期三周的扩张所需的NK细胞数量的基础上，量的扩张外周血可以是多种多样的，代表结果的详细信息部分。 （见注1） 刺激1 0天<ol><li>对于每个5X10 6外周血扩大，数量和使用在100 Gy的伽玛辐照， 照射 10X10 6 K562 CL9 mIL21。 </li><li>照射后，用PBS重悬在NK细胞扩张媒体（NKEM）洗细胞。 </li><li>种子5X10 6外周血10X10 6照射K562 CL9 mIL21（1:2比例）在40毫升的NKEM在T75烧瓶中，并放置在一个孵化器直立在37 ° C和5％的CO 2 。 </li></ol> 3和第5天<ol start="4"><li>恢复细胞在400克离心5分钟，并更换新鲜NKEM媒体的一半（整个媒体音量加入新鲜的IL - 2），并继续文化。 </li></ol>刺激2 第7天<ol><li>计数细胞的数量，在文化，在一个星期结束。 </li><li>抛开5X10 5分型流式细胞仪检测细胞（见注2） </li><li>对于每个5X10 6 restimulated细胞，数量和使用伽玛辐照在100 Gy的照射5X10 6 K562 CL9 mIL21 。 </li><li>在总额2.5 × 10 5细胞/ mL添加照射K562 CL9 mIL21（1:1比例），重悬在NKEM人数相等（见注3）。 </li><li>种子细胞在T75烧瓶（最高，每50毫升容量瓶中）。 </li></ol> 10和12天<ol start="6"><li>计数的细胞数量。 </li><li>更改与基于手机号码的新鲜NKEM的整个媒体（见注3）。 </li></ol>第14天<ol start="8"><li>在两个星期的扩张结束计数文化中的细胞数量。 </li><li>抛开5X10 5分型流式细胞仪检测细胞（见注2） </li><li>如果扩张开始从外周血NK细胞可以在这个阶段使用扩展RosetteSep净化协议（参考第四节）纯化。如果扩张开始从纯化的NK细胞进行刺激3。 </li><li>净化后预留5 × 10 5细胞表型流式细胞仪验证的NK细胞（如在第8步）的纯度。 </li><li>继续刺激使用纯化的NK细胞（见注4）3。 </li></ol>刺激3 <ol><li> NK细胞悬浮照射K562 CL9 mIL21 NKEM根据手机号码（1:1比例）（见注3）。 </li></ol> 17和19天<ol start="2"><li>计数的细胞数量。 </li><li>更改与基于手机号码的新鲜NKEM的媒体（见注3）。 </li></ol>第21天<ol start="4"><li>在三个星期的扩张结束计数文化中的细胞数量。 </li><li>恢复1 × 10 6流式细胞仪分析细胞完整的NK细胞表型抗体面板（见表1） 。 </li><li>冻结在FBS的细胞用含10％DMSO在5X10 7％，以供将来使用的小瓶细胞的最大密度。 </li></ol> 3。 NK细胞毒性试验<ol><li>在NKEM解冻小瓶NK细胞和种子，前一天到PErforming细胞毒试验，以便恢复。 </li><li>对于每个NK细胞的细胞毒作用，使用单一的目标细胞株，6x10 5 NK细胞和 3X10 5靶细胞检测的要求（见注5）。 </li><li>准备稀释钙黄绿素- AM NKEM（股票1 mg / ml的DMSO）（见注6）的CAM媒体。 </li><li>重悬在1毫升CAM媒体10 6靶细胞（见注7）。 </li><li>在37 ° C孵育1小时，偶尔晃动。 </li><li>悬浮NK细胞在1 × 10 6细胞/ ml的细胞和NK细胞悬液200uL，每个U型底部的3口井的96孔板对应到10:1 E:T比例如图1所示。 （见注8） </li><li>新增完整的媒体100uL除了"最大"的所有其余井。 </li><li>加入100uL 2％TRITON X - 100"最大"。 </li><li>执行串行稀释度为5后续电子邮件的NK细胞，T细胞转移100uL每次比例，拌匀。丢弃从上井（E:T比值为0.3125:1）100uL。 </li><li>钙黄绿素装载1小时后，在NKEM洗两次靶细胞，5分钟1200转离心。 （见注9） </li><li>重新点票的靶细胞和悬浮在1 × 10 5细胞/ mL。 </li><li>每孔加入100μL靶细胞（1 × 10 4 /以及）。离心机在百克1分钟，以启动细胞接触。 </li><li>在37 ° C和5％CO 2 4小时。 </li><li>轻轻移液100μLpipetter以均匀暂停释放的钙黄绿素混合的文化，在百克降速盘5分钟沉淀细胞和照顾，以避免气泡100μL上清转移到一个新的板块。任何弹出的气泡，可能会形成使用细针。 </li><li>用荧光酶标仪（485 nm处激发滤光片，发射滤波器530 nm）读取的板块。底部阅读建议。 </li><li>根据具体裂解百分比计算公式[（测试版自发释放）/（最大释放 - 自发释放）] × 100。 </li></ol> 4。由RosetteSep NK细胞净化<ol><li>取外周血或扩大细胞，放入50 mL的管（100:1红细胞:PBMC）的100倍，多余的红细胞。 </li><li>如果使用的新鲜红细胞直接进行下一步，如果在Alsever的解决方案中的红细胞计数，红细胞数量和洗适当（100倍超额）补充含2％FBS三次用PBS红细胞的数量，在1200转离心每次10分钟。 </li><li>结合步骤1.7或在PBS 2.10步扩 ​​大细胞的外周血红细胞+ 2％胎牛血清的1毫升每5X10 7外周血或扩大细胞的最终体积。 </li><li>添加RosetteSep1μL人类NK细胞富集外周血或扩大细胞每1 × 10 6鸡尾酒。 </li><li>搅拌均匀，在室温下孵育20分钟，轻轻搅拌，每隔5分钟。 </li><li>加入等体积PBS + 2％FBS混合轻轻上一层聚蔗糖 - 帕确顶部。 </li><li> PBMC中隔离所述的聚蔗糖帕确离心重复步骤（第一节）。 </li><li>计数净化后回收的NK细胞和预留5x105细胞通过流式细胞仪检测NK细胞纯度（步骤8）phenotpying。 </li></ol> 5。注释 注意:1。NK细胞可扩大直接从外周血单个核细胞，或从RosetteSep纯化NK细胞。我们已经注意到了类似的扩张效率，但一些捐助者可能具有非常低的NK细胞数量，造成困难净化RosetteSep前扩张。 注2:我们经常使用的表型CD56 - FITC，CD16 - PE，CD3 - PE - Cy5的扩张期间，列举那些CD3阴性，CD16或CD56阳性的NK细胞。 注3:在每个媒体的变化或刺激，悬浮细胞在2.5 × 10 5 /毫升，以保持在或低于2万每毫升外周血单核细胞/ NK细胞数量扩张的高峰阶段。这将防止营养物质的耗竭，并帮助实现最大的扩张和生存。 注4。NK的扩张速度是捐助者的依赖，在刺激1或2的细胞可以被冻结的部分月底和部分进一步扩大，根据实验的需要。我们有很好的成功，在使用冷冻细胞在稍后的时间扩展。 注5:为了让错误的空间，我们建议使用最低 7x10 5 NK细胞重悬在700 ULS NKEM和4 × 5钙黄绿素- AM染色目标4 NKEM毫升悬浮细胞设立的细胞毒试验。如果使用播种靶细胞的细胞量较高（6毫升6x10 5），可能需要的基础上正在使用的大小媒体盆地多道移液器。也是推荐的NK细胞数量是专为E:T比例显示在协议中，使用更高的发送:T比率增加NK细胞每毫升相应的号码（例如:40:1发送:T比值使用4 × 6细胞/ ml） 注6:我们建议进行了初步的首选目标细胞株的滴定钙黄绿素- AM加载，使用以下1:500，1:400稀释。 1:300，1:200和1:100，实现最大的和自发的释放之间的最佳差异。 注7:当使用贴壁细胞系作为目标，先准备单细胞悬液，使用非酶细胞分解缓冲区。如果执行的ADCC，准备在CAM媒体重复管的靶细胞。 注8，如果执行的ADCC，添加相同的NK细胞，以3口井，对应10:1 E: 为 ADCC的T。重复更多的捐助者。重复额外的靶细胞。 注9:如果执行的ADCC实验，钙黄绿素装载45分钟后，添加诱导对靶细胞的ADCC的特定抗体10ug。 15多分钟后，靶细胞完全培养基清洗两次，5分钟1200转离心。悬浮细胞在1 × 10 5细胞/ mL，并在协议的下一步骤进行。 6。代表性的成果<img src="/files/ftp_upload/2540/2540fig2.jpg" alt="图2" /> 图2当膨胀是按照计划进行上文所述，典型的NK细胞产生范围从1x10 9 10 10个细胞（捐助者的依赖变异）为起始原料，采用5x106外周血单个核细胞。图中显示NK细胞倍的扩张（N = 19）相比，在原有的产品中存在的NK细胞（中位数+ / - 四分位数）。 <img src="/files/ftp_upload/2540/2540fig3.jpg" alt="图3" /> 图3:扩大NK细胞表达不同的NK细胞受体与少数例外（CD11b表达，CD160和CD244）未展开的主要NK细胞相媲美的。 <img src="/files/ftp_upload/2540/2540fig4.jpg" alt="图4" /> 图4。外周血从Buffy大衣的恢复是捐助者的依赖，范围可以从300x10 6 800x10 6 。 NK细胞可占2％ - 18％的外周血单个核细胞。 RosetteSep扩大细胞的纯化，回收纯NK细胞从40-70％14天范围。通过以下建议扩大和净化的协议，NK细胞纯度达99％，可以预期的。 <img src="/files/ftp_upload/2540/2540fig5.jpg" alt="图5" /> 图5。扩大NK细胞都表现出对肿瘤细胞株的范围，包括神经母细胞瘤，白血病，骨肉瘤和黑色素瘤（代表反洗钱杀害％的具体裂解所示）的细胞毒性。

<ol>
	<li>McKenna, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Good+manufacturing+practices+production+of+natural+killer+cells+for+immunotherapy:+a+six-year+single-institution+experience.">Good manufacturing practices production of natural killer cells for immunotherapy: a six-year single-institution experience.</a> Transfusion. 47, 520-520 (2007).</li><li>Koehl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ex+vivo+expansion+of+highly+purified+NK+cells+for+immunotherapy+after+haploidentical+stem+cell+transplantation+in+children.">ex vivo expansion of highly purified NK cells for immunotherapy after haploidentical stem cell transplantation in children.</a> Klinische Padiatrie. 217, 345-345 (2005).</li><li>Klingemann, H. G., Martinson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ex+vivo+expansion+of+natural+killer+cells+for+clinical+applications.">ex vivo expansion of natural killer cells for clinical applications.</a> Cytotherapy. 6, 15-15 (2004).</li><li>Ayello, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cord+blood+natural+killer+and+lymphokine+activated+killer+lymphocytes+following+ex+vivo+cellular+engineering.">Characterization of cord blood natural killer and lymphokine activated killer lymphocytes following ex vivo cellular engineering.</a> Biol Blood Marrow Transplant. 12, 608-608 (2006).</li><li>Carlens, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+in+vitro+expansion+of+cytotoxic+human+CD3-CD56++natural+killer+cells.">A new method for in vitro expansion of cytotoxic human CD3-CD56+ natural killer cells.</a> Hum Immunol. 62, 1092-1092 (2001).</li><li>Boissel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Umbilical+cord+mesenchymal+stem+cells+increase+expansion+of+cord+blood+natural+killer+cells.">Umbilical cord mesenchymal stem cells increase expansion of cord blood natural killer cells.</a> Biol Blood Marrow Transplant. 14, 1031-1031 (2008).</li><li>North, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-primed+human+natural+killer+cells+lyse+NK-resistant+tumor+targets:+evidence+of+a+two-stage+process+in+resting+NK+cell+activation.">Tumor-primed human natural killer cells lyse NK-resistant tumor targets: evidence of a two-stage process in resting NK cell activation.</a> J Immunol. 178, 85-85 (2007).</li><li>Berg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical-grade+ex+vivo-expanded+human+natural+killer+cells+up-regulate+activating+receptors+and+death+receptor+ligands+and+have+enhanced+cytolytic+activity+against+tumor+cells.">Clinical-grade ex vivo-expanded human natural killer cells up-regulate activating receptors and death receptor ligands and have enhanced cytolytic activity against tumor cells.</a> Cytotherapy. 11, 341-341 (2009).</li><li>Fujisaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+highly+cytotoxic+human+natural+killer+cells+for+cancer+cell+therapy.">Expansion of highly cytotoxic human natural killer cells for cancer cell therapy.</a> Cancer Res. 69, 4010-4010 (2009).</li><li>Fujisaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicative+potential+of+human+natural+killer+cells.">Replicative potential of human natural killer cells.</a> Br J Haematol. 145, 606-606 (2009).</li><li>Gong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+expansion+of+natural+killer+cells+with+high+cytotoxicity+by+K562+cells+modified+to+co-express+major+histocompatibility+complex+class+I+chain-related+protein+A,+4-1BB+ligand,+and+interleukin-15.">Ex vivo expansion of natural killer cells with high cytotoxicity by K562 cells modified to co-express major histocompatibility complex class I chain-related protein A, 4-1BB ligand, and interleukin-15.</a> Tissue Antigens. 76, 467-467 (2010).</li></ol>

NK细胞扩增和激活介质（NKEM） <ul><li> 90％的RPMI 1640（Cellgro） </li><li> 10％胎牛血清（GIBCO） </li><li> 1X青霉素/链霉素（Cellgro） </li><li> 1X L -谷氨酰胺（Gibco公司） <ul><li>过滤消毒使用前的媒体。 </li></ul></li><li> 50 U / mL的IL - 2（Proleukin，诺华疫苗和诊断，INC） <ul><li> - 从200 IU /μL的股票摊薄。白细胞介素2添加所需的介质数量，只是每次使用前。 </li></ul></li></ul>外周血单个核细胞和NK细胞分离<ul><li>聚蔗糖帕确（GE医疗） </li><li> Alsever的解决方案（Sigma公司） </li><li> RosetteSep人类NK细胞富集酒会（干细胞技术） </li></ul> NK细胞毒性试验<ul><li>钙黄绿素- AM（Invitrogen公司） <ul><li>股票的解决方案是在1毫克/毫升，在50μL分装冷冻储存。在NKEM媒体溶解细胞染色，以达到预期的稀释。 </li></ul></li></ul>抗体下表中列出的NK细胞表型的抗体; <table border="1"><tbody><tr><td></td><td>抗体</td><td>测试卷，每</td><td>公司</td><td>目录编号</td></tr><tr><td>管1 </td><td valign="top">同型FITC 同型FITC 同型FITC 同型FITC 流式细胞仪缓冲区 总成交量</td><td valign="top"> 5 5 5 5 80 100 </td><td valign="top"> BD Pharmingen公司 BD Pharmingen公司 BD Pharmingen公司 BD Pharmingen公司</td><td valign="top"> 555748 555749 557224 340442 </td></tr><tr><td>管2 </td><td valign="top"> CD56 FITC NKp30体育 NKp44体育 NKp46体育 CD3 + PE - Cy5的 CD16 Alexa的647 流式细胞仪缓冲区 总成交量</td><td valign="top"> 5 5 5 5 5 5 70 100 </td><td valign="top"> BD Pharmingen公司 BD Pharmingen公司 BD Pharmingen公司 BD Pharmingen公司 BD Pharmingen公司 BD Pharmingen公司</td><td valign="top"> 340410 558407 558563 557991 555341 557710 </td></tr><tr><td>管3 </td><td valign="top"> CD56 FITC KIR2DL1体育 KIR2DL2 / 3 PE KIR3DL1体育 CD3 + PE - Cy5的 NKG2D的APC 流式细胞仪缓冲区 总成交量</td><td valign="top"> 5 5 5 5 5 5 70 100 </td><td valign="top"> BD Pharmingen公司 R＆D系统 美天旎生物技术公司 R＆D系统 BD Pharmingen公司 BD Pharmingen公司</td><td valign="top"> 340410 FAB1844P 130-092-618 FAB12251P 555341 558071 </td></tr><tr><td>管4 </td><td valign="top"> CD56 FITC CD11b的聚乙烯 CD3 + PE - Cy5的 CD27 APC 流式细胞仪缓冲区 总成交量</td><td valign="top"> 5 5 5 5 80 100 </td><td valign="top"> BD Pharmingen公司 BD Pharmingen公司 BD Pharmingen公司 BD Pharmingen公司</td><td valign="top"> 340410 555388 555341 558664 </td></tr><tr><td>管5 </td><td valign="top"> CD56 FITC CD266（DNAM - 1）体育 CD3 + PE - Cy5的 CD160 Alexa647 流式细胞仪缓冲区 总成交量</td><td valign="top"> 5 5 5 5 80 100 </td><td valign="top"> BD Pharmingen公司 BD Pharmingen公司 BD Pharmingen公司 eBiosciences </td><td valign="top"> 340410 559789 555341 51-1609-42 </td></tr><tr><td>管6 </td><td valign="top"> CD56 FITC CD244（2B4）PE CD3 + PE - Cy5的 CD197（CCR7）APC 流式细胞仪缓冲区 总成交量</td><td valign="top"> 5 5 5 5 80 100 </td><td valign="top"> BD Pharmingen公司 BD Pharmingen公司 BD Pharmingen公司 eBiosciences </td><td valign="top"> 340410 550816 555341 17-1979-42 </td></tr></tbody></table>

expansion, purification, and functional assessment of human peripheral blood nk cells

自然杀伤（NK）细胞，发挥对各种传染病的微生物和肿瘤免疫监视的重要作用。 NK细胞在体外的能力，扩大有限已成为制约NK细胞免疫治疗的发展。在这里，我们描述了一个方法，有效地扩大功能NK细胞体外大量使用K562细胞表达膜结合IL21，作为人工抗原提呈细胞（AAPC ）。 NK细胞收养疗法利用细胞耗尽的T细胞或NK细胞的阳性选择捐助的稳态leukapheresis获得的产品。该产品通常是在IL - 2激活过夜，然后管理翌日1。由于低频外周血NK细胞，NK细胞的数量相对较少，已交付的临床试验。 不能传播NK细胞在体外已为最佳的临床结果产生足够的细胞数量的限制因素。一些NK细胞的扩张（超过1-2周的5-10倍）通过高剂量IL - 2仅2。激活自体T细胞可以介导NK细胞扩张，想必也通过局部细胞因子3的释放。与间质的基质或人工抗原提呈细胞（AAPCS）的支持，可以支持扩展的NK细胞从外周血和脐带血 4 。结合NKp46和CD2抗体包被的磁珠激活NK细胞扩张（美天旎生物技术公司，奥本CA）是目前市场上销售，造成约100倍，在21天的扩张。 使用AAPC扩大或激活NK细胞的临床试验正在进行中，一个白血病细胞株CTV - 1素和激活NK细胞无显着扩大。一，二审利用NK细胞扩展的EB病毒拼箱，实现了平均490倍，扩大在21天6。第三，利用基于一个K562 AAPC转4 - 1BBL（CD137L）和膜结合IL - 15（MIL - 15）7，取得了在21天的平均NK扩张277倍。虽然，NK细胞扩大使用K562 - 41BBL - mIL15 AAPC高度细胞毒性， 在体外和体内未展开的NK细胞相比，参与的ADCC，其增殖是有限的，归结到端粒的缩短8由衰老。最近的NK细胞的350倍扩大使用K562细胞表达MICA，4 - 1BBL和IL15 9的报道。 我们的NK细胞扩展的方法描述，此处产生的NK细胞不衰老的迅速扩散，实现在21天的中位数为21,000倍扩张。

Watch this Scientific Journal Video about Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells at JoVE.com

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

在这里，我们描述了一种方法，有效地扩大和净化人类NK细胞的大量和评估它们的功能。

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

expansion-purification-functional-assessment-human-peripheral-blood

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

48.1K 次观看.  MD Anderson Cancer Center - University of Texas. 在这里，我们描述了一种方法，有效地扩大和净化人类NK细胞的大量和评估它们的功能。

视频: Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

特定细胞群的净化荧光激活细胞分选（FACS）

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

科研

免疫与感染

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

扩展人外周血γδT细胞使用Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

紧凑型超支化聚乙二醇接枝的膜抗原受保护的功能性红血细胞

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

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, ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.