这项工作是由意大利ASSOCIAZIONE Ricerca巨蟹座（IG＃12754）的支持。

CLL的血液样本是按照机构准则和赫尔辛基宣言得到的。 1. CLL患者的血样淋巴细胞白血病的分离<ol><li>收集在肝素钠（绿顶）管17血样。 </li><li>使1:3稀释的全血的RT 1×磷酸盐缓冲盐水（PBS）中。 </li><li>通过密度梯度离心纯化从血样外周血单核细胞（PBMC）。 <ol><li>在一15ml离心管底层5毫升密度离心介质（ 如，聚蔗糖）的，并仔细转移的10ml稀释的血液。立时离心机在1500 XG在室温下20分钟。 </li></ol></li><li>与连接到真空泵的玻璃巴斯德吸管将上清液吸出一部分，并在密度离心媒体和稀释的血浆之间的相间用5毫升吸管收集PBMC环。转移至50毫升离心管中，并用PBS洗（50米升最终体积）。离心机在500×g离心在4℃下5分钟。 </li><li>吸用玻璃巴斯德吸管取上清液，重悬在50毫升PBS中的沉淀，并用血细胞计数器计数细胞。 </li><li>用抗CD19染 ​​色外周血单个核细胞和抗体-CD5流式细胞仪对以下免疫的标准协议，并检查外周血单个核细胞亚群组成18。 注:CLL淋巴细胞CD19 + / CD5 +。 </li><li>检查CD19 + / CD5 +细胞的％使用流式细胞仪18。净化从病人的B淋巴细胞与CD19 + / CD5 +下列步骤2中为B细胞19的负隔离的详细说明白血病细胞的≥95％。 </li></ol> 2.负分离纯化白血病B细胞<ol><li>悬浮10 7的PBMC / ml的在PBS中的0.1％牛血清白蛋白（BSA）2mM EDTA的，pH值7.4（隔离缓冲液）。 </li><li>加101;鼠单克隆抗-CD3，-CD14和10 7个细胞-CD16初级抗体克孵育在4℃下30分钟。 </li><li>洗用隔离缓冲器和离心将细胞在500×g离心5分钟，4℃。 </li><li>重悬在隔离缓冲器中的细胞以10 7个PBMC中/毫升。 </li><li>与细胞温育之前，重悬羊抗小鼠IgG在小瓶磁珠。转移100微升每10 7个细胞珠的管中。 </li><li>添加1毫升分离缓冲液中，放置在管中一个磁体保持1分钟。丢弃用5毫升吸管上清液。 </li><li>从磁体取出管并在100μl分离缓冲液的重悬洗涤珠。 </li><li>孵育10 7细胞用100μl的30分钟的磁珠在4℃下温和倾斜和旋转。 </li><li>放置管在磁体保持2分钟，并与CD19 + / CD5 +未结合的细胞转移上清用5毫升吸管新管。 </li><li>在隔离协议结束时，染色用抗CD19的细胞的100微升和-CD5抗体进行流式细胞术，并在4℃下孵育30分钟。清洗细胞用3ml PBS 1％BSA的，丢弃过量上清液，并通过流再次检查B细胞纯度19流式细胞术。 注意:如果B细胞的纯度大于95％的低执行额外的阴性分离步骤。 </li></ol> 3.标准和抑制剂股票方案的制备<ol><li>为了制备AMP，称出0.00345克的AMP并溶解在5ml无血清培养基的具有AMP的2mM的标准溶液。 </li><li>为了准备ADO，掂量出0.00265克ADO和溶解在5毫升的无血清培养基中有ADO的2毫米标准的解决方案。 </li><li>为了制备INO，称出0.00268克INO的并溶解在5ml无血清培养基的具有INO的2mM的标准溶液。 </li><li>准备AMP，ADO的400μm的解决方案，INO通过在4毫升无血清培养基（5ml最终体积）的混合将1ml 2mM的储备液。 <ol><li>使1:4稀释各400μM的标准溶液通过在1500微升无血清培养基（2ml最终体积）的吸移500微升400μM溶液，得到100μM的浓度。 </li><li>制备各化合物的系列稀释液中的无血清培养基，得到以下浓度:100μM - 50μM - 25μM - 10μM - 5微米 - 2.5微米 - 1μM - 0.5μM - 0.25μM。 注意:例如，移液管1毫升1毫升的无血清培养基的100μM的溶液（1:2稀释），得到50μM的浓度，并与其余的稀释液进行。 </li></ol></li><li>制备铝塑复合板溶解在PBS中的10mM储备溶液;分装现货 - 30℃。 </li><li>制备EHNA盐酸盐，DCF达莫溶解于二甲亚砜中10mM的储备溶液（DMSO）;等分和股票-30C。 </li></ol> 4.项目的HPLC方法<ol><li>通过0.770克乙酸铵溶解在2升的双去离子水（缓冲液A）制备7 20mM乙酸铵缓冲。调整用盐酸将pH值3.0。 </li><li>准备至少含有2升超纯乙腈HPLC（缓冲液B）的玻璃瓶和连接保护柱和柱的HPLC。 注意:列和缓冲液是在RT同时在使用中。 </li><li>登录到HPLC软件，并选择"浏览项目"按钮。转到"文件"菜单，选择"新方法"，然后"仪器方法"。 </li><li>方案的平衡柱法按表1。设定为1.00毫升/分钟的流速和编程UV检测器在260nm处读取。保存仪器方法，并从"文件"菜单中再次选择"新方法"，然后"方法设置"。选择之前保存的仪器方法并保存当前方法使用相同的名称设置的。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 时间 </td><td> 流动速率（毫升/分钟） </td><td> ％一个 </td><td> ％B </td></tr><tr><td></td><td> 1.00 </td><td> 100 </td><td> 0 </td></tr><tr><td> 1.24 </td><td> 1.00 </td><td> 100 </td><td> 0 </td></tr><tr><td> 6.22 </td><td> 1.00 </td><td> 2 </td><td> 98 </td></tr><tr><td> 18.65 </td><td> 1.00 </td><td> 2 </td><td> 98 </td></tr></tbody></table> 表1: 平衡柱方法的塔的平衡溶剂变化示意图。缓冲液A:7 mM醋酸铵，pH值3.0。缓冲液B:乙腈。 <ol start="5"><li>重复步骤4.3 1.00 s的流速在表2所示的运行样本方法编程设定升/分钟和方案的UV检测器在260nm处读取。保存该仪器的方法和如在步骤4.4中所述，保存方法组重复相同的过程.. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 时间 </td><td> 流动速率（毫升/分钟） </td><td> ％一个 </td><td> ％B </td></tr><tr><td></td><td> 1.00 </td><td> 0 </td><td> 100 </td></tr><tr><td> 3.74 </td><td> 1.00 </td><td> 0 </td><td> 100 </td></tr><tr><td> 13.71 </td><td> 1.00 </td><td> 15 </td><td> 85 </td></tr><tr><td> 17.00 </td><td> 1.00 </td><td> 100 </td><td> 0 </td></tr><tr><td> 20.00 </td><td> 1.00 </td><td> 100 </td><td> 0 </td></tr></tbody></table> 表2: 运行样品的方法 。对嘌呤类物质的HPLC测定溶剂的变化示意图。浅黄色急诊室:7 mM醋酸铵，pH值3.0。缓冲液B:乙腈。 <ol start="6"><li>选择"运行样品"按钮，从"文件"菜单中选择"新的样品组方法"。 </li><li>选择"空"选项，并输入小瓶（在自动进样器）的注入量（50μL）的数量，样品名称，。选择"方法设置"栏中以前保存的方法组，并进入总运行时间（分钟）。 注:在每个样品运行前的"方法组"列中输入平衡柱法（ 表1），并进入总运行时间（分钟）。 </li></ol> 5.生成一个标准曲线各化合物的<ol><li>注入50μl的空白（特定无血清培养基中，其中不含有腺苷脱氨酶），并按照表1和表2所描述的方法的每个标准样品的。 <ol><li>使用柱平衡方法（ 表1）每个样品运行之前和在表2中描述的梯度条件，以获得足够的峰分离。请参考表3 AMP，ADO和INO这些HPLC条件下报道的保留时间。 </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td> 保留时间 </td><td> λ 最大 </td></tr><tr><td> AMP </td><td> 8.00分钟</td><td> 260 </td></tr><tr><td> INO </td><td> 11.00分</td><td> 260 </td></tr><tr><td> ADO </td><td> 11.90分</td><td> 260 </td></tr></tbody></table> 表3:嘌呤类化合物的保留时间为AMP，ADO和INO观察到典型保留时间。 UV检测器被编程在260nm阅读。 <ol start="2"><li&gt;确定AMP，ADO和INO的峰面积不同浓度的HPLC软件和旁边的"集成"选项中选择"进程"按钮。或者手动选择每个峰的起点和终点。 </li><li>暗算每个标准的标称浓度的峰面积，以获得九个点校准曲线（100，50，25，10，5，2.5，1，0.5，0.25微米）。 </li><li>计算直线的方程AMP，ADO和INO:Y = mx + b中 ， 其中x是μM浓度和y对应的峰面积。 </li></ol><img alt="图2" src="/files/ftp_upload/54124/54124fig2.jpg" /> 图2 代内部标准曲线。对于ADO代表校准标准曲线，并获得了相关方程。 <a href="https://www.jove.com/files/ftp_upload/54124/54124fig2large.jpg" target="_blank">请点击她的</a>E要查看此图的放大版本。 6.预处理与抑制剂和孵化与基材（AMP） <ol><li>为了测试AMP消耗，ADO和INO代无CD73，ADA的抑制和核苷转运，悬浮2×10 6个 CD19 + / CD5 + CLL细胞（分离步骤1的纯化和2）在250微升无血清培养基中，并板的细胞在48孔板（或在1.5ml微量离心管）。 </li><li>在培养基中1000 APCP（10毫米）的储备溶液，以获得最终浓度10μM:转向方框CD73的酶活性，稀释1。悬浮2×10 6个 CLL细胞在250微升含铝塑复合板培养基并在细胞培养培养箱中于37℃预处理60分钟。 </li><li>到方框的ADO入INO转换，稀释1:1000 EHNA盐酸盐和/或DCF的在无血清培养基中的储备溶液（均为10毫米）具有最终浓度10μM。重悬在250微升含有EHNA和/或DCF培养基为2×10 6个白血病细胞。在细胞培养培养箱温育30分钟，在37℃。 </li><li>在无血清培养基中1000达莫的储备溶液（10毫摩尔）为具有最终浓度10μM:转向方框ADO通过核苷转运的摄取，稀1。悬浮2×10 6个 CLL细胞在250微升含双嘧达莫培养基，并在细胞培养培养箱中于37℃孵育30分钟。 </li><li>准备铝塑复合板，EHNA，DCF的单一解决方案，双嘧达莫稀释1:1,000 400μMAMP。 </li><li>加入250微升400μMAMP或AMP加抑制剂，得到200μM的AMP的终浓度。包括在不存在衬底的状态，以被用作空白样。 </li><li>孵育30到37℃60分钟。 注意:实验测定AMP和孵育时间的最佳浓度。 </li><li>在t他最终的温育时间，收集500微升上清液中冷离心管（在冰上），并立即在17000 xg离心离心在4℃下5分钟。 </li><li>转移上清液在新的1.5 ml微量离心管，并立即在-80℃下存储或进行用于HPLC运行样品制备。 </li></ol> 7.样品制备HPLC <ol><li>过滤器在新的1.5 ml离心管中的上清液用0.2微米注射过滤器。使用1毫升注射器过滤。 </li><li>如果HPLC系统配备有自动进样器，准备用于HPLC玻璃瓶中，并使用0.1毫升微刀片样品的小体积。 </li><li>传送至少100微升样品在用微量或玻璃巴斯德吸管的玻璃小瓶中。小心无气泡转移和与螺旋盖关闭小瓶中。 </li><li>样品现已准备好通过RP-HPLC分析。 </li></ol> 8. HPLC Measu嘌呤rements <ol><li>选择"运行样品"按钮;从"文件"菜单中选择"新的样品组方法"。 </li><li>选择"空"选项，并指出:小瓶（在自动进样器）的注入量（50μL）的数量，样品名称，。 </li><li>选择平衡柱方法并在表1和表2中记载的运行样本方法，分别用于所有随后的空白和样品运行。 注:每个样品运行前将平衡柱法（ 表1）。 </li><li>注入50μl的空白（无血清培养基）和每个样品的。 </li><li>确定每个样品中的嘌呤的浓度，通过在表3中描述的保留时间获得的峰面积定量AMP，ADO和INO。 <ol><li>选择"进程"按钮旁边的"集成"选项。手动或者选择启动每个单峰的第二终点，获得面积测量。这是通过跟踪峰的开始和结束点之间的直线进行。 </li><li>确定μM浓度申请标准曲线得到的方程为:y = mx + b中 ，其中x表示μM浓度和y是未知样品测得的峰值的面积。 注意:ADO校准标准曲线的一个实例被报告于图2，其中:x =（γ区- 981.02）/ 40026 </li></ol></li><li>考虑到重悬于0.500毫升介质的2×10 6个细胞，计算AMP的微摩尔，ADO和INO消耗和/或通过应用下列比例10 6个白血病细胞产生的: 微摩尔:千毫升= X微摩尔:0.250毫升 注意:1,000ml中的微摩尔（数微摩尔在1升）的等同于μM浓度和x是Nu的微摩尔核苷酸或核苷由10 6个细胞中产生。 <ol><li>最后，转换成微摩尔以10 6 CLL细胞消耗和/或生产毫微摩尔: 纳摩尔=微摩尔×1,000 </li></ol></li></ol>

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J., Gandhi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+collection+methods+affect+cellular+protein+integrity:+implications+for+clinical+trial+biomarkers+and+ZAP-70+inn+CLL.">Blood collection methods affect cellular protein integrity: implications for clinical trial biomarkers and ZAP-70 inn CLL.</a> Blood. 124 (7), 1192-1195 (2014).</li><li>Kalina, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EuroFlow+standardization+of+flow+cytometer+instrument+settings+and+immunophenotyping+protocols.">EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols.</a> Leukemia. 26 (9), 1986-2010 (2012).</li><li>Deaglio, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD38+and+ZAP-70+are+functionally+linked+and+mark+CLL+cells+with+high+migratory+potential.">CD38 and ZAP-70 are functionally linked and mark CLL cells with high migratory potential.</a> Blood. 110 (12), 4012-4021 (2007).</li><li>Sachsenmeier, K. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+novel+ectonucleotidase+assay+suitable+for+high-throughput+screening.">Development of a novel ectonucleotidase assay suitable for high-throughput screening.</a> J Biomol Screen. 17 (7), 993-998 (2012).</li></ol>

The authors have nothing to disclose.

这里所描述的协议允许评价在细胞培养介质中的CD39 / CD73 adenosinergic机械从纯化的人白血病细胞的活性。通过这样的高效液相色谱法，我们可以遵循和定量测定酶生成ADO（CD73依赖型）和其随后的降解INO（CD26 / ADA相关）的。使用酶抑制剂的允许控制协议，并有内部控制。这个协议的优点和新奇是i）其可以施加到在培养生长的细胞，即ⅱ）它需要培养基少量且iii）所述的样品制备是非常容易的。 <...

腺苷（ADO）是嘌呤核苷通过糖苷键连接于核糖分子部分连接的腺嘌呤分子。当存在于细胞外的环境中，它由免疫系统的作用可以防止过度的损伤的细胞。这种作用已用不同的疾病模型，如结肠炎1，糖尿病2，哮喘3，败血症4，和局部缺血性损伤的5突出。其中一个主要的ADO功能是在肿瘤微环境的免疫应答的抑制，促进肿瘤免疫逃避6。出于这个原因，参与ADO形成和信令的机制相当的治疗利益7。 在组织微环境的ADO水平正常生理条件下，当然低于免疫细胞的灵敏度阈值相对较低。然而，缺氧时，局部缺血，炎症，感染，代谢压力和肿瘤转型，他们迅速增加8。响应于组织扰动信号升高胞外ADO水平具有双重功能:报​​告组织损伤以自分泌和旁分泌方式并产生可通常被视为细胞保护组织反应。 胞ADO可以通过多种机制，包括从由因为通过腺苷脱氨酶操作受损降解的核苷转运9或积累介导的细胞内区室释放而形成。导致增加的胞外ADO水平的主要途径包括ectonucleotidases，这是膜相关ectoenzymes通过从死亡或垂死细胞释放的核苷酸phosphohydrolysis产生的ADO级联的作用。该途径通过CD39的顺序动作前进（ectonucleoside三磷酸diphosphohydrolase-1），该转换外腺苷5&#39;-三磷酸（ATP）或腺苷5&#39;-二磷酸（ADP）腺苷5&#39;-一磷酸（AMP）和CD73（5&#39;-核苷酸）的，它转换AMP到ADO 10。 胞ADO通过结合到4个跨膜ADO受体，即A 1，A 2A，A 2B和A 3引出其生理反应。每个受体有ADO和特定的组织分布不同的亲和力。所有的受体具有七次跨膜域和是G-蛋白偶联于细胞内的GTP结合蛋白（G蛋白），即可以诱导（G蛋白）或抑制（Gi蛋白）腺苷酸环化酶活性，并随后在生产细胞内cAMP。因此，在过程中的生理反应11上的细胞内的蛋白激酶活性的胞质cAMP水平的影响的变化。在生理条件下细胞外ADO是低于1微米，它可以激活不加区别A 1，A 2A和A 3受体。然而，A2B亚型的激活需要相当高的核苷的浓度，如病理生理条件下产生的。可替代地，细胞外ADO可以被降解为肌苷（INO）腺苷脱氨酶（ADA）和CD26，一个ADA络合蛋白在细胞表面上定位ADA。另一种可能性是，ADO是由细胞通过平衡型核苷转运（ENT）和磷酸化，以AMP通过ADO激酶蛋白12,13内化。 该协议的目的是描述反相高效液相色谱法（RP-HPLC）的分析方法在单次运行来量化基板AMP和产品ADO和INO，由人淋巴细胞作为生成的。最初使用来自慢性淋巴细胞白血病（CLL）的患者，其特征为CD19 + / CD5 + B淋巴细胞组成型表达CD39 14,15的成熟群体的膨胀细胞得到我们的经验。我们大约30表现％CLL患者表达CD73胞外酶，而这表型预后不良相关16。白血病细胞共表达CD39和CD73的这种亚群可以积极借鉴ADP和/或AMP产生胞ADO。 CD73 + CLL细胞用α预孵育，β亚甲基ADP（APCP），CD73酶活性的已知抑制剂，完全阻断细胞外的ADO合成确认CD73表示级联16的瓶颈酶。 CLL细胞还表达ADA和ADA的络合蛋白CD26，这是负责ADO转化成INO。通过使用特定的ADA抑制剂，如赤-9-（2-羟基-3-壬基）我wiadenine（EHNA）盐酸盐和deoxycoformycin（DCF），它可以阻止细胞外ADO降解成INO。此外，预处理与潘生丁联合ADA抑制剂，阻止核苷转运，增强细胞积累ADO上清。 然后，我们扩展了这个协议从其他谱系，包括T淋巴细胞和骨髓细胞，证实CD73依赖ADO生产的细胞。这些结果表明，此HPLC协议是高度通用的，并且它可以被应用到不同的细胞谱系和对不同的培养条件下（ 图1）。 <img alt="图1" src="/files/ftp_upload/54124/54124fig1.jpg" /> 负责细胞外ADO生产酶促机械的 图1 示意图。腺苷5&#39;-三磷酸（ATP）和/或腺苷5&#39;-二磷酸（ADP）可通过CD39降解为腺苷5&#39;-一磷酸（AMP），其中又通过CD73转化成核苷腺苷（ADO）。一旦ADO技术是在细胞外空间中产生，它可以重新进入通过核苷转运（ENT）细胞中，被转化成肌苷（INO）或绑定到不同类型的P1 ADO受体。 <a href="https://www.jove.com/files/ftp_upload/54124/54124fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table><tbody><tr><td>Human blood</td><td></td><td></td><td></td></tr><tr><td>Milli-Q water</td><td>Millipore</td><td></td><td>double deionised water</td></tr><tr><td>Ficoll-Paque Plus</td><td>GE-Healthcare</td><td>17-1440-03</td><td></td></tr><tr><td>purified anti-CD3, -CD14, -CD16</td><td>made in-house</td><td></td><td>mouse monoclonal</td></tr><tr><td>PE-labeled anti-CD19</td><td>Miltenyi Biotec</td><td>120-014-229</td><td></td></tr><tr><td>FITC-labeled anti-CD5</td><td>Miltenyi Biotec</td><td>130-096-574</td><td></td></tr><tr><td>Dynabeads sheep anti-mouse IgG</td><td>Invitrogen</td><td>11031</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Amresco</td><td>E404-200TABS</td><td>tablets</td></tr><tr><td>bovine serum albumin (BSA)</td><td>ID bio</td><td>1000-70</td><td>standard grade</td></tr><tr><td>isolation buffer</td><td></td><td></td><td>PBS 0.1% BSA 2 mM EDTA, pH 7.4</td></tr><tr><td>AIM V serum free medium</td><td>GIBCO</td><td>12055-091</td><td>liquid (research grade)</td></tr><tr><td>adenosine 5&amp;rsquo;-diphosphate (ADP)</td><td>Sigma-Aldrich</td><td>A2754</td><td></td></tr><tr><td>adenosine 5&amp;rsquo;-monosphate (AMP)</td><td>Sigma-Aldrich</td><td>A1752</td><td></td></tr><tr><td>adenosine (ADO)</td><td>Sigma-Aldrich</td><td>A9251</td><td></td></tr><tr><td>inosine (INO)</td><td>Sigma-Aldrich</td><td>I4125</td><td></td></tr><tr><td>&amp;alpha;,&amp;beta;-methylene-ADP (APCP)</td><td>Sigma-Aldrich</td><td>M8386</td><td>CD73 inhibitor</td></tr><tr><td>EHNA hydrochloride</td><td>Sigma-Aldrich</td><td>E114</td><td>adenosine deaminase inhibitor</td></tr><tr><td>Deoxycoformycin (dCF)</td><td>Tocris</td><td>2033</td><td>adenosine deaminase inhibitor</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Dipyridamole</td><td>Sigma-Aldrich</td><td>D9766</td><td>nucleoside transporter inhibitor</td></tr><tr><td>acetonitrile (CHROMASOLV Plus)</td><td>Sigma-Aldrich</td><td>34998</td><td>HPLC-grade</td></tr><tr><td>ammonium acetate</td><td>Sigma-Aldrich</td><td>9688</td><td>7 mM, pH 3.0</td></tr><tr><td>hydrochloric acid</td><td>Sigma-Aldrich</td><td>30721-1L</td><td>min. 37%</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>B&amp;uuml;rker cell counter</td><td>VWR</td><td>631-0920</td><td>hemocytometer</td></tr><tr><td>DynaMag-15 Magnet</td><td>Invitrogen</td><td>12301D</td><td>Dynal magnetic bead separator</td></tr><tr><td>microcentrifuge safe-lock tubes</td><td>Eppendorf</td><td>030-120-0086</td><td>1.5 ml</td></tr><tr><td>PET centrifuge tubes</td><td>Corning</td><td>430053/430304</td><td>15 &amp;ndash; 50 ml</td></tr><tr><td>Minisart RC4 syringe filters</td><td>Sartorius Stedim Biotech</td><td>17821</td><td>membrane 0.2 &amp;micro;m</td></tr><tr><td>short thread vials</td><td>VWR</td><td>548-0029</td><td>1.5 ml/glass</td></tr><tr><td>micro-inserts</td><td>VWR</td><td>548-0006</td><td>0.1 ml/glass</td></tr><tr><td>screw caps</td><td>VWR</td><td>548-0085</td><td>9 mm/PP blue</td></tr><tr><td>Atlantis dC18 Column</td><td>Waters</td><td>186001344</td><td>5 &amp;micro;m, 4.6 mm x 150 mm</td></tr><tr><td>Atlantis dC18 Guard Column</td><td>Waters</td><td>186001323</td><td>5 &amp;micro;m, 4.6 mm x 20 mm</td></tr><tr><td>Waters Alliance 2965 Separations Module</td><td>Waters</td><td></td><td>HPLC separation module</td></tr><tr><td>Waters 2998 Photodiode Array (PDA) Detector</td><td>Waters</td><td></td><td>UV detector</td></tr><tr><td>Waters Empower2 software</td><td>Waters</td><td></td><td></td></tr></tbody></table>

hplc-based assay to monitor extracellular nucleotide/nucleoside metabolism in human chronic lymphocytic leukemia cells

该方法描述了一种敏感，特异的，可靠的和可重现的反相高效液相色谱法（RP-HPLC）测定法开发并验证胞外嘌呤核苷酸和核苷通过纯化的慢性淋巴细胞性白血病产生（CLL）细胞的不同培养条件下的量化。腺苷5&#39;-一磷酸的（AMP），腺苷（ADO）的色谱分离和肌苷（INO）在RT上被用于极性化合物的保留的基于二氧化硅的，反相柱进行。该方法包括一个二元流动相，它由7 mM醋酸铵和乙腈为1.00毫升/分钟的流速。洗脱液使用一光电二极管阵列UV检测器设定在260nm监测。标准校正曲线生成以计算方程式为每个嘌呤化合物的分析定量。系统控制，数据采集和分析，然后进行。运用这一协议，AMP，INO和ADO分别洗脱在7,11和11.9分钟，和总运行时间为每个样品20分钟。该协议可以使用培养基作为基质施加到不同类型的细胞和细胞系（包括悬浮液和粘附）。的优点是容易和快速的样品制备和分析少量上清液的要求。此外，使用无血清培养基中允许跳过用乙腈沉淀蛋白的步骤，影响嘌呤化合物的最终浓度。一种该方法的局限性是每个单个样品运行之前运行平衡柱的要求，使得在实验的总运行时间更长，并防止高通量筛选应用。

为了评估从一个代表性的CLL患者新鲜纯化的PBMC白血病细胞的百分比（％），将细胞标记与抗CD19和抗CD5抗体。 图3的左图表示具有活细胞上的选择性栅极的细胞荧光点图; 图3示出了从CLL患者PBMC的一个例子之前（中图）和后（右面板）的B细胞纯化。 在CD73 + CLL细胞的嘌呤化合物的高效液相色谱量?...

Watch this Scientific Journal Video about HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells at JoVE.com

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

HPLC-基于化验，以监测外核苷酸/核苷代谢的人类慢性淋巴细胞白血病细胞

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

hplc-based-assay-to-monitor-extracellular-nucleotidenucleoside

Research

JoVE Journal

Chemistry

11.0K 次观看.  University of Turin. The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

视频: HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during cancer development and progression. Despite this, the global epigenetic regulation of lncRNAs and repetitive sequences in cancer has not been well investigated, particularly in chronic lymphocytic leukemia (CLL). This study focuses on a unique approach: the immunoprecipitation-based capture of double-stranded, methylated DNA fragments using methyl-binding domain (MBD) proteins, followed by next-generation sequencing (MBD-seq). CLL patient samples belonging to two prognostic subgroups (5 IGVH mutated samples + 5 IGVH unmutated samples) were used in this study. Analysis revealed 5,800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal healthy controls. Importantly, these results identified several CLL-specific, differentially methylated lncRNAs, repetitive elements, and protein-coding genes with potential prognostic value. This work outlines a detailed protocol for an MBD-seq and bioinformatics pipeline developed for the comprehensive analysis of global methylation profiles in highly CpG-rich regions using CLL patient samples. Finally, a protein-coding gene and an lncRNA were validated using pyrosequencing, which is a highly quantitative method to analyze CpG methylation levels to further corroborate the findings from the MBD-seq protocol.

This work describes an optimized methyl-CpG-binding domain (MBD) sequencing protocol and a computational pipeline to identify differentially methylated CpG-rich regions in chronic lymphocytic leukemia (CLL) patients.

在慢性淋巴细胞性白血病患者中使用甲基CpG结合域捕获法进行综合DNA甲基化分析

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during ...

科研

遗传学

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

流式细胞仪估测原发性急性髓系白血病和患者源性移植中白血病干细胞的诊断和随访

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

癌症研究

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

一种检测慢性淋巴细胞白血病新 nfat2 靶基因的染色质免疫沉淀法

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

原发性白血病细胞代谢谱的评价

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data.

This protocol enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These samples are used to determine protein localization as well as changes in protein trafficking that take place between the nuclear and cytoplasmic compartments upon cell stimulation and drug treatment.

原发性慢性淋巴细胞白血病细胞的细胞次细胞分段监测核/细胞质蛋白贩运

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which are involved in many cell activities. Here, we describe a rapid and reliable method for the absolute qualification of nucleoside/nucleotide contents in plants. Briefly, 100 mg of homogenized plant material was extracted with 1 mL of extraction buffer (methanol, acetonitrile, and water at a ratio of 2:2:1). Later, the sample was concentrated five times in a freeze dryer and then injected into an HPLC-MS/MS. Nucleotides were separated on a porous graphitic carbon (PGC) column and nucleosides were separated on a C18 column. The mass transitions of each nucleoside and nucleotide were monitored by mass spectrometry. The contents of the nucleosides and nucleotides were quantified against their external standards (ESTDs). Using this method, therefore, researchers can easily quantify nucleosides/nucleotides in different plants.

A precise and reproducible method for in vivo nucleosides/nucleotides quantification in plants is described here. This method employs an HPLC-MS/MS.

使用 HPLC-MS/MS 进行核苷/核苷酸含量测量的工厂样品制备

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which ...

生物化学

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

免疫与感染

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, ...

Bromodeoxyuridine Pulse Labelling Assay: A Technique to Measure Cell Proliferation

Source: Welschinger R. et al. <a href="https://www.jove.com/t/52840">Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization.</a> J. Vis. Exp. (2015)
The video describes the stepwise pulse labeling assay of mammalian cells with bromodeoxyuridine (BrdU) for measuring cell proliferation. The BrdU uptake permits the temporal tracking of cells that were in the synthesis phase at a specific point in time, without requiring the cells to be synchronized.

溴脱氧尿嘧啶脉冲标记测定:一种测量细胞增殖的技术

Source: Welschinger R. et al. Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization. J. Vis. Exp. (2015)
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JoVE Encyclopedia of Experiments

白血病

In vitro techniques & assays

Extracellular Flux Assay: A Method for Measuring Metabolic Profile of Cells

Source: Hlozkova K. et al. <a href="https://www.jove.com/t/58426">Assessment of the Metabolic Profile of Primary Leukemia Cells.</a> J. Vis. Exp. (2018).
This protocol provides a biochemical approach for measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of primary leukemic cells with extracellular flux assay. The assessment of the metabolic profile of these cells helps to characterize their demands, leading to personalized therapy.

细胞外通量测定:一种测量细胞代谢谱的方法

Source: Hlozkova K. et al. Assessment of the Metabolic Profile of Primary Leukemia Cells. J. Vis. Exp. (2018).
This protocol provides a biochemical ...