这项研究得到了专业发展和研究的启动资金从大学，安大略省理工大学（UOIT）博士约翰·A.萨米和加拿大卫生研究院卫生专业学生研究奖伊琳娜·莱维特。作者承认香埃弗里特（教学及学习中心，UOIT）援助，为她准备的视频。作者承认博士迈克尔E. Nesheim（生物化学系，皇后大学，金斯顿，ON）为他的师友，洞察和有益的讨论。作者也承认郑博士福卓（罗尔德·达尔止血和血栓形成的中心，英国皇家利物浦大学医院，利物浦）提供的DIC病人等离子体研究中使用的。

1。 FV缺陷的人血浆制备<ol><li>此方法是最初Bloom 等人 4所描述的步骤的变形。 </li><li>获取整个人类的血液同意健康的成年志愿者（男或女，年龄18 - 50岁），未服用任何药物。戴乳胶手套和实验室外套，整个手术过程。 </li><li>制备100毫升的3.2％（重量/体积）的柠檬酸三钠在蒸馏水中。 6.0毫升该溶液中加载到单独的60毫升注射器。拆下空气郁闷柱塞仔细的6.0毫升的注射器上标记。 </li><li>使用酒精棉球清洁区之间的二头肌和前臂，肘正中静脉。连接20号蝴蝶装置3毫升注射器。蝴蝶针插入到肘正中静脉，轻轻画上柱塞，直到血液填充到3毫升标记。放弃与血液的注射器。执行此步骤，以消除任何组织因子在针从损坏的内皮细胞释放可激活凝血过程中的损伤部位和干扰的准备FV缺陷等离子。 </li><li>连接蝴蝶针60 ml注射器&#39;加载&#39;与6.0毫升的柠檬酸三钠和绘制轻轻地放在柱塞，直到血液到达的60毫升注射器上标记。 （决赛的柠檬酸浓度10.9毫米）。 </li><li>将注射器从蝴蝶针，轻轻地混合注射器，注射器上的插座，同时保持一个戴着手套的手指或拇指。 </li><li>继续抽血成60毫升注射器各填充有如上所述的6.0毫升的3.2％柠檬酸三钠。人们应该预期回收约50％的柠檬酸血液体积，离心后，需要每个样品50μl的FV-缺陷血浆中的微孔板检测血浆。我们的实验室常规处理300-420毫升血液含柠檬酸，在每次使用5-7×60毫升注射器6.0毫升3.2％柠檬酸钠/注射器。这是缺乏足够的FV-等离子约3,000微板试验（见下文）。 </li><li>志愿者的手臂，然后按删除蝴蝶针静脉穿刺部位用无菌纱布垫1.0分钟。量静脉穿刺部位用无菌绷带。 </li><li>在3,300 xg离心20分钟，在室温下离心柠檬酸血液。恢复黄河上游等离子层塑料烧杯中搅拌棒用塑料移液管小心不要打扰下红和白血细胞层。 </li><li>测量体积的血浆毫升，保留100μL血浆前冰EDTA的添加对未来的凝血酶原的血块的时间（PT）测试（见下文）。 </li><li>慢慢加入固体与温和的搅拌，在室温下实现5mM的EDTA枸橼酸血浆（终浓度）。一旦已经溶解的EDTA，pH调节到7.0，通过温和搅拌下滴加1.0M的NaOH。 </li><li>量烧杯中，用保鲜膜，并培育8-10小时无搅拌，在37°C。 </li><li>每2个小时，恢复100微升EDTA-处理过的血浆在冰上，并确定PT在孵化用EDTA的时间和比较血浆的PT之前到EDTA此外（见上文）。 </li><li> PT测定，地方可移动的8的良好immunomodule条到塑料模板的持有人，并插入酶标仪。 </li><li>在酶标仪东方immunomodule条:空，空，样品，样品，空，空，空，空由左到右的模板支架，以尽量减少光的散射干扰邻近的样品含带。此外，使用井2-7 8的好immunomodule条在每个从顶部向底部的塑料模板支架的边缘，以减少散射光的干扰。 </li><li> FV微孔板检测是能测量血纤维蛋白凝块的形成，在至少12个不同的样本，同时（6超过2 immunomodule条）样本每immunomodule条。 </li><li>使用多道移液器，加入50μl哈佛商学院（20MM HEPES，pH值7.4，150mM NaCl的）每个微孔板以及为每个待测定的样品。使用一个单一的吸移管中，添加50微升正常人体汇集参考血浆（NHP）或血浆加入EDTA之前或在培养不同时间后，加入EDTA至微孔板分开。 </li><li>使用多道移液器，加入50μl凝血活酶（请参阅下面的制备方法）的所有微孔板与血浆进行检测。酶标仪孵育在25℃下1分钟，程序酶标仪摇动板孵育1分钟15-25秒期间。 </li><li>使用酶标仪，设施SoftMax Pro软件微孔板的应用程序接口与计算机。设置读者的吸光度，波长405nm处，模式动力学，温度至25℃，和程序酶标仪动摇，持续5秒，和6分钟，在405nm处读取吸光度每5秒。 </li><li>使用多道移液器，添加氯化钙（50微升25mm的蒸馏水，2.1MM国际泳联升浓度），所有的微孔板，等待15秒，，微孔板的SoftMax Pro菜单，然后按"开始"图标，开始试验。 </li><li>确定点的吸光度随时间变化曲线中的SoftMax Pro的时间达到半最大增幅，在405nm处的吸光度（中点后产生的凝血活酶和氯化钙此外吸光度随时间变化曲线的S形。以下， 图1）。 </li><li>计算的FV 1阶段的活动从登陆血块时间（秒）与登陆FV活性（单位/ ml），如下图所示，在图2A中。对于定量的目的被定义为，1台的FV活性的活性存在于故意激活与加入凝血酶（见下文，与FV 1阶段和2阶段微孔板凝固分析含量测定人血浆样品）前1.0毫升NHP。 </li><li>温和搅拌下在蒸馏水中与含有0.15M NaCl的20mM HEPES4.0升做准备。调整pH值至7.4，缓慢dropw伊势，加入5.0M的NaOH（HBS，pH 7.4）中。 </li><li>获得足够的透析管的长度，并用蒸馏水彻底冲洗。管的一端打结。转让EDTA处理血浆透析管，用塑料移液管，排除空气，并在管的另一端打结。 </li><li>小心地挂在哈佛商学院的EDTA处理的等离子管，轻轻搅拌。量与保鲜膜，并在4℃下缓慢搅拌下的14 - 16小时透析</li><li>小心地取出EDTA-treated/dialyzed的血浆中15.0毫升螺旋盖管，并保留"最终"冰FV缺陷等离子PT测定的100微升3.0毫升等分。的FV-缺陷血浆储存在-80℃直至使用。在上述条件下，PT的增加，从10-15秒之前EDTA此外90-100秒8-10小时后EDTA此外。由这种方法制备的FV-缺陷血浆常规包含小于0.1％的FV的活性存在于起始新鲜人血浆。 </li></ol> 2。制备凝血活酶<ol><li>透析是必需的，以消除任何钙从该试剂中，以允许精确地定时从氯化钙时，除了启动凝血过程的纤维蛋白凝块的形成。 </li><li>制备100毫升和4.0升的20mM HEPES，0.15M NaCl的蒸馏水中，在缓慢搅拌下在塑料烧杯中。调节pH值至7.4，用5.0M NaOH（HBS，pH值7.4）中缓慢滴加。 </li><li>各瓶中加入6.0毫升，pH值7.4 HBS的凝血活酶试剂，更换盖子，轻轻摇晃溶解。孵育15分钟，在室温下干燥的材料溶解试剂封端的小瓶。轻轻振荡至完全溶解。 </li><li>切一个足够长的透析管，用蒸馏水冲洗，并配合管的一端与一个结。凝血活酶转移到透析管，排除空气，并配合另一端的管道与结。 </li><li>钙refully挂在哈佛商学院，pH值7.44.0升的透析管，轻轻搅拌均匀覆盖着保鲜膜在4°C为14-16小时。 </li><li> 3.0毫升等分试样的透析的凝血活酶转移至15毫升螺旋盖管和存储于-80℃直至使用。 </li></ol> 3。制备FV-微孔板凝固分析活动标准曲线<ol><li>解冻FV-缺陷血浆，NHP，和凝血活酶在37℃下10分钟。轻轻地混合血浆和凝血活酶缺乏FV-独立，转移到塑料清洗托盘分开，并在冰上孵育。请将NHP上冰后轻轻搅拌。商店氯化钙（在蒸馏水中的25mM）在一个单独的洗涤，在室温下的托盘。 </li><li>准备各200微升2倍系列稀释液，NHP从0 - ，2 - ，4 - ，8 - ，16 - ，32 - ，64 - ，128 - ，256 - ，512倍，在HBS中，pH值7.4用1.5 ml微量离心管。涡和冰上孵育。 </li><li>将可移动的8个很好immunomodule条到plastic模板支架，并插入酶标仪。 </li><li>在酶标仪东方immunomodule条:空，空，样品，样品，空，空，空，空由左到右的模板支架，以尽量减少光的散射干扰邻近的样品含带。此外，使用井2-7 8的好immunomodule条在每个从顶部向底部的塑料模板支架的边缘，以减少散射光的干扰。 </li><li> FV微孔板检测是能测量血纤维蛋白凝块的形成，在至少12个不同的样本，同时（6超过2 immunomodule条）样本每immunomodule条。 </li><li>打开酶标仪访问的SoftMax Pro微孔板应用软件。 </li><li>使用多通道移液管，使FV-缺陷血浆（50微升）的同时增加到所有样本微孔板。使用一个单一的移液管中，加入50μl的10系列稀释液制备的NHP上述分离微孔板。</li><li>使用多道移液器，添加促凝血酶原激酶（50微升）的所有样品井。酶标仪在在25℃下孵育1分钟，程序酶标仪在15-25秒的孵育1分钟，摇动板。 </li><li>使用酶标仪，设施SoftMax Pro软件微孔板的应用程序接口与计算机。设置读者吸光度，波长405nm处，模式动力学，温度为25°C，和程序的酶标仪振摇在第5秒后添加氯化钙，并且在405nm处测量吸光度每5秒后的6分钟。 5秒的振荡间隔，不包含在所计算的凝块时间（见下文）。 </li><li>使用多道移液器，添加氯化钙（50μL蒸馏水为25mm;终浓度为3.5毫米），所有样品微孔板，等待15秒，按"开始"图标，在微孔板的SoftMax Pro的应用程序从计算机开始法。 </li><li> Ţ他血块倍的时间，以秒计算的促凝血酶原激酶和氯化钙的添加后，在405nm处达到的中点之间的最小和最大吸光度。 </li><li>在405nm处的吸光度的增加速率（mUnits /分钟），包围第1凝块形成的5个时间点的吸光度与时间的关系曲线的线性部分计算为凝块形成的初始速度。 </li><li>凝块形成的程度被计算为在405nm处（单位）的最大和最小之间的吸光度的差异的凝块形成事件期间取得。 </li></ol> 4。分析人体血浆样本FV 1级和2级微孔板凝固分析<ol><li>解冻FV-缺陷血浆，NHP，样品的等离子体，并促凝血酶原激酶，在37℃下10分钟。轻轻地混合血浆和凝血活酶缺乏FV-独立，转移到单独的塑料ELISA清洗托盘，和在冰上培育。存储NHP和样品等离子体冰后轻轻搅拌。商店氯化钙（在蒸馏水中的25mM）在一个单独的ELISA洗涤，在室温下的托盘。 </li><li>准备NHP和样品的等离子体在HBS中，pH为7.4，使用1.5毫升微量离心管中的40倍稀释液各200微升。涡和冰上孵育。 </li><li>将可移动8的良好immunomodule条塑料模板支架插入酶标仪。 </li><li>的备用空，空，样品，空，空，样品，空，空immunomodule的条由左到右的模板支架，以尽量减少光的散射干扰邻近的样品含带。只能使用2-7井从顶部底部在每个immunomodule条，以尽量减少光的散射干扰的塑料模板支架的边缘。 </li><li>打开酶标仪访问的SoftMax Pro微孔板应用软件。 </li><li>使用多道移液器，FV-缺陷血浆（50微升）添加到所有样本微孔板。使用SINGLË移液管中，加入50μl的40倍稀释的NHP或样品等离子体上述制备分离微孔板。 </li><li>使用多道移液器，添加促凝血酶原激酶（50微升）的所有样品井。酶标仪在在25℃下孵育1分钟，程序酶标仪在15-25秒的孵育1分钟，摇动板。 </li><li>使用酶标仪，设施SoftMax Pro软件微孔板的应用程序接口与计算机。设置读者吸光度，波长405nm处，模式动力学，温度为25°C，和程序的酶标仪振摇在第5秒后添加氯化钙，并且在405nm处测量吸光度每5秒后的6分钟。 5秒的振荡间隔，不包含在所计算的凝块时间（见下文）。 </li><li>使用多道移液器，添加氯化钙（50微升为25mm，在蒸馏水的6.25mM最终浓度）的所有样品微孔板，并立即公关ESS的"开始"图标，在微孔板的SoftMax Pro的应用程序从计算机开始检测。 </li><li>在运行结束时，确定的时间，初始从吸光率随时间的公司中的SoftMax Pro中稀释40倍的NHP和样品的等离子体的凝块形成的速率和程度。 </li><li>考虑到40倍稀释，使用时间为每个样品的血块，计算FV活动的单位/ ml的FV 1阶段的活动日志血块时间与登录的FV活动（ 图2A）的标准曲线。这代表了FV-1阶段的活动，如果没有"故意"激活凝血酶。 </li><li> FVA 1，与凝血酶的加法和孵化后的第二检测由于FV被激活与凝血酶的活性的辅因子，是至关重要的精确测量的FV 2级的血浆样品的活性（随着"有意添加凝血酶激活）。 </li><li> FV 2阶段的试验，准备200-300μl的纯化凝血酶18一哈佛商学院，pH值7.4吨100nm的孵化冰。计划使用10微升稀释的凝血酶与FV 2级测定待测定每个血浆样品。 </li><li>稀释120微升上述NHP和样品的等离子体从40倍到100倍，用170微升，pH值7.4的HBS含有2.8MM氯化钙。加入10μl的凝血酶（10nm的最终浓度），各样品。涡混合，并在37°C孵育1分钟。 </li><li> NHP和样品等离子体稀释至500倍，加入400微升的HBS，pH值7.4，涡流以及，测定各血浆样品的50微升的FV 1阶段微孔板法如上所述。 </li><li>确定NHP和各血浆样品测定的吸光度随时间变化曲线中的SoftMax Pro的血液凝块的时间。考虑到500倍稀释液，使用血液凝块的时间为每个样本计算FV 2阶段活动的FV 1级标准曲线的日志血块时间与登录FV活动的的（ 图2A）。 </li><li>总FV活动然后可被计算为FV 2级活动 - FV 1阶段活动性。 </li></ol>

<ol>
	<li>Orfeo, T., Brufatto, N., Nesheim, M. E., Xu, H., Butenas, S., Mann, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+factor+V+activation+paradox.">The factor V activation paradox.</a> J. Biol. Chem. 279, 19580-19591 (2004).</li><li>Bukys, M. A., Blum, M. A., Kim, P. Y., Brufatto, N., Nesheim, M. E., Kalafatis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+factor+Va+into+prothrombinase+is+required+for+coordinated+cleavage+of+prothrombin+by+factor+Xa.">Incorporation of factor Va into prothrombinase is required for coordinated cleavage of prothrombin by factor Xa.</a> J. Biol. Chem. 280, 27393-273401 (2005).</li><li>Nesheim, M. E., Katzmann, J. A., Tracy, P. B., Mann, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factor+V.">Factor V.</a> Methods. Enzymol. 80, 249-274 (1981).</li><li>Bloom, J. W., Nesheim, M. E., Mann, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+technique+for+the+preparation+of+factor+V+deficient+plasma.">A rapid technique for the preparation of factor V deficient plasma.</a> Thromb. Res. 15, 595-599 (1979).</li><li>Samis, J. A., Stewart, K. A., Nesheim, M. E., Taylor, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factor+V+cleavage+and+inactivation+are+temporally+associated+with+elevated+elastase+during+experimental+sepsis.">Factor V cleavage and inactivation are temporally associated with elevated elastase during experimental sepsis.</a> J. Thromb. Haemost. 5, 2559-2561 (2007).</li><li>Samis, J. A., Stewart, K. A., Toh, C. H., Day, A., Downey, C., Nesheim, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+changes+in+factors+associated+with+neutrophil+elastase+and+coagulation+in+intensive+care+patients+with+a+biphasic+waveform+and+disseminated+intravascular+coagulation.">Temporal changes in factors associated with neutrophil elastase and coagulation in intensive care patients with a biphasic waveform and disseminated intravascular coagulation.</a> J. Thromb. Haemost. 2, 1535-1544 (2004).</li><li>Cutler, J. A., Patel, R., Rangarajan, S., Tait, R. C., Mitchell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+11+novel+mutations+in+patients+with+heterozygous+and+homozygous+FV+deficiency.">Molecular characterization of 11 novel mutations in patients with heterozygous and homozygous FV deficiency.</a> Haemophilia. 16, 937-942 (2010).</li><li>Ihara, M., Suzuki, T., Kobayashi, N., Goto, J., Ueda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-sandwich+enzyme+immunoassay+for+one-step+noncompetitive+detection+of+corticosteroid+11-deoxycortisol.">Open-sandwich enzyme immunoassay for one-step noncompetitive detection of corticosteroid 11-deoxycortisol.</a> Anal. Chem. 81, 8298-8304 (2009).</li><li>Janke, R., Genzel, Y., Wahl, A., Reichl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+key+metabolic+enzyme+activities+in+mammalian+cells+using+rapid+and+sensitive+microplate-based+assays.">Measurement of key metabolic enzyme activities in mammalian cells using rapid and sensitive microplate-based assays.</a> Biotechnol. Bioeng. 107, 566-581 (2010).</li><li>Beebe, D. P., Aronson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+fibrinolytic+assay+performed+in+microtiter+plates.">An automated fibrinolytic assay performed in microtiter plates.</a> Thromb. Res. 47, 123-128 (1987).</li><li>Frantantoni, J. C., Poindexter, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+platelet+aggregation+with+microplate+reader.+A+new+technical+approach+to+platelet+aggregation+studies.">Measuring platelet aggregation with microplate reader. A new technical approach to platelet aggregation studies.</a> Am. J. Clin. Pathol. 94, 613-617 (1990).</li><li>Pratt, C. W., Monroe, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplate+coagulation+assays.">Microplate coagulation assays.</a> Biotechniques. 13, 430-433 (1992).</li><li>Tilley, D., Levit, I., Samis, J. A. <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+microplate+coagulation+assay+for+Factor+V+in+human+plasma.">Development of a microplate coagulation assay for Factor V in human plasma.</a> Thromb. J. 9, 11-16 (2011).</li><li>Toh, C. H., Downey, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Back+to+the+future:+Testing+in+disseminated+intravascular+coagulation.">Back to the future: Testing in disseminated intravascular coagulation.</a> Blood Coagul. Fibrinolysis. 16, 535-542 (2005).</li><li>Spero, J. A., Lewis, J. H., Hasiba, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disseminated+intravascular+coagulation:+Findings+in+346+patients.">Disseminated intravascular coagulation: Findings in 346 patients.</a> Thromb. Haemost. 43, 28-33 (1980).</li><li>Tracy, P. B., Eide, L. L., Bowie, E. J., Mann, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioimmunoassay+of+factor+V+in+human+plasma+and+platelets.">Radioimmunoassay of factor V in human plasma and platelets.</a> Blood. 60, 59-63 (1982).</li><li>Krishnaswamy, S., Nesheim, M. E., Pryzdial, E. L. G., Mann, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+prothrombinase+complex.">Assembly of prothrombinase complex.</a> Meths. Enzymol. 222 (Part A), 261-280 (1993).</li><li>Bajzar, L., Manuel, R., Nesheim, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+TAFI,+a+thrombin-activable+fibrinolysis+inhibitor.">Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor.</a> J. Biol. Chem. 270, 14477-14784 (1995).</li><li>Mammen, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disseminated+intravascular+coagulation+(DIC).">Disseminated intravascular coagulation (DIC).</a> Clin. Lab. Sci. 13, 239-2345 (2000).</li><li>Dahlb&#228;ck, B., Carlsson, M., Svensson, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Familial+thrombophilia+due+to+a+previously+unrecognized+mechanism+characterized+by+poor+anticoagulant+response+to+activated+protein+C:+prediction+of+a+cofactor+to+activated+protein+C.">Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C.</a> PNAS. 90, 1004-108 (1993).</li><li>Dahlb&#228;ck, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+understanding+pathogenic+mechanisms+of+thrombophilic+disorders.">Advances in understanding pathogenic mechanisms of thrombophilic disorders.</a> Blood. 112, 19-27 (2008).</li></ol>

没有利益冲突的声明。

动力学微孔板测定方法概述FV凝血活性的测量的人血浆样品中，没有（FV 1阶段测定）或一种新型的，快速，廉价，和方便的技术的发展已被有意地激活与增值凝血酶（FV 2阶段的测定）。该试验利用动能酶标仪和所有所需的原料和试剂是市售的，或者可以由在内部。该试验连续监测:在405nm处的血纤维蛋白凝块的形成过程中的等离子体中的光散射的变化。微孔板法使用小的血浆样品体积（μl）的，具有的优点是适合用于分析多个样品的同时（最多12个）。这些测定属性是有利的，当使用昂贵的设备和试剂（自动分析仪和因子缺陷等离子体）;只有小样品体积可用（病人等离子体）或分析大量的样品（在FV纯化期间from的血浆）。 FV微孔板法具有额外的优点，即它是方便，快捷，并且不需要分离和纯化所需的构成成分。的FV微孔板法具有与手动倾斜管3，4和自动化已报道的5-7 FV检测相比，比较有用的范围凝块的时间（25-75秒）和相应的FV样品中的水平（0.5-0.005单位/毫升），和灵敏度/检测限（20-80pM）。 FV微孔板法与手动倾斜管FV检测遭受从主观视觉评估凝块形成3，4的相比，允许客观和定量测量的凝块的时间，初始速率和程度的血纤维蛋白凝块的形成。与遭受的要求，昂贵的分析仪及试剂5-7的自动化的FV分析，比较，FV微孔板检测试剂和材料价格便宜，可能是选购电脑所需的所有材料sed的商业或内部。手动倾斜管3，4和自动化5-7 FV检测一般只提供凝块形成的时间;不在的时候，初始速率和程度都准确地测定分光光度法与FV微孔板法的血纤维蛋白凝块的形成。最后，手动倾斜管3,4和自动化5-7 FV检测患有唯一能测量血浆样品一次的的FV活动中;，而FV微孔板法是适合于高吞吐量和12个样品可能同时分析。 微孔板法是用来证明，在9 DIC病人等离子体的FV是较不活跃因为延迟血块的时间和较低的初始速度在FV 1阶段测定的凝块形成的组合中NHP。从NHP FV 2阶段试验在DIC病人等离子体血凝块形成的初始速度没有改变。在DIC患者年龄血凝块形成的程度吨等离子电视也没有改变NHP FV 1级和2级检测。减少FV 1级，2级，和总的活动可能是由于增加FV消费19和/或失活6按照与其他研究在此取得的血液疾病的发病机制。鉴于在DIC等离子体凝块形成的程度是相同的观察NHP，此变量是最有可能的FV-缺陷血浆中的纤维蛋白原浓度的结果，而不是相关的病人的等离子体的特性。 从微孔板检测的结果表明，FV的人血浆样品中的活性的测量可以得到从时间和初始从FV 1阶段测定凝块形成速率和凝块形成的时间和总活性从FV 2 - 阶段试验。这是不可能的，以获得定量测定血浆样品中的FV活性凝块形成的程度，在t的基础上他FV 1 - 和2 - 阶段实验或凝块形成的FV 2阶段的测定中的初始速率。鉴于所有的反应达到约相同程度的凝块形成为0.35 - 0.45单位之间的初始吸光度前和后凝血活酶的最大吸光度和氯化钙此外，测量凝块形成的程度，在不提供一个定量的评估的FV活性给定的血浆样品。 新颖的微孔板法会发现及其从人类和动物的血浆中的纯化的人类血浆和组分在样品中的FV活性的测量使用在研究和临床设置。微孔板法可用于测量的时间，初始的凝块形成的速率和程度;参数一般不同时监控在手动倾斜管检测和自动凝血分析仪。在血块形成事件信息提供了更多的参与FV一个完整的定量评估人血浆中比以前已经3-7。此信息是特别重要的，在研究和临床实验室测量时，显着变化在FV的血浆样品中的活性或与纯化的FV或FVA。 FV微孔板法也可用于表征和测量的化合物，可激活或灭活FV和FVA，静脉血栓形成的风险，测量患者的FV活性FV Leiden突变的结果为20，21，并监测的活性FV在其从血浆中提纯。 FV微孔板法是很容易适应测量外在的，内在的，或共同途径凝血因子的活性的任何使用适当的因子-缺陷血浆和发起了血纤维蛋白形成的试剂。该法将提高我们的的FV生物化学的了解，通过更准确，更完整的测量，其活动的研究和临床实验室。最终LY，此信息将产生积极影响医疗环境中，通过更有效的治疗患有凝血功能障碍，如DIC的早期诊断和发展。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>动力学酶标仪</td><td> Molecular Devices公司</td><td>光谱最大190 </td><td> SOFTMAX PRO 4.3 LS软件</td></tr><tr><td>普通合并的人参考血浆</td><td>精密生物</td><td> CCN-10 </td><td></td></tr><tr><td>柠檬酸钠</td><td>贝克顿迪肯森</td><td> B10242 </td><td></td></tr><tr><td> EDTA </td><td>贝克顿迪肯森</td><td> B10093 </td><td></td></tr><tr><td> FV缺陷的人血浆4 </td><td></td><td></td><td>由新鲜人血浆和存储以等分试样在-70℃。 </td></tr><tr><td> 3和60ml注射器</td><td>贝克顿迪肯森</td><td> 309585和136898 </td><td></td></tr><tr><td>蝴蝶设备</td><td>真空采血管</td><td> 367251 </td><td> 20号</td></tr><tr><td> HEPES </td><td>适马/爱秩序</td><td> H-3375 </td><td></td></tr><tr><td>氯化钠</td><td>费舍尔</td><td> BP358-212 </td><td></td></tr><tr><td>凝血活酶</td><td>三位一体生物技术</td><td> T1106 </td><td>透析过的与HBS，pH 7.4和存储中的等分试样在-70℃下</td></tr><tr><td>氯化钙</td><td>贝克顿迪肯森</td><td> B10070 </td><td></td></tr><tr><td>人凝血酶15 </td><td></td><td></td><td>从人血浆中，储存在-20℃下在50％（体积/体积）甘油。 </td></tr><tr><td>透析膜</td><td>费舍尔</td><td> 21-152-6 </td><td> 6 8kDa截留分子量;为50mm x3米。 </td></tr><tr><td>模板支架和可移动的8孔条。 </td><td> Nunc公司</td><td> 14-245-63和469957 </td><td></td></tr><tr><td> ELISA清洗托盘</td><td> BioRad公司</td><td> 224-4872 </td><td></td></tr><tr><td> 1.5 ml离心和15毫升螺旋盖管。 </td><td>萨尔斯塔特</td><td> 72690和62554205 </td><td></td></tr><tr><td>多道移液器</td><td>费舍尔</td><td> 2703630 </td><td> 8信道模型。 </td></tr></tbody></table>

measurement of factor v activity in human plasma using a microplate coagulation assay

以响应于损伤，血液凝固被激活，并在产生凝血蛋白酶，凝血酶的结果。的凝血酶裂解纤维蛋白原，纤维蛋白形成不溶性凝块，停止出血。其活化形式，FVA，凝血因子V（FV）是一个重要的辅助因子的蛋白酶的FXa和加速器的凝血酶生成期间为凝血酶原酶 1，2的一部分的纤维蛋白凝块的形成。手册FV检测已被描述3，4，但它们是费时的和主观的。自动FV检测已报道5-7，但分析仪和试剂是昂贵的，一般只提供血块的时间，而不是纤维蛋白形成的速率和程度。微孔板平台优选酶催化的事件，这是因为测量的方便，时间，成本，体积小，连续监测，和高吞吐量的8，9。已报告的血块溶解，血小板聚集微孔板检测11，凝血因子12，但不适用于人体血浆中的FV活动。我们的目标的方法是开发一个微孔板检测人血浆中纤维蛋白的​​形成过程中，测量FV活动。 这种新颖的的微孔板方法，勾勒出简单，价格低廉，快速检测人血浆中的FV活动。该试验利用动能酶标仪在人血浆中的纤维蛋白的形成（ 图1）13 中，以监测在405nm处的吸光度变化。该检测精确测量的时间，初始速率和程度的血纤维蛋白凝块的形成。它仅需要微升量的血浆，在6分钟完成，具有高吞吐量，是敏感24-80pM FV，和测量的量无意中激活（1阶段的活性）和凝血酶活化的FV（2级活动），以获得一个完整的评估，其总的功能活性（2级活动 - 1阶段的活动）。 弥漫性intravas所辖内凝血（DIC）是一种获得性凝血功能障碍，最常见的开发从已存在的感染14。 DIC以上的预现有的病理与预后差，增加死亡率。检测显示，9例DIC，FV 1级，2级，总活动减少，平均而言，54％，44％，42％，分别比正常汇集人类参考血浆（NHP）。 FV微孔板法测量任何凝血因子的活性是很容易适应。该法将提高我们的的FV生物化学的了解，通过更准确，更完整的测量，其活动的研究和临床。这些信息将医疗环境，通过更有效的治疗凝血功能障碍，如DIC的早期诊断和发展产生积极的影响。

代表性的成果 - 1级FV微孔板凝血试验活动的标准曲线的制备在FV随着时间的推移产生的酶标仪测定血纤维蛋白凝块形成的代表性例子在图1中示出。的FV测定精确测量的时间，初始速率和程度的血纤维蛋白凝块的形成。检查井试验完成后出现凝块的形成。所有的反应达到了血块的形成大致相同的程度，一般为0.35在405nm处的吸光度变化 - 0.45单位之间的起始吸光度之前和促凝血酶原激酶和氯化钙的除了最大后的吸光度。典型的例子FV 1级活动标准曲线的日志血块时间（以秒为单位）与登录FV活动（单位/毫升），并登录初始凝块形成速率（在mUnits /分）与登录FV活动（单位/毫升NHP系列稀释液）中示出<strong&gt;的图2A和图2B中 ，分别拟合log-log图凝块时间与FV 1活动表明了牢固的合作关系，这些变量之间的线性回归分析（ 图2A，R 2 = 0.980）。拟合log-log图的血块形成与FV活动的的初始速度也表明了牢固的合作关系后，这些变量之间的线性回归分析（ 图2B，R 2 = 0.983）。保持线性关系的两个日志凝块时间和登录血凝块形成的初始速率与登录FV活动NHP稀释到512倍。由于FV循环NHP在约12-40nm的16，微孔板检测是敏感，约24 80pM FV NHP的。 FV微孔板法鉴于FVA-FXa的-类脂在凝血酶原酶的相互作用的解离常数是约1nm的17，是完全适合的中的FV电平的测量的生理relevan吨纳米范围内为FVA功能凝血酶原。 使用FV微孔板检测，也有人FV 1级活性测定15名健康控制等离子体（男性和女性，年龄18 - 20yrs）的FV活动中的正常范围为（平均值±标准偏差范围）: 0.96±0.14U/ml; 0.68-1.11U/ml。同意与正常健康的FV活动和的范围（0.66-1.14U/ml）Cutler 等人 。 FV 1级活动自动分析仪7。批内变异的时间，程度和血凝块形成FV 1阶段试验在不同的8天6口井的初始速度分别为3.4％，4.4％和3.1％。批间变异的时间，程度，FV 1阶段试验在8个不同的实验，不同的8天6口井的血凝块形成的初始速度分别为7.1％，7.8％和9.2％。因此，这三个measu内和批间变异红色的变量是在一个可接受水平之内和之间微孔板同时检测多个样品（最多12个）的强大的检测性能。 代表性的成果 - 分析人体血浆样本FV 1级和2级微孔板凝固分析日志血块时间与日志FV活动的标准曲线（ 图2A）是用来衡量NHP 9 DIC病人等离子体不是故意激活增值凝血酶（FV 1级活动），或故意激活FV活动加入凝血酶（FV的2级活动），其结果示于表1中。全部9 DIC病人等离子体表现出FV 1阶段的活动和血凝块形成的初始速度平均分别下降54％和18％，分别从NHP。在DIC患者FV 1阶段试验的血凝块形成的程度没有很大的不同，NHP，平均增加了13％，从NHP。 <html> NHP与凝血酶的激活产生的近似8倍的增长，在FV的FV 1阶段的活性（表1）以上的2 - 阶段活动。这表明，在NHP的公允价值，主要是在未激活的形式存在，并同意与先前报告的结果，使用手动倾斜管FV分析3，4，自动FV分析5-7。在DIC患者的平均FV 2级和总活性也下降，由44％和42％，分别从NHP。初始在FV 2 - 阶段检测在DIC患者的凝块形成的速率以及程度，没有显着不同，NHP，平均变化，由约9％和4％，分别为，从所观察到的与NHP。这些结果表明，相对于在较长的时间内，在NHP FV，FV的DIC病人等离子体中产生的纤维蛋白凝块的形成率下降，患者FV平均而言，只有56％的激活凝血酶。 ENT"FO:保持together.within"页面="总是"&gt; <img src="/files/ftp_upload/3822/3822fig1.jpg" alt="图1" /> 图1在正常混合人与动能微孔板FV 1阶段的凝固分析测得的参考血浆的血凝块形成。使用动能酶标仪在405nm的纤维蛋白凝块的形成NHP连续监测。该地块是6分钟反应的32倍稀释的NHP，FV-缺陷血浆，促凝血酶原激酶，和氯化钙的微孔板以及酶标仪输出。垂直轴表示作为一个结果，在血浆中的纤维蛋白的​​形成发生在405nm处的吸光度变化。血纤维蛋白形成的时间的时间到达的半最大光吸收增加，或曲线的中点（36.40秒）被定义为。最初的凝块形成速率被定义为在405nm处的吸光度的变化率在所述第一5个时间点的曲线的吸光度部（611.88 mUnits /分钟）的线性增加。这位前被定义为在405nm处（0.35单位）的最大值和最小值之间的吸光度的差异帐篷凝块形成。 <img src="/files/ftp_upload/3822/3822fig2.jpg" alt="图2" /> 图2。凝块形成对比使用的FV 1阶段微孔板法在正常汇集人参考血浆因子V活性的时间和初始速率的标准曲线。 NHP连续稀释（0 - 在HBS 512倍），在协议中所描述的FV 1阶段微孔板法测定。登陆登陆地块线性回归建模后的面板中的数据A和B，分别显示的时间和初始速度，在1阶段的FV微孔板法凝块形成对FV活性。 <table border="1"><tbody><tr><td> 样品 </td><td> 1级测定活动（单位/ ml） </td><td> 1级检测范围（单位） </td><tD&gt; 1阶段测定初始评估（mUnits /分钟） </td><td> 2级测定活（单位/ ml） </td><td> 2级检测范围（单位） </td><td> 2级测定初始评估（mUnits /分钟） </td><td> 总活性（单位/ ml） </td></tr><tr><td> NHP </td><td> 1.02 </td><td> 0.363 </td><td> 744.96 </td><td> 7.93 </td><td> 0.433 </td><td> 375.84 </td><td> 6.91 </td></tr><tr><td> 患者1 </td><td> 0.36 </td><td> 0.404 </td><td> 583.80 </td><td> 3.84 </td><td> 0.432 </td><td> 249.96 </td><td> 3.48 </td></tr><tr><td> 患者2 </td><td> 0.67 </td><td> 0.416 </td><td> 704.04 </td><td> 5.28 </td><td> 0.469 </td><td> 323.16 </td><td> 4.61 </td></tr><tr><td><strong&gt;患者3 </td><td> 0.31 </td><td> 0.435 </td><td> 562.08 </td><td> 3.92 </td><td> 0.453 </td><td> 294.96 </td><td> 3.61 </td></tr><tr><td> 患者4 </td><td> 0.39 </td><td> 0.435 </td><td> 617.04 </td><td> 4.14 </td><td> 0.462 </td><td> 372.60 </td><td> 3.75 </td></tr><tr><td> 患者5 </td><td> 0.73 </td><td> 0.401 </td><td> 641.40 </td><td> 5.91 </td><td> 0.433 </td><td> 354.24 </td><td> 5.18 </td></tr><tr><td> 患者6 </td><td> 0.45 </td><td> 0.403 </td><td> 600.72 </td><td> 4.16 </td><td> 0.445 </td><td> 393.96 </td><td> 3.71 </td></tr><tr><td> 患者7 </td><td> 0.49 </td><td> 0.395 </td><td> 575.40 </td><td> 4.89 </td><td> 0.449 </td><td> 357.48 </td><td> 4.40 </td></tr><tr><td> 患者8 </td><td> 0.19 </td><td> 0.448 </td><td> 489.00 </td><td> 2.89 </td><td> 0.450 </td><td> 330.48 </td><td> 2.70 </td></tr><tr><td> 患者9 </td><td> 0.64 </td><td> 0.423 </td><td> 699.48 </td><td> 5.11 </td><td> 0.455 </td><td> 417.24 </td><td> 4.47 </td></tr></tbody></table> 表1 FV活动NHP和开发弥散性血管内凝血（DIC）9例 FV 1级，2级和NHP与DIC病人等离子体9的总活性测定FV 1阶段微孔板检测标准。曲线对比FV活性（ 图2A）的凝块形成的时间，并且给定的单位/ ml。初始速度（初始速率增加A405nm前五个时间点在mUnits /分钟）和程度（最大A405nm - 最少A40为5nm）的凝块形成的FV是1 - 和2 - 阶段微孔板法中也示。

Watch this Scientific Journal Video about Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay at JoVE.com

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

本研究描述了一种新颖的微孔板法测量FV凝血活性在人血浆中的纤维蛋白凝块的形成还没有被以前的报告。该方法使用一个动能酶标仪人血浆中的纤维蛋白凝块的形成过程中，连续地测量在405nm处的吸光度变化。

测量人体血浆中的凝血因子V活性，用酶联凝固分析

 In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin ...

measurement-factor-v-activity-human-plasma-using-microplate

Research

JoVE Journal

Immunology and Infection

18.9K 次观看.  University of Ontario Institute of Technology. 本研究描述了一种新颖的微孔板法测量FV凝血活性在人血浆中的纤维蛋白凝块的形成还没有被以前的报告。该方法使用一个动能酶标仪人血浆中的纤维蛋白凝块的形成过程中，连续地测量在405nm处的吸光度变化。

视频: Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

科研

免疫与感染

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

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

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

人外周血NK细胞的扩展，纯化和功能评估

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

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

荧<em在原位</em&gt;蚊虫的有丝分裂染色体上的杂交

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

微孔板的检测，以评估RBL-2H3肥大细胞脱颗粒:三氯生的影响不使用有机溶剂的化学效应

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

激活和NLRP3炎性活性的人单核细胞来源的树突状细胞用IL-1β测量

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

巨噬细胞集落刺激因子分化的人单核细胞衍生的巨噬细胞的培养

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

人体血液中测粒及单核细胞吞噬功能和呼吸爆发活动

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

测量<em&gt;体外</em&gt; HIV-1 Preintegration配合物的整合活动

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...