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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#8217;-diphosphate (ADP)</td>
			<td>Sigma-Aldrich</td>
			<td>A2754</td>
			<td></td>
		</tr>
		<tr>
			<td>adenosine 5&#8217;-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>&#945;,&#946;-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 height="21">
			<td height="21" style="height:21px;width:315px;">Name</td>
			<td style="width:168px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:235px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Equipment</td>
			<td style="width:168px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B&#252;rker cell counter</td>
			<td>VWR</td>
			<td style="width:119px;">631-0920</td>
			<td>hemocytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DynaMag-15 Magnet</td>
			<td>Invitrogen</td>
			<td style="width:119px;">12301D</td>
			<td>Dynal magnetic bead separator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microcentrifuge safe-lock tubes</td>
			<td>Eppendorf</td>
			<td>030-120-0086</td>
			<td>1.5 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PET centrifuge tubes</td>
			<td>Corning</td>
			<td>430053/430304</td>
			<td>15 &#8211; 50 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minisart RC4 syringe filters</td>
			<td>Sartorius Stedim Biotech</td>
			<td>17821</td>
			<td>membrane 0.2 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">short thread vials</td>
			<td>VWR</td>
			<td>548-0029</td>
			<td>1.5 ml/glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micro-inserts</td>
			<td>VWR</td>
			<td>548-0006</td>
			<td>0.1 ml/glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">screw caps</td>
			<td>VWR</td>
			<td>548-0085</td>
			<td>9 mm/PP blue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Atlantis dC18 Column</td>
			<td style="width:168px;">Waters</td>
			<td style="width:119px;">186001344</td>
			<td>5 &#181;m, 4.6 x 150 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Atlantis dC18 Guard Column</td>
			<td style="width:168px;">Waters</td>
			<td style="width:119px;">186001323</td>
			<td>5 &#181;m, 4.6 x 20 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waters Alliance 2965 Separations Module</td>
			<td style="width:168px;">Waters</td>
			<td style="width:119px;"></td>
			<td>HPLC separation module</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waters 2998 Photodiode Array (PDA) Detector</td>
			<td style="width:168px;">Waters</td>
			<td style="width:119px;"></td>
			<td>UV detector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Waters Empower2 software</td>
			<td style="width:168px;">Waters</td>
			<td style="width:119px;"></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

10.9K 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

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

科研

化学

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

使用气相色谱怀孕期间分析脂肪酸组成的变化在大鼠肝组织

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

合成与盘状磷酸盐锆剥落获取胶体液晶

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

免疫染色磷酸化表位整装斑马鱼胚胎纤毛器官

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

毛细管电泳监视肽接枝壳聚糖膜实时

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

由 F− 310 Nm 支队所示的阴离子光电子成像

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests phase of drug developing process because they are genetically human and recapitulate the interplay among tissues. In this study, the human intestinal barrier (emulated by a co-culture of Caco-2 and HT-29 cells) and the liver equivalent (emulated by spheroids made of differentiated HepaRG cells and human hepatic stellate cells) were integrated into a two-organ chip (2-OC) microfluidic device to assess some acetaminophen (APAP) pharmacokinetic (PK) and toxicological properties. The MPS had three assemblies: Intestine only 2-OC, Liver only 2-OC, and Intestine/Liver 2-OC with the same media perfusing both organoids. For PK assessments, we dosed the APAP in the media at preset timepoints after administering it either over the intestinal barrier (emulating the oral route) or in the media (emulating the intravenous route), at 12 &#181;M and 2 &#181;M respectively. The media samples were analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Organoids were analyzed for gene expression, for TEER values, for protein expression and activity, and then collected, fixed, and submitted to a set of morphological evaluations. The MTT technique performed well in assessing the organoid viability, but the high content analyses (HCA) were able to detect very early toxic events in response to APAP treatment. We verified that the media flow does not significantly affect the APAP absorption whereas it significantly improves the liver equivalent functionality. The APAP human intestinal absorption and hepatic metabolism could be emulated in the MPS. The association between MPS data and in silico modeling has great potential to improve the predictability of the in vitro methods and provide better accuracy than animal models in pharmacokinetic and toxicological studies.

We exposed a microphysiological system (MPS) with intestine and liver organoids to acetaminophen (APAP). This article describes the methods for organoid production and APAP pharmacokinetic and toxicological property assessments in the MPS. It also describes the tissue functionality analyses necessary to validate the results.

药物药代动力学和毒理学评估的肠道/肝脏微生理系统

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests ...

Mass Spectrometry-Guided Genome Mining as a Tool to Uncover Novel Natural Products

The chemical space covered by natural products is immense and widely unrecognized. Therefore, convenient methodologies to perform wide-ranging evaluation of their functions in nature and potential human benefits (e.g., for drug discovery applications) are desired. This protocol describes the combination of genome mining (GM) and molecular networking (MN), two contemporary approaches that match gene cluster-encoded annotations in whole genome sequencing with chemical structure signatures from crude metabolic extracts. This is the first step towards the discovery of new natural entities. These concepts, when applied together, are defined here as MS-guided genome mining. In this method, the main components are previously designated (using MN), and structurally related new candidates are associated with genome sequence annotations (using GM). Combining GM and MN is a profitable strategy to target new molecule backbones or harvest metabolic profiles in order to identify analogues from already known compounds.

A mass spectrometry-guided genome mining protocol is established and described here. It is based on genome sequence information and LC-MS/MS analysis and aims to facilitate identification of molecules from complex microbial and plant extracts.

质谱导览基因组挖掘作为发现新天然产品的工具

The chemical space covered by natural products is immense and widely unrecognized. Therefore, convenient methodologies to perform wide-ranging evaluation ...