作者感谢朱医生曼海姆，瓦尔特·埃利奇曼，拉莫纳·斯特姆，丰达礁，丹尼尔·布卡拉，马伦·哈兰特以及纳塔利·阿尔特迈耶的实验和数据分析过程中的支持。这项工作是由沃纳西门子基础上，通过SFB685东风集团（项目B6）和财富（2309-0-0）的支持。

安全注意事项:当处理放射性，存储后面的2英寸厚的铅砖64 Cu和使用各自的屏蔽，用于携带活性的所有容器。使用适当的工具来间接处理非屏蔽来源，以避免直接用手接触，并尽量减少暴露在放射性物质。始终佩戴辐射剂量监测徽章和个人防护装备，并检查自己和污染的工作区域要立即解决这一问题。留在使用放射性物质的区域之前丢弃可能被污染的个人保护装备。直到放射性64将Cu前充分的处置衰减（大约10半衰期= 127 1H）存储后面铅屏蔽整个放射性废物。 1. 64铜生产注:放射性同位素64的Cu经由64的Ni（P，N）使用PETT 64铜核反应产生的根据McCarthy 等人的修改的协议种族回旋加速器。 18。 <ol><li>对于64铜生产，照射64的Ni，其被电镀在铂/铱板（90/10）中，用30μA为12.4兆电子伏的质子束6小时。 </li><li>加热该铂/铱靶至100℃，在一个专用的聚醚醚酮（PEEK）腔室和在2mL浓HCl孵育20分钟以溶解64铜/镍64。 </li><li>添加浓HCl的另一个1 mL和孵育10分钟。 </li><li>蒸发的HCl使用氩气气流和冷却腔室至室温。 </li><li>用3mL的4％的0.2M的HCl和96％甲醇（体积/体积）冲洗室和转移该溶液到离子交换柱，这是预调节用4％的0.2M HCl的甲醇中至少15分钟。流过的可用于回收64的Ni。 </li><li>洗64的Cu保留在柱中，用4％的0.2M的HCl我Ñ甲醇。 </li><li>洗脱64的Cu，用70％1.3M的盐酸/ 30％异丙醇（V / V）到收集小瓶中，蒸发在氩气流的溶液，并让管形瓶冷却至室温。 </li><li>溶解64的Cu在0.1M HCl中的140-210μL。 </li></ol> 2.抗体缀合与DOTA和随后的64的Cu-放射性标记注:螯合剂DOTA将由N-羟基琥珀酰亚胺（NHS）酯化学链接到mAb的氨基官能团和缀合物将与64铜19随后被放射性标记。 <ol><li>调整KJ1-26-mAb的浓度为8毫克/毫升和mAb中的溶液渗滤1针对与1.2克使用分子量螯合离子交换树脂的/ L处理的0.1M乙酸钠2 HPO 4 pH为7.5溶液切断（ MWCO 30 kDa）的离心过滤装置。应用3个随后的洗涤步骤用14毫升的缓冲液中。最终的洗涤步骤后，再次浓缩溶液至1mL。量化通过OD 280nm处的测量的抗体浓度。 </li><li>以10毫克/毫升在临使用之前制备浓度在超纯或PCR级水中的DOTA-NHS溶液。让管形瓶调整至室温打开之前，以避免水冷凝，然后小心地使用塑料抹刀除去DOTA-NHS。添加216μL此DOTA-NHS溶液至8mg的渗滤KJ1-26单抗溶液中的，调匀，并在4℃下在转鼓混合器孵育24小时。 </li><li>渗滤液的DOTA-KJ1-26单抗针对0.25M的乙酸铵，pH为7.0，用螯合离子交换树脂为1.2g / L处理，使用分子量截断（MWCO 30 kDa）的离心过滤装置。申请7个洗涤步骤。浓缩的mAb为1毫升的最终体积，并再次测量由OD 280nm处的测量蛋白质浓度。 </li><li>放射性标记之前， 经由尺寸-EXC交换DOTA-KJ1-26-mAb的缓冲器以PBSlusion色谱法，使用凝胶过滤柱。这也消除潜在的小分子杂质。 </li><li>制备100在10mM盐酸活度64的CuCl 2溶液中并用10倍PBS将pH调节至6-7。加入DOTA-KJ1-26单抗的200微克。在37℃下孵育60分钟。 </li><li>对于质量控制，用0.1M柠檬酸钠（pH值为5）为流动相进行薄层色谱法，通过放射自显影分析。至少活性90％应被结合到抗体上，从而在开始点来检测。未结合的活性通过基于柠檬酸盐的流动相和条纹到溶剂前沿螯合。用于参考，使用64的CuCl 2的建议。 </li></ol> 3.鸡卵清蛋白特异性TH1（科瓦-TH1）细胞分离和扩展注:TH1细胞的培养是根据先前公布的研究16所描述的，17。 <ol><li> 隔离脾和从DO11.10小鼠腹膜外淋巴结（LNS）。 <ol><li>根据联邦法规牺牲鼠标。消毒用70％乙醇的动物和具有用于取出脾和LN的胶带固定它。做一个正中切口，并通过其横向拉动到每个站点从腹膜皮肤分离。 </li><li>找到颈椎，轴向，肱动脉和腹股沟淋巴结。钝钳除去它们，并将它们放在1％胎牛血清（FCS）/ PBS缓冲液中。 </li><li>打开腹膜并找到脾。从胰腺和结缔组织中分离脾，并将其放置在1％FCS / PBS缓冲液中。进一步指导见参考文献20,21。 </li></ol></li><li>通过40微米的过滤器到50mL剁碎脾和LN中使用注射器的柱塞螺旋帽试管中。冲洗用10mL 1％FCS / PBS缓冲液的过滤器。 </li><li>离心在400×g离心5分钟，去除上清。 SubsequenTLY，加入氯化铵 - 钾（ACK）裂解缓冲液（每供体动物1.5mL）中，在室温下5分钟进行红细胞的裂解。添加8.5毫升1％FCS / PBS缓冲液（每供体动物）。 </li><li>离心细胞，在400×g离心5分钟，去除上清。清洗细胞在10毫升1％FCS / PBS缓冲液，离心机在400×g离心5分钟，去除上清。 </li><li>对于磁性细胞分离，重悬在1个％FCS / PBS缓冲液将细胞并添加CD4 +微珠。请参阅制造商的说明缓冲体积和CD4 +微珠的使用量。 </li><li>孵育在4℃下20分钟。再加入1个％FCS / PBS缓冲液补足到50ml，离心细胞，在400×g离心5分钟，并除去上清液。 </li><li>使用商业磁性分离柱，并根据制造商的方案的相应的磁体支架与CD4 + T细胞分离继续进行。调整洗脱CD4 + T细胞，以助ncentration的10个6个细胞/ mL，在Dulbecco改良的Eagle氏培养基用10％热灭活的FCS，2.5％青霉素/链霉素，1％HEPES缓冲液，1％MEM氨基酸，1％丙酮酸钠和0.2％2-巯基乙醇和存储他们在4℃下。 </li><li>收集柱放电在50mL螺旋盖管以制备抗原呈递细胞（APC）。离心柱放于400×g离心5分钟，去除上清。 </li><li>添加10μg/ mL的抗CD4（克隆:GK1.5），10微克/毫升抗CD8（克隆:5367.2）和10微克/毫升小鼠抗大鼠单克隆抗体（MAR18.5）和1.5毫升兔补体。在37℃下孵育45分钟。 </li><li>离心细胞，在400×g离心5分钟，除去上清液，加入3mL的培养基。上照射总共用30 Gy的γ射线或X射线源的APC。然后，调整的APC至5×10 6个细胞/ mL的浓度。 </li><li>添加100个CD4 + T的μL细胞和100μL的APC上的96孔PLATE连同10μg/ mL的科瓦323-339肽，10微克/毫升的抗IL-4单抗，0.3μMCPG1668寡核苷酸和5U / mL的IL-2。 </li><li>添加50U / ml的IL-2每隔一天。后3 - 4天后，从96孔板转移到细胞的24孔板。从96孔板结合3-5孔中一个井在24孔板中。比为1:在一个1添加100U / mL的IL-2的含平台。 </li><li>接连2-3天后，从48孔板转移细胞至175厘米2细胞培养烧瓶（每个烧瓶中1×48孔板）。比为1:在一个1介质填充。添加50U / ml的IL-2每隔一天。 </li><li>根据细胞密度分裂细胞，并添加50U / ml的IL-2每隔一天。在这种方式下，培养细胞另外10天。 </li></ol> 4.科瓦-TH1细胞放射性标记注:64的Cu-DOTA-KJ1-26单抗将应用于培养的科瓦-TH1细胞，以使细胞内放射性标记。 <ol><li>对于COVA-TH1 CELL放射性标记，绘制37 MBq的放射性标记的64的Cu-DOTA-KJ1-26单抗的在注射器不使用剂量校准器死体积。加入1个毫升盐水成接收37活度/ mL的溶液。 </li><li>悬浮在介质科瓦-TH1细胞以2×10 6个细胞/ mL，然后添加细胞悬浮液的0.5毫升至每个孔在48孔板中。 </li><li>添加新鲜制备的37个活度的20μL/ mL的64的Cu-DOTA-KJ1-26单抗溶液的48孔板的每个孔中。用于标记的最终比例为每10个6个细胞0.7活度在520μL的体积。孵育在37℃和7.5％CO 2的30分钟。 </li><li>温育后，小心地重悬在每个细胞孔，并从48孔板转移细胞悬液至50mL螺旋帽试管中。为了最大限度地减少细胞丧失，与预热介质冲洗每个孔中。 </li><li>离心科瓦-TH1细胞悬浮液在400×g离心5分钟，弃上清，重悬在10mL PBS预热的细胞。删除一个将细胞悬浮液进行细胞计数的liquot。重复洗涤步骤。 </li><li>伯爵在适当的稀释与台盼蓝染色可行COVA-TH1细胞。 </li><li>调节细胞浓度至5×10 7个细胞/ mL的10 7个细胞在200μlPBS的腹膜内（ip）注射。 </li><li> 重复步骤4.3-4.5放射性标记的科瓦-TH1细胞与增加活性的量， 例如 ，1.4或活度每10个6个细胞2.1活度。 <ol><li>放射性标记的细胞用每10个6个细胞1.4活度，使用37个活度/ mL的64的Cu-DOTA-KJ1-26单抗溶液和60μL的40μL用每10个6个细胞2.1 MBq的放射性标记。执行这些步骤4.3-4.7 0.7活度，活度1.4和2.1活度64的Cu-PTSM但增加标记时间3小时进行比较调查17。 </li><li>进行到步骤5，以确定活性的最佳量。 </li></ol></li></ol> 5. 在的放射性标记的上科瓦-TH1细胞影响的体外评价注:在TH1细胞的放射性标记的影响的表征通过台盼蓝排除测定的生存力进行的，IFN-γELISA用于评估功能和PE膜联蛋白V染色的细胞凋亡16,17的诱导。胞内摄取和放射性的流出的测定也描述如下。作为比较，也可以使用64的Cu-PTSM标记科瓦-TH1细胞。 <ol><li> 通过台盼蓝染色上的可行性影响 <ol><li>调整至少18×10 6科瓦-TH1细胞的放射性标记用0.7活度/ 10 6个细胞，1.4活度/ 10 6个细胞和2.1活度/ 10 6个细胞分别以2×10 6细胞/ mL的浓度，并执行步骤5.1。 2-5.1.7每个活动剂量。使用非radioacti已经KJ1-26单抗标记科瓦-TH1细胞和未标记的科瓦-TH1细胞作为对照。 </li><li>吸取1mL的细胞溶液到9米的孔24孔板中的。 </li><li>收集3个孔的内容转换成3个独立的15毫升螺旋盖管中，初始放射性标记后3个小时。 </li><li>冲洗现在空孔用预温热培养基以最小化细胞损失和介质添加到相应的15毫升从步骤5.1.3螺旋帽试管中。 </li><li>离心15 mL管，在400×g离心5分钟，重悬所得的细胞沉淀在1ml预热培养基。 </li><li>算两个活细胞和死细胞的台盼蓝分别为每个15mL的管中，计算活细胞的百分比。 </li><li>重复步骤5.1.3-5.1.6 24和48小时后64的Cu-DOTA-KJ1-26单抗放射性标记。 </li></ol></li><li> 上的功能效果通过IFN-γELISA测定 注意:为了评估64的Cu-DOTA-KJ1 26单抗放射性标记对IFN-γ生产中的作用N作为功能性的标记物，从商业供应商执行IFN-γELISA和参考参考17的进一步细节。 <ol><li>分散在96孔板中，3小时后64科瓦-TH1细胞用0.7活度，活度1.4或2.1活度的铜-DOTA-KJ1-26放射性标记在100μL培养基的10 5个活细胞。使用未标记的，无放射性KJ1-26单抗标记的或64的Cu-PTSM标记科瓦-TH1细胞作为对照。 </li><li>刺激10 5 64的Cu-DOTA-KJ1-26单抗标记的科瓦-TH1细胞在100μL培养基IFN-γ产生用10μg科瓦肽，5U / mL的IL-2和5×10 5的APCs在100 /毫升在37℃下24个小时培养基和7.5％CO 2的μL。进一步控制可能是不加入科瓦肽的其他条件。 </li><li>根据制造商的说明通过ELISA分析上清液。 </li><li>重复步骤5.2.2。 5.2.3。在标记过程后24个48小时。 </li></ol></li><li> 细胞凋亡的诱导通过PE-膜联蛋白V染色用于流式细胞术确定 注意:要由64的Cu-DOTA-KJ1-26单抗放射性标记评估细胞凋亡诱导，可使用市售的试剂盒用于流式细胞术和制备细胞样品根据生产商的说明在细胞膜上与膜联蛋白V染色磷脂酰丝氨酸的论述之前。 <ol><li>在3,24和48小时后64科瓦-TH1细胞的Cu-DOTA-KJ1-26单抗放射性标记，一式三份污渍具有至少10 6 64的Cu-DOTA-KJ1-26单抗标记的科瓦-TH1细胞与根据制造商的说明书和膜联蛋白V的试剂盒在一个小时内分析。使用非放射性KJ1-26单抗标记，64的Cu-PTSM标记和未标记的科瓦-TH1细胞作为对照。请参阅参考资料17进一步的细节。 </li></ol></li><li> 摄取和排出 注:64铜DOTA-的摄取KJ1-26单抗进入细胞是在剂量校准器测量和放射性的流出是在γ计数测定0，5，24测量，并且放射性标记后48个小时。 <ol><li>准备各10γ计数管64的Cu-DOTA-KJ1-26单抗标记的科瓦-TH1细胞，各上清液和洗涤步骤。 </li><li>将10 6 64的Cu-DOTA-KJ1-26单抗标记的科瓦-TH1细胞在1mL培养基到每个10γ计数管的放射性标记后直接。对于每个时间点5，24和放射性标记后48个小时的，保持至少10 7 64的Cu-DOTA-KJ1-26单抗标记在介质科瓦-TH1细胞在24孔细胞培养板中，在37的培养箱℃，7.5％CO 2。 </li><li>离心γ计数管中，在400×g离心5分钟并将上清液转移到新的10γ计数管中。 </li><li>在1ml培养基洗涤细胞一次以除去64的Cu-DOTA-KJ1-26单抗一个的未结合部分ð收集在10新γ计数管中的上清液。 </li><li> 1mL的新介质添加到科瓦-TH1细胞和确定在剂量校准器的初始摄取值。所述管也可以在37℃下储存的培养箱和7.5％的CO 2。 </li><li>重复步骤5.4.2根据制造商的方案5中，24和48小时后，以和5.4.5测量所有管在γ计数器。为了校正放射性衰变和定量分析，使用标准的具有限定的放射性剂量。 </li></ol></li></ol> 6. OVA诱导的急性气管炎DTHR 注:迁移动力学和过继转移和放射性标记的科瓦-TH1细胞炎症部位的归巢模式将被可视化和在动物模型中对于科瓦诱导的气道-DTHR 16,17量化。 <ol><li>注入8周大BALB / c小鼠用150μL人的混合物的ipuminum凝胶/ 50μL科瓦溶液（10微克在50μlPBS）来免疫小鼠。 </li><li>免疫后两周，麻醉小鼠用100mg / kg氯胺酮和5mg / kg的甲苯噻嗪通过腹膜内注射。放置在他们的后面实验小鼠和吸管慢慢地溶解在50μLPBS入动物的鼻孔100微克科瓦。这些动物将被吸入一滴一滴的解决方案。 </li><li> 24小时后重复。对于强大的气道DTHR归纳，48小时后再次重复。 </li><li>为了分析转科瓦-TH1细胞的特定迁移，诱发气道DTHR与火鸡-或野鸡-OVA作为对照。因此，重复通过使用各自的OVA蛋白步骤6.1-6.3。 </li></ol> 7. 体内成像使用PET / CT 注意: 在 64的Cu-DOTA-KJ1-26单抗标记的科瓦-TH1细胞在科瓦-DTHR的体内成像患病小鼠和对照同窝演示特异性归巢和MI该科瓦-TH1细胞的格雷申动力学。因此，获得静态PET扫描和解剖CT顺序地扫描3，24和48小时后过继细胞转移。 <ol><li>放射性标记后直接，调整64的Cu-DOTA-KJ1-26单抗标记的科瓦-TH1细胞至5×10 7个细胞/ mL的10 7个细胞在200μl的腹膜内注射。 </li><li>使用1ml注射器和30号针头，绘制64的Cu-DOTA-KJ1-26单抗标记的科瓦-TH1细胞悬浮液的200μL，并注入细胞的ip到4之间的科瓦-DTHR患病动物和第 5乳头。为了确定放射性的总注射量，注射前和注射后测量在剂量校准器的注射器。 </li><li>在温度受控的麻醉箱:麻醉小鼠，所需的摄取时间之前10分钟，用100％氧气1.5％异氟烷（0.7升/分钟流速）。 </li><li> 在此期间，为方便合作登记图像分析过程中PET和CT图像的，固定含有小鼠床下64的Cu-DOTA-KJ1-26单抗溶液或免费64的CuCl 2溶液的玻璃毛细管。 <ol><li>因此，制备的0.37活度/ mL的溶液和填充玻璃毛细管10μL的此溶液的体积。由于细胞来源的放射性信号本质上是弱者，不要用大量的活动，以确保标记不与小鼠细胞衍生的信号干扰。 </li></ol></li><li>到达手术耐受后，通过后肢的踏板撤回反射的损失所指示的，小鼠转移到配备有合适的管道系统，以维持麻醉的PET-CT和兼容小动物床。 </li><li>固定使用棉签和医用胶带的小动物床鼠标。应用眼膏，以避免眼睛干燥。 </li><li>鼠标床转移到PET扫描仪，具有focu中心的视场S于肺和获取与350-650千电子伏的能量窗20分钟静态PET扫描。 </li><li>鼠标床转移到了CT扫描仪。通过侦察视图，居中的观点对肺部的领域。在"步骤和拍"模式转动360°与350毫秒的曝光时间和分级因子4期间获取经由360个突起的平面CT图像。 </li></ol> 8.图像分析<ol><li>通过用75微米的像素尺寸应用统计迭代有序子集期望最大化（OSEM）2D算法和CT扫描到3D图像重建PET列表模式数据。 </li><li>用于在适当的图像分析软件的图像配准（ 例如 Inveon研究工作场所或PMOD），使用自动配准工具。如果失败，则使用玻璃毛细管鼠标床下为指导，在轴向，冠状和矢状视图空间上共注册重建后的PET和CT图像。 </li><li> 纠正PET图像对于使用放射性衰变法，并在12.7小时的放射性核素64 Cu的一半时间的放射性衰变。对于图像归一化，选择的一个图像（A）作为基准，调整图像显示的强度相应的图像分析软件。 <ol><li>利用对其他影像只是设置针对参考图像（A）的值，注入的活性（A）和所注入的活性（X），归一化使用以下等式其它图像:（注射的活性（X）/注射的活性（甲））×强度值（A）。 </li></ol></li><li>画出对肺和淋巴结perithymic的归一化PET信号兴趣（VOI）的3D体积。 </li><li>确定每cm 3使用下面的公式（％ID /厘米3）的百分比注射剂量:（平均在小鼠VOI /总活性活性）×100进一步指导有关的图像分析，参见参考文献22，23。 </li></ol>

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作者什么都没有透露。

该协议提出了一种可靠和简单的方法来通过PET稳定地放射性标记用于体内跟踪细胞。利用这种方法，科瓦-TH1细胞，分离的和在从供体小鼠体外扩增，可能是放射性标记的具有64的Cu-DOTA-KJ1-26单抗和它们的归巢被跟踪到肺和淋巴结perithymic如在科瓦呈现的网站COVA引起的急性呼吸道DTHR。 与螯合剂的mAb的修改需要快速高效的工作和无胺的痕迹使用超纯解决方案。将溶液用螯合离子交换树脂处理，以确保DOTA-NHS活性酯的正确缀合至胺mAb的残基。螯合剂 - 缀合的mAb作为细胞内放射性标记具有优于使用非特异性细胞标记试剂的数个优点。首先，将单克隆抗体提供了极好的特异性允许限定的细胞群体的靶向标记。该单抗本身不是细胞毒性的，并且对细胞的生存力或功能没有不利影响。接着，从细胞的放射性的流出物显著降低改善PET图像17，24的信号-背景比率。通过选择一个mAb具有不同的特异性，该方法是容易移植到其他感兴趣的细胞群，例如CD8 + TIL或CD14 +单核细胞。此外，这种细胞标记方法可用于在人类炎性疾病（ 例如 ，类风湿性关节炎和支气管哮喘）或不同的人类癌症类型的动物模型的各种动物模型中细胞跟踪研究。这种标记方法，但是，仅限于膜结合受体或分化标志物作为靶，因为单克隆抗体不被动跨细胞膜扩散。目标或，激活诱导的内简单地说，膜 - 梭动是有利的。一个的单克隆抗体与靶之间高亲和力，然而，也可能是足够的用于体内成像，可能具有降低的信号背景比。 DOTA作为我们的实验螯合剂的选择是基于使用64铜25的早期工作。然而，在此期间DOTA被证明不适合这种同位素的最佳螯合剂，导致由transchelation肝脏相当高的活性摄取到超氧化物歧化酶26。虽然使用放射性标记的抗体，然后可继转移应该降低64铜剥离和运输到肝脏的风险的细胞在体外标记，对于64的Cu优化现代螯合剂，如1,4,7-三氮杂环，1-戊二酸-4,7-二乙酸（NODAGA）或1,4,7-三氮杂环壬三乙酸（NOTA），可以用我们的方法可以优选使用，以进一步增强所获得的数据27的特异性</s了&gt;。此外，其它的活性酯可用于64的Cu（ 例如 N-氯代琥珀酰亚胺（NCS））或其它同位素，诸如89的Zr（ 例如去铁胺; DFO）。 64 Cu作为放射性同位素的选择是基于这样的事实，其在体内细胞迁移和归巢是一个相当慢的过程相比，通常由PET成像代谢变化。 64铜12.7小时的半衰期时间使细胞迁移的超过48小时后注射重复成像。对于64铜生产，它是使用不痕量金属离子高档解决方案，以确保64 Cu的纯度很重要的。此外，64 Cu的纯度是用于自多价金属离子杂质的DOTA-mAb与放射性标记高重要性可能导致64铜溶液的减少的特异性活性至排除蛋白放射性标记，并因此成像水平。虽然64的Cu产生SI高能，细胞毒性的俄歇电子的gnificant百分比衰减28的情况下，放射性同位素的调整剂量以及由细胞耐受如通过放射性标记后的生存能力和功能性和低细胞凋亡诱导的影响最小表示。然而，它显然是可取的最初滴定用于放射性标记为其它细胞类型的活性剂量和调整放射性标记协议。快速和容易的体外评价方法已被证明在协议，包括存活力通过台盼蓝排除和染色用PE膜联蛋白V的细胞凋亡馏分的判定的判定。这两种方法都相对便宜，使用容易获得的实验室设备。结合PE-膜联蛋白V染色的生活和如7-氨基放线菌素D（7-AAD）的死细胞的鉴别的染料将允许活力和细胞凋亡的测定在一个实验STE页。作为替代，以评估标记的T细胞的存活力，3-（4,5-二甲基吡啶-2-基）-2,5-二苯基溴化四（MTT-）测定可用于评估细胞的代谢活性。与剂量校准和γ-计数器的摄取和排出值的评价都比较先进，但仍然快速和廉价的实现的，因为几乎所有的实验室，放射性工作已在现场各自的硬件。基因测序或基于微芯片的方法会产生更详细的数据。这些方法，但是，并不总是容易获得，需要相应的专业知识和最经常与更高的成本。 虽然PET不能达到像显微成像技术29的蜂窝的分辨率，它提供了在皮摩尔范围内用于检测甚至小动物或人8的内部细胞的数量的优异的灵敏度。相比于其它非侵入性成像技术如。光学升成像，PET是定量的，而不是在穿透深度的限制，提供了具有三维分辨率的高画质的图像。用CT的解剖精度组合PET的高灵敏度允许细胞迁移的高精度的成像。此外， 在体内的数据可以容易地通过几种体外方法，包括γ计数用于生物分布分析验证。冷冻切片和冰冻切片的后续的组织学染色的放射自显影可以叠加到放射自显影活动图与由苏木精和曙红染色识别的免疫细胞浸润相关。

非侵入性细胞跟踪是监视在体内细胞的功能，迁移和归巢的多功能工具。最近细胞跟踪研究在再生医学中，自体外周血白细胞中在对抗癌症3,4过继细胞疗法的炎症或T淋巴细胞的上下文中都集中于间充质1,2或骨髓来源的干细胞3。作用部位和细胞疗法的基本生物学原理的阐明是极其重要的。 CD8 +细胞毒性T淋巴细胞，遗传工程嵌合抗原受体（CAR）的T细胞或肿瘤浸润淋巴细胞（TIL的）被广泛认为是金标准。然而，肿瘤相关抗原特异性T H 1细胞已被证明是一种有效的替代治疗方案4，&lt;/ SUP&gt; 5，6，7。 如在炎症的主要参与者，器官特异性自身免疫性疾病（ 例如类风湿性关节炎或支气管哮喘），并在癌症免疫疗法高度感兴趣的细胞，它表征TH1细胞的时间分布和归巢模式是重要的。非侵入性体内通过PET成像提出了一种定量的，高度敏感的方法8检测细胞迁移模式， 在体内归巢，和的T细胞作用和响应过程中的炎症，过敏，感染或肿瘤排斥9，10，11中的位点。 在临床上，在111-喔星用于白细胞显像对炎症和感染12的辨别，而2-脱氧-2-（18F）氟-D-葡萄糖（18 F-FDG）由PET 3,13通常用于细胞跟踪研究。此PET示踪剂的一个主要缺点，但是，是的放射性核素（18）F在109.7分钟，阻碍在稍后的时间点成像后过继细胞转移的低细胞内稳定性的半衰期短。用于在由PET 体内细胞跟踪研究长期来看，虽然在不稳定的细胞中，64的Cu-PTSM经常用于非特异性地标记细胞14，15与T细胞活力最小化有害作用和功能16。 这个协议描述了一种方法，以进一步减少使用T细胞受体（TCR）特异性的放射性标记的mAb对细胞存活力和功能的不利影响。首先，生产放射性同位素64的Cu，所述的mAb KJ1-26与第共轭的ë螯合剂DOTA，和随后的64的Cu-放射性标记被示出。在第二步骤，分离和DO11.10供体小鼠的科瓦-TH1细胞的扩增，并用64的Cu-加载DOTA-缀合的mAb KJ1-26（64的Cu-DOTA-KJ1-26）放射性标记进行详细说明。通过γ计数，分别摄取值，并用一个剂量校准器的放射性流出的和评估，以及对64的Cu-放射性标记对细胞活力通过台盼蓝排除和功能用IFN-γELISA呈现效果的评价。对于体内细胞跟踪非侵入性的，由PET / CT过继细胞转移后科瓦引起的急性呼吸道DTHR和图像采集的小鼠模型的启发进行说明。 此外，这种标记方法可以被转移到不同的疾病模型，小鼠T细胞与不同的TCR或与膜结合受体或表达擦伤兴趣一般细胞KERS底层连续膜穿梭17。

<table><tbody><tr><td>HCl, Suprapur</td><td>Merck, Darmstadt, Germany</td><td>1.00318</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>Methanol, Suprapur</td><td>Merck, Darmstadt, Germany</td><td>1.06007</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>Isopropanol, Suprapur</td><td>Merck, Darmstadt, Germany</td><td>1.0104</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>Pt/Ir (90/10) plate</td><td>&amp;Ouml;gussa</td><td>Custom made</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>PEEK chamber</td><td>WKL</td><td>Custom made</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>&lt;sup&gt;64&lt;/sup&gt;Ni</td><td>Chemotrade</td><td></td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>Polygram SIL G/UV 254 plate</td><td>Macherey-Nagel</td><td>805021</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>Ion exchange column</td><td>BioRad</td><td>AG1-X8</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>Solid state target system for PETtrace</td><td>WKL</td><td>costum made</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>&lt;sup&gt;64&lt;/sup&gt;Cu work-up module</td><td>WKL</td><td>costum made</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>Dose calibrator</td><td>Capintec</td><td>CRC-25R</td><td></td></tr><tr><td>PETtrace cyclotron</td><td>General Electric Medical Systems</td><td></td><td></td></tr><tr><td>DOTA-NHS</td><td>Macrocyclics</td><td>B-280</td><td>DOTA-conjugation</td></tr><tr><td>Anti-cOVA-TCR antibody (KJ1-26)</td><td></td><td>Isolated from hybridoma cell culture</td><td>DOTA-conjugation</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>71633</td><td>DOTA-conjugation</td></tr><tr><td>H&lt;sup&gt;+&lt;/sup&gt; Chelex 100</td><td>Sigma-Aldrich</td><td>C7901</td><td>DOTA-conjugation</td></tr><tr><td>Amicon Ultra-15 filter unit</td><td>Merck Millipore</td><td>UFC910008</td><td>DOTA-conjugation</td></tr><tr><td>Rotipuran ultrapure water</td><td>Carl Roth</td><td>HN68.3</td><td>DOTA-conjugation</td></tr><tr><td>Ammonium acetate</td><td>Sigma-Aldrich</td><td>32301</td><td>DOTA-conjugation</td></tr><tr><td>PBS</td><td>University Tuebingen</td><td></td><td>DOTA-conjugation</td></tr><tr><td>Micro Bio-spin P-6 column</td><td>Bio-Rad Laboratories</td><td>7326221</td><td>DOTA-conjugation</td></tr><tr><td>Sodium citrate</td><td>Sigma-Aldrich</td><td>71497</td><td>DOTA-conjugation</td></tr><tr><td>Cyclone Plus PhosphorImager&amp;nbsp;</td><td>Perkin-Elmer</td><td>L2250116</td><td>DOTA-conjugation</td></tr><tr><td>DMEM</td><td>Merck Millipore</td><td>102568</td><td>ingredient for T cell medium&amp;nbsp;</td></tr><tr><td>FCS</td><td>Merck Millipore</td><td>S0115/1004B</td><td>ingredient for T cell medium&amp;nbsp;</td></tr><tr><td>Sodium pyruvate</td><td>Merck Millipore</td><td>L0473</td><td>ingredient for T cell medium&amp;nbsp;</td></tr><tr><td>MEM-amino acids</td><td>Merck Millipore</td><td>K0293</td><td>ingredient for T cell medium&amp;nbsp;</td></tr><tr><td>HEPES&amp;nbsp;</td><td>Merck Millipore</td><td>L 1613</td><td>ingredient for T cell medium&amp;nbsp;</td></tr><tr><td>&amp;nbsp;Penicillin/Streptomycin</td><td>Merck Millipore</td><td>A2212</td><td>ingredient for T cell medium&amp;nbsp;</td></tr><tr><td>0.05 mM 2-&amp;beta;-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td>ingredient for T cell medium&amp;nbsp;</td></tr><tr><td>DO11.10 mice</td><td></td><td>in-house breeding</td><td>TH1 cell culture</td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190144</td><td>TH1 cell culture</td></tr><tr><td>Cell strainer 40 &amp;micro;m&amp;nbsp;</td><td>Corning</td><td>352340</td><td>TH1 cell culture</td></tr><tr><td>ACK Lysing Buffer</td><td>Lonza</td><td>10-548E</td><td>TH1 cell culture</td></tr><tr><td>CD4 MicroBeads, mouse</td><td>Miltenyi Biotech</td><td>130-097-145</td><td>TH1 cell culture</td></tr><tr><td>QuadroMACS separator</td><td>Miltenyi Biotech</td><td>130-090-976</td><td>TH1 cell culture</td></tr><tr><td>LS column</td><td>Miltenyi Biotech</td><td>130-042-401</td><td>TH1 cell culture</td></tr><tr><td>anti-CD4 antibody (Gk1.5)</td><td></td><td>Isolated from hybridoma cell culture</td><td>TH1 cell culture</td></tr><tr><td>anti-CD8 antibody (5367.2)</td><td></td><td>Isolated from hybridoma cell culture</td><td>TH1 cell culture</td></tr><tr><td>Anti-rat antibody (MAR18.5)</td><td></td><td>Isolated from hybridoma cell culture</td><td>TH1 cell culture</td></tr><tr><td>Rabbit complement MA</td><td>tebu-Bio</td><td>CL3221</td><td>TH1 cell culture</td></tr><tr><td>Anti-IL-4 antibody (11B11)</td><td></td><td>Isolated from hybridoma cell culture</td><td>TH1 cell culture</td></tr><tr><td>cOVA 323-339-peptide&amp;nbsp;</td><td>EMC-micro-collections</td><td>Custom order</td><td>TH1 cell culture</td></tr><tr><td>CPG1668-oligonucleotides</td><td>Eurofins MWG Operon</td><td>Custom order</td><td>TH1 cell culture</td></tr><tr><td>IL-2</td><td>Novartis</td><td>65483-116-07</td><td>TH1 cell culture</td></tr><tr><td>96-well plates</td><td>Greiner&amp;nbsp;</td><td>655180</td><td>TH1 cell culture</td></tr><tr><td>24-well plates</td><td>Greiner&amp;nbsp;</td><td>662160</td><td>TH1 cell culture</td></tr><tr><td>cell culture flask</td><td>Greiner&amp;nbsp;</td><td>660175</td><td>TH1 cell culture</td></tr><tr><td>48-well plates</td><td>Greiner&amp;nbsp;</td><td>677 180</td><td>cell labeling</td></tr><tr><td>Gammacell 1000</td><td>Best Theratronics</td><td>&lt;em&gt;via&lt;/em&gt; inquiry&amp;nbsp;</td><td></td></tr><tr><td>Gulmay RT225</td><td>Gulmay</td><td>&lt;em&gt;via&lt;/em&gt; inquiry&amp;nbsp;</td><td></td></tr><tr><td>Trypan blue</td><td>Merck Millipore</td><td>L6323</td><td>&lt;em&gt;in vitro&lt;/em&gt; evaluation</td></tr><tr><td>Mouse IFN-&amp;gamma; ELISA</td><td>BD Biosciences</td><td>558258</td><td>&lt;em&gt;in vitro&lt;/em&gt; evaluation</td></tr><tr><td>PE Annexin V Apoptosis Detection Kit&amp;nbsp;</td><td>BD Biosciences</td><td>559763</td><td>&lt;em&gt;in vitro&lt;/em&gt; evaluation</td></tr><tr><td>Tube 5 mL</td><td>Sarstedt</td><td>55.476</td><td>&lt;em&gt;in vitro&lt;/em&gt; evaluation</td></tr><tr><td>Round-bottom tubes&amp;nbsp;</td><td>BD Biosciences</td><td>352008</td><td>&lt;em&gt;in vitro&lt;/em&gt; evaluation</td></tr><tr><td>Wizard &amp;gamma;-counter</td><td>Perkin-Elmer</td><td>2480-0010</td><td>&lt;em&gt;in vitro&lt;/em&gt; evaluation</td></tr><tr><td>ELISA Reader MultiscanEX</td><td>Thermo Fisher Scientific</td><td>51118177</td><td>&lt;em&gt;in vitro&lt;/em&gt; evaluation</td></tr><tr><td>Microscope</td><td>Leica</td><td>&lt;em&gt;via&lt;/em&gt; inquiry&amp;nbsp;</td><td>&lt;em&gt;in vitro&lt;/em&gt; evaluation</td></tr><tr><td>BD LSRII&amp;nbsp;</td><td>BD Biosciences</td><td>&lt;em&gt;via&lt;/em&gt; inquiry&amp;nbsp;</td><td>&lt;em&gt;in vitro&lt;/em&gt;evaluation</td></tr><tr><td>BALB/c mice</td><td>Charles River</td><td>028</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking</td></tr><tr><td>Aluminum gel</td><td>Serva Electrophoresis</td><td>12261.01</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking</td></tr><tr><td>Xylazine</td><td>Bayer HealthCare</td><td>Ordered &lt;em&gt;via&lt;/em&gt; University hospital</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking</td></tr><tr><td>Ketamine</td><td>Ratiopharm</td><td>Ordered &lt;em&gt;via&lt;/em&gt; University hospital</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking</td></tr><tr><td>Isoflurane</td><td>CP-Pharma</td><td>Ordered &lt;em&gt;via&lt;/em&gt; University hospital</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking</td></tr><tr><td>30 G needle</td><td>BD Biosciences</td><td>304000</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking</td></tr><tr><td>Syringe</td><td>BD Biosciences</td><td>11612491</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking</td></tr><tr><td>Capillaries 10 &amp;micro;L</td><td>VWR</td><td>612-2439</td><td></td></tr><tr><td>Inveon PET scanner</td><td>Siemens Healthineers</td><td>no longer available</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking, alternative companies: Bruker, Mediso&amp;nbsp;</td></tr><tr><td>Inveon SPECT/CT scanner</td><td>Siemens Healthineers</td><td>no longer available</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking</td></tr><tr><td>Inveon Research Workplace</td><td>Siemens Healthineers</td><td></td><td>image analysis, alternative software: Pmod</td></tr></tbody></table>

murine lymphocyte labeling by 64cu-antibody receptor targeting for in vivo cell trafficking by pet/ct

该协议说明了生产64的Cu和随后的鼠淋巴细胞的细胞培养和64的Cu-抗体受体的细胞的靶向缀合的螯合剂/单克隆抗体（mAb）的放射性标记。 在的在气道迟发型超敏反应（DTHR）由PET / CT的动物模型的放射性标记和体内细胞跟踪非侵入性地描述体外评价。 详细地说，与螯合剂1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸（DOTA）mAb的共轭被示出。以下生产放射性64的Cu，所述DOTA缀合的mAb的放射性标记的描述。接着，鸡卵清蛋白（科瓦） -特异性CD4 +干扰素的膨胀（IFN）-γ的T辅助细胞（科瓦-TH1）和科瓦-TH1细胞的放射性标记随后被描绘。各种体外技术被提出来评价EF对细胞64的Cu-放射性标记的fects，如细胞活力的测定通过台盼蓝排除，染色的细胞凋亡与膜联蛋白V用于流式细胞术，和功能通过IFN-γ酶联免疫吸附测定法评估（ELISA） 。此外，放射性摄取进入细胞和标记稳定性的确定被详细描述。该协议还描述了如何在动物模型中进行小区跟踪研究为气道DTHR和，因此，在BALB / c小鼠科瓦引起的急性呼吸道DHTR的诱导是包括在内。最后，一个强大的PET / CT工作流程，包括图像采集，重建和分析被呈现。 相比普通PET-示踪剂细胞标记与随后受体内在64的Cu-抗体受体靶向方法提供高特异性和稳定性，降低的细胞毒性，和低流出速率， 例如 64铜-pyruvaldehyde双（N4-甲基氨基硫脲）（64的Cu-PTSM）。最后，我们的方法使得能够在由PET / CT 体内细胞跟踪非侵入性具有最佳信号-背景比为48小时。此实验方法可以被转移到不同的动物模型和细胞类型与被内化的膜结合受体。

图1总结了科瓦-TH1细胞与64的Cu-DOTA-KJ1-26单抗和实验设计用于在体外和在包括在本协议体内研究的标记。 <img alt="图1" src="/files/ftp_upload/55270/55270fig1.jpg" /> 图1:64的Cu-DOTA-KJ1-26单抗贴标过程＆实验设计。放射性细胞标记的（A）示意性表示具有64的Cu-DOTA-KJ1-26单抗。 64的Cu-DOTA-KJ1-26-mAb结合到TH1细胞的细胞表面上的科瓦-TCR受体和被内用24小时内的受体。単后COVA-的TCR再表达24个小时。 （B）CD4 + T细胞分离，扩大和64的Cu-DOTA-KJ1-26单抗在体外放射性标记。摄取和排出值疫情周报e。通过剂量校准和γ计数来确定。对细胞活力的放射性抗体的影响用台盼蓝染色进行了评估，在功能上与IFN-γELISA，和对细胞凋亡的诱导与藻红蛋白（PE）-Annexin V染色。 （C）科瓦气道DHTR在雌性BALB / c小鼠中诱导。 10 7细胞TH1放射性标记后直接过继转移到科瓦气道DHTR患病的动物。 PET / CT图像进行采集3，24和48小时后过继细胞转移。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55270/55270fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 的TH1细胞与64的Cu-DOTA-KJ1-26-mAb的成功细胞内标记物通过免疫组织化学（ 图2A）证实。 64的Cu-DOTA-KJ1-26抗体（绿色）为共定位与CD3在放射性标记后，细胞膜3小时，而mAb的在细胞膜上的信号是仅在第24个小时（黄色）微弱。在放射性标记后48个小时，将64的Cu-DOTA-KJ1-26-TCR复合物通过细胞内吞作用内在化与在TH1细胞的细胞质中的强烈信号。所施加的放射性剂量的0.7活度由只有8％减少了TH1细胞的活力，而较高剂量的活度1.4和2.1活度64的Cu-DOTA-KJ1-26单抗有更显着的效果。相比于用64的Cu-PTSM，然而，观察到64的Cu-DOTA-KJ1-26单抗的无显著优点（ 图2B）放射性标记。 64的Cu-抗体放射性标记的细胞表现出的功能几乎没有损失如通过IFN-γ分泌（ 图2C）中所示，减少放射性的流出（ 图2D），并与64的Cu-PTSM标记TH1细胞凋亡减少（ 图2E）的感应细胞。 图2: 在放射性TH1细胞标记的体外评价与64的Cu-DOTA-KJ1-26单抗。 （A）共聚焦显微镜证明64的Cu-DOTA-KJ1-26-单抗（绿色）在科瓦-TH1细胞（细胞膜;红色）的细胞内摄取初始标记后24小时内（该图已经从参考17修改） 。通过锥虫蓝排除24小时后标记所检测的（B）放射性标记的剂量64的Cu-DOTA-KJ1-26单抗或64的Cu-PTSM的滴定揭示的存活力的影响相等。 0.7活度为细胞标记的使用诱导的生存力的最低损伤（平均值±SD百分比;该图已经从引用16,17改性（C）放射性标记的滴定做64的Cu-DOTA-KJ1-26单抗或64的Cu-PTSM的SES揭示的功能性的较强的损伤后64的Cu-PTSM贴标，通过IFN-γ酶联免疫吸附24小时后标记所检测。 64的Cu-DOTA-KJ1-26单抗对细胞标记的剂量增加对功能没有任何影响（平均值±百分比SD;统计:学生t检验; * P &lt;0.05，** P &lt;0.01，** * p &lt;0.001;该图已经从引用16改性，17）。放射性标记的细胞（γ计数）的（D）流出的测量表明相比64的Cu-PTSM 5小时和24小时后标记64的Cu-DOTA-KJ1-26单抗的更高稳定性标记（平均值±百分比SD;统计:学生t检验; *** p &lt;0.001;该图已经从17修改）。 （E）各流式细胞仪（PE-膜联蛋白V）的图指示较高的APoptosis诱导后64的Cu-PTSM标记24个小时相比，64的Cu-DOTA-KJ1-26单抗标记（该图已经从参考17修改）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55270/55270fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 过继性转移10 7 64的Cu-DOTA-KJ1-26单抗标记的科瓦-TH1细胞进入科瓦-DTHR患病动物和未处理的对照组，高分辨率静态PET和解剖CT图像后获得的。用于图像分析，图像与含有64的Cu-DOTA-KJ1-26单抗（ 图3A）的少量的玻璃毛细管的帮助冠状的，轴向和矢状视图融合。的VOI上绘制的衰变校正和归一化的PET图像的肺和淋巴结perithymic来计算百分比注射每cm 3的剂量（ 图3B）。 <img alt="图3" src="/files/ftp_upload/55270/55270fig3.jpg" /> 图3:PET / CT-配准和分析。 （A）PET和CT图像进行共登记在图像分析软件。如果由软件自动注册失败，将图像通过叠加玻璃毛细管（标记），填充有放射性和固定动物床下的PET和CT信号融合。 （B）的VOI放置在perityhmic和肺LN的校正和归一PET信号。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55270/55270fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> COVA-TH1细胞迁移跟踪到肺和perithymic LN如气道DTHR COVA-演示站点。 体内 PET信号解源性从64的Cu-DOTA-KJ1-26单抗标记的科瓦-TH1细胞比从64的Cu-PTSM标记的细胞（ 图4A）的信号高得多。在肺和淋巴结perithymic科瓦-TH1细胞摄取值进行比较，以未经处理的对照同窝（ 图4B）在DTHR患病动物显著增加。 <img alt="图4" src="/files/ftp_upload/55270/55270fig4.jpg" /> 图4:在气道DTHR模型科瓦-TH1细胞追踪通过体内 PET / CT。 （A）的过继相应PET / CT图像转印科瓦-TH1细胞中，其预先用64的Cu-DOTA-KJ1-26单抗或64的Cu-PTSM标记，进科瓦-DTHR患病或未经处理的小鼠。所传送的科瓦-TH1细胞归属到肺和淋巴结perithymic。 （B）的科瓦-TH1细胞归巢位点的定量分析证明了一个较高的h1oming到发炎的组织（平均值±SD;统计:学生t检验; * P &lt;0.05，** P &lt;0.01）。与64的Cu-DOTA-KJ1-26单抗的标记证明了更高的灵敏度相比，64的Cu-PTSM在不同归巢位点（增加％ID /厘米3）（平均值±SD，统计:学生t检验; P | &lt;0.05，## p &lt;0.01，### p &lt;0.001）。该图已被从基准16,17改性。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55270/55270fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT at JoVE.com

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

以下64的Cu改性的单克隆抗体结合到转基因小鼠的T细胞受体的制备中，T细胞在体内的放射性标记的，用于存活力，功能，标记稳定性和细胞凋亡分析，并过继转移到小鼠的气道迟发型超敏反应用于非侵入性成像通过正电子发射断层成像/计算机断层扫描（PET / CT）反应。

小鼠淋巴细胞通过标签<sup&gt; 64</sup&gt;的Cu-抗体受体靶向用于<em&gt;体内</em&gt;由PET / CT细胞运输

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

murine-lymphocyte-labeling-64cu-antibody-receptor-targeting-for-vivo

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

8.7K 次观看.  Eberhard Karls University Tübingen. 以下64的Cu改性的单克隆抗体结合到转基因小鼠的T细胞受体的制备中，T细胞在体内的放射性标记的，用于存活力，功能，标记稳定性和细胞凋亡分析，并过继转移到小鼠的气道迟发型超敏反应用于非侵入性成像通过正电子发射断层成像/计算机断层扫描（PET / CT）反应。 

视频: Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

在Vivo免疫荧光定位中用于评估癌症研究中的治疗和诊断抗体生物分布

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

科研

癌症研究

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

利用生物正交反电子需求的Diels-Alder环的预靶向PET成像

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

生物工程

Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for various cancers. Despite significant progress having been made in these areas, there is still more to be learned to better understand the pharmacokinetics and pharmacodynamics of the administered therapeutic cells in the living system. For noninvasive, in vivo tracking of cells with positron emission tomography (PET), a novel [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN)-mediated cell radiolabeling method has been developed utilizing 89Zr (t1/2 78.4 h). The present protocol describes a [89Zr]Zr-DBN-mediated, ready-to-use, radiolabeling synthon for direct radiolabeling of variety of cells, including mesenchymal stem cells, lineage-guided cardiopoietic stem cells, liver regenerating hepatocytes, white blood cells, melanoma cells, and dendritic cells. The developed methodology enables noninvasive PET imaging of cell trafficking for up to 7 days post-administration without affecting the nature or the function of the radiolabeled cells. Additionally, this protocol describes a stepwise method for the radiosynthesis of [89Zr]Zr-DBN, biocompatible formulation of [89Zr]Zr-DBN, preparation of cells for radiolabeling, and finally the radiolabeling of cells with [89Zr]Zr-DBN, including all the intricate details needed for the successful radiolabeling of cells.

Presented here is a protocol to radiolabel cells with a positron emission tomography (PET) radioisotope, 89Zr (t1/2 78.4 h), using a ready-to-use radiolabeling synthon, [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN). Radiolabeling cells with [89Zr]Zr-DBN allows noninvasive tracking and imaging of administered radiolabeled cells in the body with PET for up to 7 days post-administration.

细胞运输的正电子发射断层扫描成像:一种细胞放射性标记方法

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

病毒衍生肽修饰抗体结合物对增强细胞积累和改善肿瘤靶向性的初步评价

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

免疫与感染

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects.
This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

使用正电子发射断层扫描对实验性自身免疫性脑脊髓炎小鼠模型中的 CD19+ B 细胞进行成像

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

神经科学

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

使用前 Vivo 共聚焦显微镜可视化淋巴节点结构和细胞定位

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

生物学

An In Vivo PET Imaging Technique to Detect Tumors in a Murine Model Using Radiolabeled Antibodies

Source: Zeglis, B. M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52521">The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies.</a> J. Vis. Exp. (2015).
This video demonstrates a technique involving a radioimmunoconjugate for the in vivo imaging of a tumor in a mouse model. Upon injection of the radioimmunoconjugate, the antibodies of the conjugate bind to a specific membrane antigen on the cancer cells. The radiotracer of the conjugate emits positrons that interact with neighboring electrons to produce energy, which is detected by a positron emission tomography (PET) scanner to create an image of the tumor.

一种使用放射性标记抗体检测小鼠模型中肿瘤的体内 PET 成像技术

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. In ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

Labeling Murine T Helper Lymphocytes using Radiolabeled Monoclonal Antibodies

Source: Hoffmann, S. H. L., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55270">Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the intracellular radiolabeling of T helper cells using radioactive isotope-conjugated monoclonal antibodies for non-invasive positron emission tomography imaging.

使用放射性标记的单克隆抗体标记小鼠 T 辅助性淋巴细胞

Source: Hoffmann, S. H. L., et al., Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT. J. Vis. Exp. ...

Immune Systems and Components

Assays and Functional Profiling

An In Vivo Technique to Track Bispecific Antibody-Induced T Cell Trafficking to Tumors

After anesthetizing the mouse using a 26-gauge needle, inject 20 million T cells in 100 microliters of media retro-orbitally. Administer 1,000 units of recombinant IL-2 subcutaneously to support T cell survival in vivo. Then, administer the bispecific antibody retro-orbitally or intraperitoneally.
For in vivo imaging, administer 100 microliters of three milligrams of D-luciferin by retro-orbital injection with a 26-gauge needle in an anesthetized mouse. To capture images, place the mouse in the light-tight chamber of the imager, such that the flank bearing the xenograft is facing up toward the camera. Using the acquisition control panel, select luminescent, photograph, and overlay. Set the exposure time to auto, the binning to medium, and F-stop to one, and acquire images.
After the first image, set the exposure time to match that automatically calculated for the first image so that subsequent images may be compared directly. Take another set of images with the mouse supine to evaluate T cell presence in the lungs.

小鼠淋巴细胞通过标签

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