我们感谢淋巴组织的恶性肿瘤单位，Elisa的10克勤，肥姐Scarfò，马西莫·阿莱西奥，安东尼奥·孔蒂，安吉拉八尺，和Angela郭居静。 该项目支持:ASSOCIAZIONE ITALIANA每拉Ricerca SUL巨蟹座AIRC（调查员补助金和特别节目分子肿瘤学 - 5千分数＃9965） CS和BA设计的研究，进行了实验，分析数据和撰写文章。 MTSB，UR，FBPR协助撰写稿件。 PG和FCC的设计提供了研究患者的标本和临床资料。

注:所有组织样品与圣拉斐尔医院（米兰，意大利）的机构审查委员会批准获得。 1，人体组织样本和细胞纯化（图1） 注:淋巴细胞白血病从CLL患者的外周血（PB）根据Mulligan 等人获得，确诊9。 1.1）从PB白血病细胞分离<ol><li>与肝素钠真空采血管的血液收集管收集的PB。 </li><li>传输血液入15ml离心管，以50微升/毫升的全血添加人B细胞富集鸡尾酒。搅拌均匀，保温20分钟，在室温下。 </li><li>稀释样品用PBS +2％FBS等体积，轻轻混匀，小心叠加血在聚蔗糖，确保不突破的接口。使用至少1份聚蔗糖的2份稀释样品（ 即 4毫升聚蔗糖+10毫升血液在15ml吨UBE）。 </li><li>离心机在400 XG，在室温（20℃）20分钟，用不加制动。单核细胞会在界面处。 </li><li>小心吸出以回收细胞并将其放置到一个新的管的接口。一旦用PBS离心300 XG在黑暗中5分钟，洗涤细胞，4℃。沉淀会在底部。 </li><li>弃上清。通过轻弹管来回用手指悬浮在剩余的上清液中沉淀。稀释，用10ml的完全RPMI培养基中的小球（RPMI 1640补充有10％胎牛血清和15毫克/毫升庆大霉素）和计数用台盼蓝排除法的细胞。 </li></ol> 1.2）纯度评价注:所有制剂纯度必须始终在99％以上。 <ol><li>孵育100微升纯化的细胞悬浮液与下列抗体（市售;见表材料）:CD3 FITC（5微升/ TES吨），CD14 PE（5微升/试验），CD19 ECD（6微升/试验），CD16-CD56 PC5（2微升/试验），CD5 PC7在（4微升/试验），进行20分钟，在4℃下流式细胞仪管。 </li><li>洗用4ml的PBS（离心5分钟，在300 XG）的样品，并检查其纯度通过流式细胞cytometry.Check在剧情CLL细胞（&gt; 99％），其共表达CD19和CD5在其表面上作为％在图1中示出（6）。确保准备工作几乎毫无NK细胞，T淋巴细胞和单核细胞在情节，这应该是接近零检测CD3 +，CD14和CD16-56的％。 </li></ol> 1.3）样品储存<ol><li>用于蛋白组学研究中，收集25×10 6个细胞在1.5ml管中，离心分离细胞，在300×g离心10分钟，弃去上清液并冷冻干燥的颗粒4小时。保持在-80℃直到使用。 </li><li>进行验证试验，重悬细胞完全RPMI（数量取决于进一步的分析是选择），并继续进行步骤4。 </li></ol> 2，双向电泳（2-DE）（图2） 2.1）等电聚焦电泳（IEF） <ol><li>溶解CLL细胞颗粒用380微升2-DE缓冲液（344微升的RBthio解决方案的最终体积（9M尿素，10毫米的Tris，4％CHAPS），30微升65毫米的DTT，6微升2％IPG缓冲两性电解质pH为4-7）。 </li><li>适用于蛋白质样品（1毫克），以18 IPG胶条（pH为4-7），并进行IEF以下标准协议如由帝等人 10 </li><li>补充有新鲜的DTT 2％，在RT15分钟:（50含有6M尿素，30％甘油，2％SDS毫pH为8.8的Tris-HCl缓冲液EB）在2毫升平衡缓冲液中平衡的条。放弃EB + DTT和加入2ml的EB辅以2.5％碘乙酰胺。孵育在室温下15分钟的摇动平台上。 </li></ol> 2.2），SDS-PAGE <ol><li>干在纸条而不触及凝胶去除多余的EB。加载每个条上的9-16％梯度SDS-PAGE凝胶的顶部。加入小体积的SDS-电泳缓冲液（Tris 25mM的，甘氨酸192毫米，SDS 0.1％重量/体积）上的凝胶的顶部，以促进负载的钢带的。 </li><li>加入〜加入5ml琼脂溶液（0.8％），以防止浮带的密封胶，然后组装的电泳单元和填充用SDS-电泳缓冲液中。在4℃下进行运行在60毫安/凝胶4小时。 </li><li>从电泳装置中取出凝胶，并将其放置在适当的染色中。 </li></ol> 2.3）银染色制备凝胶<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td>定影液</td><td> 50％甲醇12％乙酸 0,05％的福尔马林</td><td> 2小时（或过夜）* </td></tr><tr><td>洗涤缓冲液</td><td> 35％Ehanol </td><td> 20分钟（重复步骤三次） </td></tr><tr><td>增敏</td><td> 0,02％硫代硫酸钠</td><td> 2分钟</td></tr><tr><td>洗</td><td> H 2 O的</td><td> 5分钟（重复步骤三次） </td></tr><tr><td>硝酸银</td><td> 0,2％硝酸银0076％福尔马林</td><td> 20分钟</td></tr><tr><td>洗</td><td> H 2 O的</td><td> 1分钟（重复此步骤二次） </td></tr><tr><td>开发者</td><td> 6％的碳酸钠0.0004％硫代硫酸钠 0,05％的福尔马林</td><td>直到染色充足</td></tr><tr><td>停止</td><td> 50％甲醇</td><td> 30分钟</td></tr><tr><td></td><td> *需要2小时的最小时间为蛋白质固定</td><td></td></tr></tbody></table> 表1。 注:蛋白质斑点可以通过凝胶染色与MS兼容的银染11可视化。所有的解决方案所需为银染色列于表1。 <ol><li>制备约200毫升每一溶液/凝胶中，并按照表1中所示，小心地添加各溶液到含有凝胶的染色盒。完成摇摆的平台上所有的孵化。 </li><li>染色后，取出一站式解决方案，并使凝胶1％醋酸溶液，直到采集。 </li></ol> 2.4）凝胶采集与分析<ol><li>使用内染色3天密度仪获得高分辨率的图像。 </li><li>通过使用软件进行2-DE图谱分析2-D蛋白质模式。 注:该软件允许快速和可靠的图像比较。 <ol><li>通过选择"创建新项目"从上下文菜单中创建一个项目。创建一个文件夹并选择"导入凝胶图像"与.TIF，巴纽导入​​凝胶。或.gel扩展。 </li><li>一旦凝胶导入并添加到worksp王牌，通过在菜单中选择凝胶进行当场检测，并选择"编辑&gt;现场&gt;检测"执行自动当场检测。 </li><li>其次，从菜单中选择"减法背景"选择执行背景减除。注意:在此之后就可以比较多个凝胶并检测通过定量和/或定性分析的差异或相似性。 </li></ol></li></ol> 3，蛋白质鉴定通过MALDI-TOF MS分析（图3） 3.1），蛋白质的消化<ol><li>无论是手动（用干净的手术刀切割）或自动切除术用清水冲洗和消费点兴趣凝胶的凝胶。 </li><li>与水（100-150微升）洗凝胶粒子，自旋向下（30秒400 XG）除去液体。 </li><li>加入的CH 3 CN等体积（根据凝胶体积）的凝胶片，并等待10-15分钟，直至凝胶碎片收缩。干凝胶颗粒í呐真空离心机。 </li><li>添加75-100微升10毫米二硫赤藓醇稀释在50毫米的NH 4 HCO 3孵育30分钟，56℃（还原步骤）。再次收缩的凝胶片，用CH 3 CN，如步骤3.1.3中描述。 </li><li>通过加入溶液55 mM的碘乙酰胺稀释于50mM NH 4 HCO 3，孵育20分钟，在室温下在黑暗中的75-100微升进行烷基化。 </li><li>使用足够量如步骤3.1.3中所述，以覆盖凝胶块，用CH 3 CN收缩一次的凝胶片。干式真空离心机。 </li><li>在含25mM NH 4 HCO 3，5mM的氯化钙 ， 和12.5毫微克胰蛋白酶/微升，在4℃下进行20分钟的缓冲液再水合粒子。使用足够量的缓冲来覆盖凝胶块。从凝胶块中取出的未吸收的上清液，加入25 mM的NH 4 HCO 3，5毫米氯化钙2没有胰蛋白酶COV尔的凝胶。 </li><li>离开样品在37℃下至少3小时（所需的肽消化的最短时间）至过夜。 </li></ol> 3.2）质谱分析（干滴法） <ol><li>每个样品到不锈钢MALDI板上的斑点1微升等份和混合用1μl的α-氰基-4 -羟基肉桂酸作为基质，在50％CH 3 CN，0.1％TFA的制备。 </li><li>得到的质谱上的MALDI-TOF质谱仪。累积每点约200光谱，调节激光强度，以防止信号饱和或增加信号。通过数据浏览器软件过程的光谱如下所述: <ol><li>导入dat档，并通过选择"程序&gt;基线校正"进行基线校正。通过选择"程序&gt;质量校正&gt;手动校准校准用胰蛋白酶自溶的产品和基质峰群众;选择峰值接近M / Z 855.1和2163.1 bŸ左拖动并选择相应的参考群众，然后单击"绘图"，并在手动校准窗口"应用校准"。 </li><li>调整阈值的峰值检测（"峰&gt;峰值检测"），重复的活动轨迹（"显示器&gt;复制活跃的踪迹"），选择新的跟踪和执行deisotoping（"峰&gt; deisotoping"）。使用宏"getpeaklist"，产生群众用于识别列表。如果需要，手动删除采摘的高峰清洗名单，并获得更好的识别噪声信号。 </li></ol></li><li>通过搜索用深刻而吉祥物的搜索引擎12进行全面的非冗余蛋白质数据库鉴定蛋白质。 <ol><li>使用以下参数在所有的搜索:一遗漏每肽裂解，氧化蛋氨酸作为修饰的变量，半胱氨酸carbamidomethylation固定的​​修改和initi50ppm的人的质量偏差。 </li></ol></li></ol> 4，细胞骨架的活性测定（图4） 注:将细胞再悬浮于步骤1中的完全RPMI（10 6个细胞/ 200微升）进行纯化。 4.1）迁移实验。该测试可分析的细胞（淋巴细胞）的迁移能力的定量。使用6.5mm的直径和5.0微米的孔径的transwell小室并以一式三份进行测定。在不同类型的细胞的情况下优化孔径和迁移时间（步骤4.1.3）。 <ol><li>通过加入600微升的完全RPMI中具有或不具有特异性刺激（ 即 ，SDF-1）分别测量诱导的自发迁移准备下部腔室。 </li><li>将上部腔室就可以了，并让系统平衡30分钟，在37℃培养箱中。 </li><li>种子10 6细胞/200μl的在上部腔室（总细胞数），并培育在孵化器中的板4小时，在37℃。 </li><li>除去上部腔室，收集在下部腔室中的介质，并用1ml的PBS洗涤下部腔室一次。收集的PBS中的管。 </li><li>离心5分钟300 XG，丢弃在500μl的PBS上清，重悬。 </li><li>通过流式细胞术，计数采集的1分钟后的迁移细胞的数目。在壳体的分离的细胞群，没有必要添加任何表面抗体。查中的双维散点图考虑到细胞的侧散射和可视化在1分钟内，这是用于计算的数目的事件数的细胞数。 </li><li>计算迁移指数（MI）为: <img alt="式（1）" fo:content-width="3.5in" src="/files/ftp_upload/51942/51942eq1.jpg" /></li></ol> 4.2）附着力试验。此测定法允许测量所述细胞的粘附能力。一式三份进行检测。 <ol><li>预涂96 WELLS板（平底）用50μl/孔的任2％BSA-PBS（作为背景）或1微克ICAM-1的稀释于PBS中，过夜，在4℃。 </li><li>用PBS洗涤平板两次，并通过添加2％BSA-PBS（100μl/孔）1小时，在37℃下阻断非特异性位点。 </li><li>在完全RPMI同时悬浮细胞以5×10 6 / ml时，标记他们用1毫米CMFDA绿色电池跟踪器和孵化30分钟，在37℃。 </li><li>加入1×10 6个细胞/孔的预包被的孔中弃去封闭液（步骤4.2.2）后，孵育1小时，在37℃下。 </li><li>用酶标仪定量附着力（激发滤光片:485 nm，发射滤波器:535牛米）。在总输入（INPUT）进行第一次测量。 </li><li>用2％BSA-PBS轻轻洗涤该板（200μl/孔），4倍（倍）。每个清洗步骤（粘附倍）后进行板的读数。 </li><li>经过背景扣除每次测量，计算具体adhes离子（附着力）: <img alt="式（1）" fo:content-width="2in" src="/files/ftp_upload/51942/51942eq2.jpg" /></li></ol> 4.3）聚合试验。这是一个比色测定，量化的F-肌动蛋白聚合。一式三份进行检测。 <ol><li>重悬1×10 ^ 6个细胞于100μl预热的完全RPMI中，并添加特定的刺激（ 即 α-IgM的20微克/毫升）10分钟。 </li><li>停止用400μl的4％多聚甲醛的反应和固定的细胞在室温下10分钟。 </li><li>用PBS（离心机在300×g离心5分钟，4℃）洗涤3次。 </li><li>弃去上清液并通透细胞，在冰上的PBS-甙0.2％（200微升），持续2分钟。 </li><li>洗3次，用PBS-甙0.1％（离心机在100×g离心5分钟，4℃），弃于100μl的PBS-甙0.1％的上清，重悬。 </li><li>为30分钟，在室温吨染色用鬼笔环肽-Alexafluor 488（3微克/毫升）emperature在黑暗中。 </li><li>用PBS-0.1％皂苷洗3次。丢弃在300μL的PBS上清，重悬。 </li><li>量化的F-肌动蛋白的流动量术，计算所述鬼笔环肽的平均荧光的强度（MFI）。为了产生基于荧光强度的直方图中的单个维度的情节所产生的数据进行分析时，MFI值显示在图形报告。 </li><li>测量的F-肌动蛋白聚合（ΔF-肌动蛋白）为: <img alt="式（1）" fo:content-width="2in" src="/files/ftp_upload/51942/51942eq3.jpg" /></li></ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProFound:+an+expert+system+for+protein+identification+using+mass+spectrometric+peptide+mapping+information.">ProFound: an expert system for protein identification using mass spectrometric peptide mapping information.</a> Anal Chem. 72, 2482-2489 (2000).</li><li>Scielzo, C., 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=HS1+protein+is+differentially+expressed+in+chronic+lymphocytic+leukemia+patient+subsets+with+good+or+poor+prognoses.">HS1 protein is differentially expressed in chronic lymphocytic leukemia patient subsets with good or poor prognoses.</a> J Clin Invest. 115, 1644-1650 (2005).</li><li>Muzio, M., 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=HS1+complexes+with+cytoskeleton+adapters+in+normal+and+malignant+chronic+lymphocytic+leukemia+B+cells.">HS1 complexes with cytoskeleton adapters in normal and malignant chronic lymphocytic leukemia B cells.</a> Leukemia. 21 (9), 2067-2070 (2007).</li><li>Scielzo, C., 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=HS1+has+a+central+role+in+the+trafficking+and+homing+of+leukemic+B+cells.">HS1 has a central role in the trafficking and homing of leukemic B cells.</a> Blood. 116, 3537-3546 (2010).</li><li>Kashuba, E., 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=Proteomic+analysis+of+B-cell+receptor+signaling+in+chronic+lymphocytic+leukaemia+reveals+a+possible+role+for+kininogen.">Proteomic analysis of B-cell receptor signaling in chronic lymphocytic leukaemia reveals a possible role for kininogen.</a> J Proteomics. 91, 478-485 (2013).</li><li>Dhamoon, A. S., Kohn, E. C., Azad, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ongoing+evolution+of+proteomics+in+malignancy.">The ongoing evolution of proteomics in malignancy.</a> Drug Discov Today. 12, 700-708 (2007).</li><li>Ummanni, R., 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=Identification+of+clinically+relevant+protein+targets+in+prostate+cancer+with+2D-DIGE+coupled+mass+spectrometry+and+systems+biology+network+platform.">Identification of clinically relevant protein targets in prostate cancer with 2D-DIGE coupled mass spectrometry and systems biology network platform.</a> PLoS One. 6, (2011).</li></ol>

The authors have nothing to disclose.

<html>该原稿的目的在于描述一个优化的协议中涉及白血病的发病机制中的分子的识别，即使这种方法可以扩展到其它的疾病和/或其他类型的样品16-18。通过比较2亚CLL患者，良好与不良预后19的蛋白质组曲线，我们表明，它们在HS1的磷酸化状态的不同，它的激活影响白血病细胞的迁移和粘附能力。 相对于其他方法，在手稿，2DE凝胶和MS给出的蛋白质组技?...

慢性淋巴细胞白血病（CLL），在西方国家中最常见的成人白血病，来源于单克隆肿瘤CD5 + B淋巴细胞的积累和在临床上是非常不均匀的。它可以作为一个预白血病形式，定义为单克隆B细胞淋巴细胞增多（MBL）1,2,3。在其他情况下这种疾病可以出现任何与懒惰的临床过程，可以无需治疗前数年保持稳定，或作为一个渐进的chemorefractory疾病，预后极差，尽管治疗。最后，它可能会发展成一种经常致命的高恶性度淋巴瘤（里氏综合征-RS）2,3,4。患者通常被分为两个主要亚群:好的和坏的预后，根据一组预后因素，提供对疾病预后和生存预测因素的补充信息。临床异质性可能反映了生物异质性。许多基因，微环境和CEllular因素已被证明同意对疾病发病机理虽然没有统一的机制，已发现5。详细地说，一些研究已经表明，在疾病的临床过程的差别可以通过的具有预后价值6,7,8某些生物标记物的存在（或不存在）被部分地说明。这些数据表明，更好的了解该疾病的生物学可用于病理学的临床管理提供额外的提示，由两个预后标志物和治疗靶的鉴定。 本研究的目的是为了表明如何不同的蛋白质组技术的组合可用于参与CLL发作和进展的蛋白质的鉴定。我们的做法表明，它有可能联合起来基本蛋白质组学和临床资料5。 在本工作流程中，主从选择的患者的白血病细胞（良好与不良的预后）被分离和细胞裂解物，然后用于获得蛋白质的地图。 2DE允许的细胞群体，包括翻译后修饰的蛋白质分布图的特性，从而使每个蛋白的生物学活性的间接信息。全细胞裂解物通过基于等电点的第一维运行，随即通过上是基于其分子量解析蛋白质在聚丙烯酰胺凝胶上运行的第二尺寸。蛋白质组图谱进行比较分析允许差异表达的蛋白质的识别（无论是在数量和翻译后修饰而言）作为凝胶上的斑点可被切割并通过质谱法进行分析。每名候选人的角色可以由不同的试验来再利用。 这种方法中，只限于CLL在当前的手稿，可以很容易地扩展到其他疾病/样品，从而提供关于proteomi信息C /两组之间的生物学差异（ 即病理性与正常，刺激比未受刺激，野生型与击倒）。

<table><tbody><tr><td>Acetonitril</td><td>MERCK</td><td>61830025001730&amp;nbsp;</td><td></td></tr><tr><td>Ammonium Bicarbonate</td><td>SIGMA</td><td>A6141-500G</td><td></td></tr><tr><td>1,4-Dithioerythritol</td><td>SIGMA</td><td>D9680-5G</td><td></td></tr><tr><td>Iodoacetamide</td><td>SIGMA</td><td>I6125-25G</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>SIGMA</td><td>223506-25G</td><td></td></tr><tr><td>Trypsin, Sequencing Grade</td><td>ROCHE</td><td>11418475001</td><td></td></tr><tr><td>&amp;alpha;-cyano-4-hydroxycinnamic acid</td><td>SIGMA</td><td>C2020-10G</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>SIGMA</td><td>T6580</td><td></td></tr><tr><td>ZipTip&lt;sub&gt;&amp;micro;-C18&lt;/sub&gt;</td><td>MILLIPORE</td><td>ZTC18M096</td><td></td></tr><tr><td>RosetteSep Human B Cell Enrichment Cocktail</td><td>STEMCELL</td><td>15064</td><td></td></tr><tr><td>PBS</td><td>EUROCLONE</td><td>ECB4004L</td><td></td></tr><tr><td>FBS</td><td>DOMINIQUE DUTSCHER</td><td>S1810</td><td></td></tr><tr><td>Lymphoprep</td><td>SENTINEL DIAGNOSTICS</td><td>1114547</td><td></td></tr><tr><td>Trypan Blue</td><td>SIGMA</td><td>T8154</td><td></td></tr><tr><td>CD19</td><td>BECKMAN COULTER</td><td>082A07770</td><td>ECD</td></tr><tr><td>CD5</td><td>BECKMAN COULTER</td><td>082A07754</td><td>PC5</td></tr><tr><td>CD3</td><td>BECKMAN COULTER</td><td>082A07746</td><td>FITC</td></tr><tr><td>CD14</td><td>BD</td><td>345785</td><td>PE</td></tr><tr><td>CD16</td><td>BECKMAN COULTER</td><td>082A07767</td><td>PC5</td></tr><tr><td>CD56</td><td>BECKMAN COULTER</td><td>082A07789</td><td>PC5</td></tr><tr><td>Ettan IPGphor 3 Isoelectric Focusing Unit</td><td>GE-Healthcare</td><td>11-0033-64&amp;nbsp;</td><td></td></tr><tr><td>Urea</td><td>SIGMA</td><td>U6504</td><td></td></tr><tr><td>Tris-Hcl Buffer</td><td>BIO-RAD</td><td>161-0799</td><td></td></tr><tr><td>CHAPS</td><td>SIGMA</td><td>C2020-10G</td><td></td></tr><tr><td>DTT</td><td>SIGMA</td><td>D9163-5G</td><td></td></tr><tr><td>IPGstrips</td><td>GE-Healthcare</td><td>17-1233-01&amp;nbsp;</td><td>Linear pH 4-7 18cm</td></tr><tr><td>IPGbuffer</td><td>GE-Healthcare</td><td>17-6000-86</td><td>pH 4-7</td></tr><tr><td>Glycerol</td><td>SIGMA</td><td>G6279</td><td></td></tr><tr><td>PROTEAN II XL Cell</td><td>BIO-RAD</td><td>165-3188</td><td></td></tr><tr><td>SDS</td><td>INVITROGEN</td><td>NP0001</td><td></td></tr><tr><td>Acrilamide</td><td>BIO-RAD</td><td>161-0156</td><td></td></tr><tr><td>Agarosio</td><td>INVITROGEN</td><td>16500</td><td></td></tr><tr><td>Methanol</td><td>SIGMA</td><td>32213</td><td></td></tr><tr><td>Acetic Acid</td><td>VWR</td><td>631000</td><td></td></tr><tr><td>Formalin</td><td>BIO-OPTICAL</td><td>05-K01009</td><td></td></tr><tr><td>Ethanol</td><td>VWR</td><td>9832500</td><td></td></tr><tr><td>Sodium Thiosulfate</td><td>FLUKA</td><td>72049</td><td></td></tr><tr><td>Silver Nitrate</td><td>FLUKA</td><td>85228</td><td></td></tr><tr><td>Molecular Dynamics Personal SI Laser Densitometer</td><td>Amersham Biosciences</td><td></td><td></td></tr><tr><td>ImageMaster 2D Platinum 5.0</td><td>Amersham Biosciences</td><td></td><td></td></tr><tr><td>Sodium Carbonate</td><td>MERCK</td><td>A0250292</td><td></td></tr><tr><td>MALDI-TOF Voyager-DE STR</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>Data Explorer software (version 3.2)&amp;nbsp;</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>transwell chambre 6.6 mm diameter</td><td>CORNING</td><td>3421</td><td></td></tr><tr><td>RPMI</td><td>EUROCLONE</td><td>ECB2000L</td><td>WITH L-GLUTAMINE</td></tr><tr><td>Gentamicin</td><td>SIGMA</td><td>G1397</td><td></td></tr><tr><td>SDF-1</td><td>PREPROTECH</td><td>300-28A</td><td></td></tr><tr><td>Flat-bottom 96-well plate</td><td>BECTON DICKINSON</td><td>353072</td><td></td></tr><tr><td>BSA</td><td>SANTA CRUZ</td><td>9048-46-8</td><td></td></tr><tr><td>ICAM-1</td><td>R&amp;amp;D Systems</td><td>720-IC</td><td></td></tr><tr><td>CMFDA</td><td>Life Thechnology</td><td>C7025</td><td>Green</td></tr><tr><td>IgM</td><td>SOUTHERNBIOTECH</td><td>2020-02</td><td></td></tr><tr><td>Paraformaldehyde</td><td>SIGMA</td><td>P6148</td><td></td></tr><tr><td>Saponine</td><td>SIGMA</td><td>S-7900</td><td></td></tr><tr><td>Phalloidin&amp;nbsp;</td><td>Life Thechnology</td><td>A12379</td><td>Alexa fluor 488</td></tr></tbody></table>

from a 2de-gel spot to protein function: lesson learned from hs1 in chronic lymphocytic leukemia

参与肿瘤发生和发展的分子的鉴定是基本的理解疾病的生物学和，其结果是，对于患者的临床管理。在目前的工作中，我们将介绍对参与慢性淋巴细胞白血病（CLL）的进展分子识别的优化蛋白质组学的方法。具体而言，白血病细胞裂解液是由二维电泳（2DE）解决，如可视化的双向凝胶电泳凝胶"点"。蛋白质组图谱进行比较分析允许差异表达的蛋白质的鉴定（在数量和翻译后修饰而言），该被拾取，分离并鉴定的质谱（MS）。所识别的候选者的生物学功能可以通过不同的测定（ 即迁移，粘附和F-肌动蛋白聚合）进行测试，我们已经对原代白血病细胞中进行优化。

我们分离的原代白血病B细胞形成CLL患者的PB和我们分析蛋白质组学地图。样品（n = 104），根据每个患者的（坏的预后与预后良好），并进行双向凝胶电泳凝胶的对比分析，临床特征主要在两个子集进行分组。 允许的分析鉴定斑两个子集之间的差异表达的（在存在/不存在或移凝胶上的术语，意味着改变翻译后修饰; N≈16）。我们切下选定斑点从2DE凝胶和被还原和烷基化?...

Watch this Scientific Journal Video about From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia at JoVE.com

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

在这里，我们描述了一种协议，它把两个蛋白质组学技术，即2维电泳（2DE）和质谱（MS）来识别差异表达/翻译后修饰的蛋白质中的两个或多个基团主样品。这种做法，再加上功能性实验，允许预后标志物/治疗靶点的鉴定和表征。

从2DE凝胶点在蛋白质功能:在课慢性淋巴细胞白血病学到HS1

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

from-2de-gel-spot-to-protein-function-lesson-learned-from-hs1-chronic

Research

JoVE Journal

Medicine

13.7K 次观看.  IRCCS, San Raffaele Scientific Institute. 在这里，我们描述了一种协议，它把两个蛋白质组学技术，即2维电泳（2DE）和质谱（MS）来识别差异表达/翻译后修饰的蛋白质中的两个或多个基团主样品。这种做法，再加上功能性实验，允许预后标志物/治疗靶点的鉴定和表征。 

视频: From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically comprise less than 1% of the tumor mass. Material derived from bulk tumor contains tumor content at a concentration insufficient for characterization. Therefore, fluorescence activated cell sorting using eight antibodies, as well as side- and forward-scatter, is described here as a method of rapidly separating and concentrating with high purity thousands of HRS cells from the tumor for subsequent study. At the same time, because standard protocols for exome sequencing typically require 100-1,000 ng of input DNA, which is often too high, even with flow sorting, we also provide an optimized, low-input library construction protocol capable of producing high-quality data from as little as 10 ng of input DNA. This combination is capable of producing next-generation libraries suitable for hybridization capture of whole-exome baits or more focused targeted panels, as desired. Exome sequencing of the HRS cells, when compared against healthy intratumor T or B cells, can identify somatic alterations, including mutations, insertions and deletions, and copy number alterations. These findings elucidate the molecular biology of HRS cells and may reveal avenues for targeted drug treatments.

Here, we describe a combined flow cytometric cell sorting and low-input, next-generation library construction protocol designed to produce high-quality, whole-exome data from the Hodgkin Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (CHL).

经典霍奇金淋巴瘤的芦苇细胞的流分选和外显子测序

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically ...

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

Selecting Multiple Biomarker Subsets with Similarly Effective Binary Classification Performances

Biomarker detection is one of the more important biomedical questions for high-throughput &#39;omics&#39; researchers, and almost all existing biomarker detection algorithms generate one biomarker subset with the optimized performance measurement for a given dataset. However, a recent study demonstrated the existence of multiple biomarker subsets with similarly effective or even identical classification performances. This protocol presents a simple and straightforward methodology for detecting biomarker subsets with binary classification performances, better than a user-defined cutoff. The protocol consists of data preparation and loading, baseline information summarization, parameter tuning, biomarker screening, result visualization and interpretation, biomarker gene annotations, and result and visualization exportation at publication quality. The proposed biomarker screening strategy is intuitive and demonstrates a general rule for developing biomarker detection algorithms. A user-friendly graphical user interface (GUI) was developed using the programming language Python, allowing biomedical researchers to have direct access to their results. The source code and manual of kSolutionVis can be downloaded from http://www.healthinformaticslab.org/supp/resources.php.

Existing algorithms generate one solution for a biomarker detection dataset. This protocol demonstrates the existence of multiple similarly effective solutions and presents a user-friendly software to help biomedical researchers investigate their datasets for the proposed challenge. Computer scientists may also provide this feature in their biomarker detection algorithms.

用相似有效的二元分类性能选择多个生物标记子集

Biomarker detection is one of the more important biomedical questions for high-throughput &#39;omics&#39; researchers, and almost all existing biomarker ...

Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia: From Patient Material To Sequence Interpretation

During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a unique DNA sequence within each individual B cell. Since B cell malignancies result from the clonal expansion of a single cell, IG genes represent a unique molecular signature common to all the malignant cells within an individual patient; thus, IG gene rearrangements can be used as clonal markers. In addition to serving as an important clonal identifier, the IG gene sequence can act as a &#39;molecular timeline&#39; since it is associated with specific developmental stages and hence reflects the history of the B cell involved in the neoplastic transformation. Moreover, for certain malignancies, in particular chronic lymphocytic leukemia (CLL), the IG gene sequence holds prognostic and potentially predictive capabilities. That said, extrapolating meaningful conclusions from IG gene sequence analysis would be impossible if robust methods and tools were not available to aid in their analysis. This article, drawing on the vast experience of the European Research Initiative on CLL (ERIC), details the technical aspects and essential requirements necessary to ensure reliable and reproducible IG gene sequence analysis in CLL, a test that is now recommended for all CLL patients prior to treatment. More specifically, the various analytical stages are described ranging from the identification of the clonotypic IG gene rearrangement and the determination of the nucleotide sequence to the accurate clinical interpretation of the IG gene sequence data.

Herein, we present a protocol that details the technical aspects and essential requirements to ensure robust IG gene sequence analysis in patients with chronic lymphocytic leukemia (CLL), based on the accumulated experience of the European Research initiative on CLL (ERIC).

慢性淋巴细胞白血病免疫球蛋白基因序列分析: 从患者材料到序列解释

During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a ...

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 ...

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

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

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 ...

化学

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 ...

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

Traction Force Microscopy to Study B Lymphocyte Activation

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor. Gel rigidity, bead density, and protein functionalization must be optimized for the study of relatively small cells (~ 6 &#181;m) that interact with, and respond specifically to ligands for cell surface receptors.

Here we present a protocol used to perform traction force microscopy experiments on B cells. We describe the preparation of soft polyacrylamide gels and their functionalization, as well as data acquisition at the microscope and a summary of data analysis.

牵引力显微镜研究B淋巴细胞激活

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...

生物工程

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or mucosa and ensure humoral immunity. Due to their low frequency and location, the study of PCs is difficult in human. We reported a B to PC in vitro differentiation model using selected combinations of cytokines and activation molecules that allow to reproduce the sequential cell differentiation occurring in vivo. In this in vitro model, memory B cells (MBCs) will differentiate into pre-plasmablasts (prePBs), plasmablasts (PBs), early PCs and finally, into long-lived PCs, with a phenotype close to their counterparts in healthy individuals.&#160;We also built an open access bioinformatics tools to analyze the most prominent information from GEP data related to PC differentiation. These resources can be used to study human B to PC differentiation and in the current study, we investigated the gene expression regulation of epigenetic factors during human B to PC differentiation.

Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation model.

人正常记忆 b 细胞与长寿浆细胞体外分化模型

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...