Name: high yield purification of plasmodium falciparum merozoites for use in opsonizing antibody assays
Uploaded: 2014-07-17T15:00:00.000+00:00
Duration: 10 min 38 s
Description: Watch this Scientific Journal Video about High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays at JoVE.com

作者要承认儿童和成人血浆者，工作人员在医学研究巴布亚新几内亚研究所。作者要感谢阿曼迪妮乙Carmagnac，凯瑟琳Q聂，丹尼·W·威尔逊，伊沃·穆勒和戴安娜S汉森他们对这项技术的发展作出的贡献，并感谢澳大利亚红十字会血液和血清包。这项工作成为可能，通过维州政府运营基础设施的支撑和澳大利亚政府NHMRC IRIISS。这项工作是由国家健康和医学研究委员会支持授予＃1031212和＃637406，和国家卫生研究院授予＃AI089686。

注：所有步骤，除了离心分离和流式细胞术，应该在层流罩内进行，以保持无菌。确保适当的预防措施，有关人体标本的处理。该法是少量的等离子非常敏感。提供的稀释液是最优的解决对策介乎5 - 78％，自巴布亚新几内亚（PNG）半儿童免疫血浆。最佳稀释度可以与等离子体套被测试而变化，因此，建议等离子体被滴定，以确定试验条件下对每一个应用程序。确保列入2阴性对照THP-1细胞在缺乏血浆的裂殖子;和THP-1细胞的裂殖子调理与血浆疟疾天真的个人池。这将允许控制对裂殖子粘附到THP-1细胞，并使细胞吞噬活动严格流式细胞门控。 使用PNG血浆样品是经医学研究咨询委员会，卫生巴布亚新几内亚部，瓦尔特和Eliza霍尔学院人类研究伦理委员会（项目编号04/04号）。书面同意从所有参与者的父母/监护人取得。 1，THP-1培养<ol><li>维持人类单核细胞系THP-1中添加了10％胎牛血清（FCS）和55μM2 - mercapthoethanol（THP-1培养基）的RPMI-1640培养基中，并在37℃下在5％CO 2的湿润培养箱。 </li><li>维持细胞在低于5×10 5细胞/ ml的密度。注意：过度生长会导致分化为粘附细胞和下调的Fc受体。一旦细胞已传代约10次，解冻一个新的小瓶，以保持细胞的表型。 </li></ol> 2，抗体样品制备和稀释<ol><li>热灭活血浆孵育在56℃进行测试和疟疾天真控制等离子;℃30分钟</li><li>连续稀释血浆样本的1/2，000 THP-1介质。 </li><li>在-20℃下存储稀释血浆</li></ol>高度同步的P的3。文化恶性疟原虫 注：表达GFP的寄生虫线D10-PfPHG 27是利用由于其48小时的生命周期这有助于寄生虫的同步和E64另外的控制时序。此外，因为该行的GFP表达在细胞质中，这条线可以检测寄生虫和免费裂殖子通过流式细胞仪检测GFP的。但是，GFP荧光强度是不够的THP-1细胞内提供的可视化，并且因此，裂殖子被counterstained用溴化乙锭（EtBr的）。其他寄生虫菌株可以利用，只要严格的同步性是可以实现的。使用山梨糖醇处理的组合以裂解成熟形式28和肝素抑制侵袭15同步的寄生虫</s了&gt;。 <ol><li>维护体育恶性疟原虫在RPMI-1640培养基3％的血细胞比容（pH 7.4）中添加了25毫克/毫升HEPES，50μg/ ml的次黄嘌呤，10％合并的人血清，2毫克/毫升碳酸氢钠寄生在O +人红细胞（RBC），及20微克/毫升的庆大霉素（寄生虫介质）。添加5毫克/毫升的杀稻瘟菌素S-盐酸加入到培养基中以选择的GFP +寄生虫。 </li><li>在1％的O 2，4％CO 2和95％N 2的气氛中孵育培养物在气密箱或替代双密封培养瓶中，在37℃。 </li><li>制备薄涂片，在100％甲醇中，持续10秒固定，染色并在6.7毫米（pH值7.1）10分钟来监测寄生虫血症的磷酸缓冲液（吉姆萨液）10％吉姆萨溶液。染色后，冲洗幻灯片水和空气干燥。使用100X油镜评估原虫。 </li><li>维持寄生虫培养在5％以下感染的红细胞一个由原虫文化分裂和增加感染红细胞的要求。 </li><li>与肝素同步，加医疗级肝素20 IU / ml的响阶段的文化。 </li><li>当大部分寄生虫是在裂殖体阶段，沉淀细胞，在300×g离心5分钟，除去含有肝素的培养基中重悬在寄生虫介质，以允许裂殖体破裂和裂殖子侵入。 </li><li> 4小时后，加入20 IU / ml的肝素回到文化，阻止任何进一步的裂殖子入侵。注：可能需要山梨醇和肝素治疗的几个周期之前寄生虫充分同步。要生成合适的数量裂殖子，准备150毫升寄生虫文化在3 - 5％寄生虫。 </li></ol>晚期P的4。隔离恶性疟原虫滋养体<ol><li>返回肝素培养后三十六个小时，沉淀培养的细胞在300×g离心5分钟，并在25％的血细胞比容重新悬浮颗粒在介质中的寄生虫。 </li><li>附上一个大的磁柱（6.3基质体积毫升），以一个磁体，并平衡与寄生虫介质的柱，并确保所有的气泡都被删除。 </li><li>添加悬浮体育恶性疟原虫文化柱，并调整流量，以每秒一滴。 </li><li>一旦培养已通过该柱，用寄生虫介质洗柱，直到流过运行清楚。 </li><li>从30毫升37℃寄生虫介质的柱中流出的寄生虫。 </li><li>准备一个薄寄生虫涂片，在100％甲醇，持续10秒修正，并与染色3分钟姬姆萨液。检查在显微镜下的幻灯片，并确保寄生虫几乎填补了红血细胞，而且似乎是在早期裂殖体阶段。如果寄生虫都没有达到相应的成熟阶段，回归纯净寄生虫到37℃培养箱中1％的O 2，4％CO 2和95％N 2的气氛中，直到适当展开。是必需的纯度大于90％的受感染的红细胞，以确保注：注的BC不会污染裂殖子的准备。 </li><li>对待分离的裂殖用10μME64（Epoxysuccinyl-L-leucylamido（4 - 胍基）丁烷（E64）为6 - 10小时注：停止孵育时间与E64是可能的，但是产生的裂殖子将不再侵入。 </li></ol> 5，隔离P的恶性疟原虫裂殖子<ol><li>温育后用E64，涂抹寄生虫，固定的细胞用100％甲醇中，染色用吉姆萨溶液以评估膜封闭的裂殖子的形成。注意：如果寄生虫大于50％已经形成膜封闭裂殖子裂殖子的一个很好的收益会得到。 </li><li>以1,900×g离心8分钟颗粒E64处理裂殖。用50毫升室温的RPMI-1640培养基（pH 7.4）中添加了25毫克/毫升HEPES，50μg/ ml的次黄嘌呤，2毫克/毫升的碳酸氢钠，和20微克/毫升庆大霉素（洗涤培养基）洗以除去剩余的人血清。 </li><li>重悬浮颗粒在2毫升的洗涤介质。注：使用FCS的THP-1媒体会产生过滤过程中起泡。 </li><li>通过1.2μm/32毫米注射器过滤器过滤器2毫升重悬E64-裂殖体。收集的滤液在10ml的试管中。注意：滤液含裂殖子和疟原虫色素晶体，具有收集在过滤器中未破裂的裂殖体和碎片。 </li><li>小磁列附加到一个磁体，并平衡用500μlTHP-1培养基中。 </li><li>通过滤液在磁列。收集流过。注：疟原虫色素会绑定到列，而裂殖子会通过。 </li><li>通过流过比该柱两次以除去疟原虫色素，通过每次收集的流动。 </li><li>用2毫升RT THP-1介质冲洗色谱柱，以收集任何剩余的裂殖子。 </li><li>加溴化乙锭在10微克/毫升（最终浓度）和染色30分钟，在RT。避光，防止漂白。注意：确保所有溴化乙锭污染液体和p城塑料废物在适宜的细胞毒性的化学物质的方式处置。裂殖子不再侵入下面的这个孵化。 </li><li>降速裂殖子在4000×g离心10分钟。 </li><li>弃上清到适当的废物容器。 </li><li>重悬裂殖子沉淀在4毫升的THP-1中和降速于4000×g离心10分钟。 </li><li>添加THP-1中型高达15毫升的体积，避光。 </li></ol> 6，量化裂殖子浓度流式细胞仪<ol><li>把计数珠至室温。 </li><li>制备PBS与0.5％BSA稀释的裂殖子。 </li><li>裂殖子数为3的稀释; 1 100; 1 50和1 25。准备3 FACS管与940，930和910微升PBS +0.5％BSA的分别。加10，20和40微升每管纯化的裂殖子，分别为。 </li><li>涡流计数珠，持续30秒，然后加入50微升每含有稀释的裂殖子的FACS管珠。 </li><li>运行三稀上的流uted裂殖子样本流式细胞仪配备有488nm的激光。基于溴化乙锭和GFP荧光双荧光裂殖子，和栅极计数珠的荧光在一个单独的通道上设置一个严格的门。收购事件，直到2000珠被收集。注意：这些指令是指计数的GFP +裂殖子已被反沾上溴化乙锭。如果不利用绿色荧光蛋白表达的寄生虫然后设置基于侧向散射和溴化乙锭荧光裂殖子门。 </li><li>确定裂殖子的比率：通过分割的裂殖子事件的数量由2000计数珠事件珠，计算每个稀释度的比率。使用计数珠子批次规格来确定在50μl的珠子每管中加入小珠的数量。 </li><li>通过稀释因子和裂殖子倍增的小珠的数量在50微升：珠比率为稀释因子。这个数目相当于每毫升裂殖子的浓度。执行日ESE每个稀释步骤。 </li><li>平均跨越三个稀释度测定的裂殖子的浓度。注：超过10％的标准偏差为100，1 50确定为1的浓度，并在1 25稀释表明技术上的不准确编制计数样品。 </li><li>裂殖子悬浮于8×10 6个裂殖子/在THP-1毫升培养基中，以确保每THP-1细胞的裂殖子4的最终比例。注：也可以使用其它裂殖子，以THP-1的比率，和浓度应作相应修改。 </li></ol> 7，吞噬分析<ol><li>加入200μl每96孔U型底板以及散热无效的FCS，并在室温O / N离开阻断板。孵育后，取出FCS和200微升无菌PBS洗两次井。在4℃下除去PBS及储存板，直到需要。 </li><li>计算测试血浆样品和对照所需的THP-1细胞的数目，从而允许每个样品一式三份孔中以1×10 <su对&gt; 5 THP-1细胞以每孔。 </li><li>通过加载到血球滑板10微升培养物测定THP-1培养浓度。计数细胞中存在的4套的血球计16角落正方形，在显微镜下数，然后平均计数。乘以平均计数由万计算每毫升的细胞数。 </li><li>降速THP-1细胞所需数量的，在500×g离心5分钟，并重新悬浮于6.7×10 5细胞/在THP-1培养基毫升。 </li><li>加入150μlTHP-1细胞以每孔THP-1培养基中，以一个FCS封闭96孔U型底的板，导致10 5个细胞/孔。准备每个样品3井进行测试。返回板孵化器，直到准备添加裂殖子。 </li><li>在8×10 6个/ ml的每加入150μl裂殖子的制备以及一个单独的FCS封闭96孔U型底的板。每个样品测试准备一台好</li><li>加入10微升准备稀释的血浆样品，每孔，到t他板包含裂殖子，拌匀，以保证均匀的溶液。 </li><li>从培养箱中取出的THP-1块板，并加入50微升的裂殖子/血浆溶液，每孔含有THP-1细胞，以每血浆样品一式三份孔。 </li><li>拌匀，并在5％的CO 2，饱和湿度培养箱中培养40分钟，在37°C。盖上铝箔避光。 </li><li> 40分钟后，在4℃下离心样品5分钟，在500 XG离心机预冷逮捕吞噬。 </li><li>除去上清液，并在冰冷的PBS 0.5％BSA 2毫摩尔EDTA（FACS缓冲液）洗涤细胞两次。注意：EDTA将有助于在维持单细胞悬浮液用于流式细胞术分析。 </li><li>修正了在PBS +0.5％BSA +2毫米EDTA（FACS固定液）90微升2％多聚甲醛（PFA）的细胞。置于冰上，直到采集，避光。 </li></ol> 8，流式细胞仪<ol><li>使用流式细胞仪配备无线网络获得的样品TH 488 nm激光和高通量酶标仪附件。注意：这将使多达400血浆样品从一个裂殖子准备进行测试。细胞也可以转移到管手工上样。 </li><li>门上可行的THP-1细胞通过前向和侧向散射。 </li><li>设置EtBr的正栅极基于“THP-1细胞与裂殖子和没有等离子体的控制范围。 </li><li>获得至少每个样品10,000个事件。 </li></ol> 9。数据分析<ol><li>用流式细胞仪分析软件，并设置严格的闸门溴化乙锭正面事件分析数据。 </li><li>计算百分比吞噬每个样品：溴化乙锭+细胞的样品减去与非免疫血浆的％阳性细胞％。 </li><li>跨越式三份的平均结果。注：标准偏差超过10％表示一式三份实验误差。一些背景EtBr的阳性事件可以被观察为merozoi的结果特密合性THP-1细胞的表面，但应该是一致的两端含有裂殖子的所有样品。设置栅极基于“THP-1细胞与裂殖子和没有等离子体&#39;控制，然后减去”THP-1细胞与非免疫血浆&#39;控制的任何背景将导致％吞噬作用，反映了荧光由于内在/吞噬仅裂殖子。 </li></ol>

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作者什么都没有透露。

为了测量裂殖子的吞噬能力，​​熟练掌握两种技术是必需的：裂殖子和THP-1细胞吞噬功能测定的净化。为结合这两种技术中最关键的步骤是：1）高度同步的寄生虫; 2）添加E64在正确的时间，得到膜封闭的裂殖子; 3）删除疟原虫色素，避免聚集; 4）用流式细胞仪精确的裂殖子计数; 5）用于血浆的稀释;和6）保持低的细胞密度和THP-1细胞的传代次数。仔细考虑这些关键方面将确保强大的吞噬反应观察。 虽然，这里描述的是制备裂殖子用于评估吞噬作用，该技术可被用于广泛的应用范围。不论往下流的方法，最优裂殖子制剂依赖于正确的定时E64除了以产生裂殖子。这里所描述的E64方法已被证明为yIELD侵入的裂殖子的入侵事件及药物敏感试验15-20高分辨率显微镜的使用。而裂殖子生存能力不是吞噬必不可少的，裂殖子表面涂层的完整性是必需的。因此，这里所描述的E64方法允许产生用于评估抗体应答的表面涂层高质量的裂殖子。作为概括在图1中，如果E64加入太早或太晚膜封闭裂殖子不形成。出于这个原因，高度同步的寄生虫培养物是必需的。这里，使用山梨糖醇及肝素治疗，以紧密地同步D10-GFP的寄生虫培养物，以2小时的窗口进行说明。肝素能促进gametocytogenesis在其他实验室分离，因此应谨慎使用。替代同步方法，例如丙氨酸也可使用，其前提是紧密同步的寄生虫产生29。如果E64添加到异步的寄生虫，较低的道具膜封闭裂殖子钮用来将生产和剩余的寄生虫感染的红细胞要么常破裂或发展形态异常。寄生虫还没有发展成膜包围的裂殖子的存在将导致在过滤器的堵塞和显著降低得到的裂殖子产率。 中的膜封闭裂殖子过滤，疟原虫色素是从消化液泡解放，是作为游离的晶体在溶液中。疟原虫色素是高度致炎和已报道调节单核细胞和巨噬细胞吞噬反应30,31。除了 ​​调节的吞噬作用， 如图2中 ，疟原虫色素可以形成聚集体与溶液中的裂殖子。由于THP-1细胞可以吞噬这些聚集，这可能是抗体介导的吞噬作用的分辨率的主要混杂因素。因此，清除疟原虫色素是必要的，这个实验到AV疟原虫色素对巨噬细胞生物学OID额外的复杂性。此外，未能消除疟原虫色素也可以使用这种技术时，产生游离的裂殖子用于其他应用，尤其是在裂殖子的定量要求是有害的。 如在图4中概述的，加入到测定裂殖子的数目可以由THP-1细胞吞噬作用的影响的程度。虽然流式细胞术允许裂殖子浓度，小心吸液和复制裂殖子的计数列举有必要以提高准确性。如果多个寄生虫线侧由端进行测试，这一点尤为重要。以前已经证明，从PNG半免疫儿童等离子体可显著稀释反应下降23之前。这里描述的是等离子体的最佳稀释度（1/120，等离子体000最终稀释度）为5队列 - 12岁的儿童，PNG，产生的大范围phagocyt的，在图5中描述的尘埃沉着病回应此范围允许的响应分层分为四组（0 - 19％，20 - 39％，40 - 59％，和60 - 79％的吞噬作用），以及回归建模显示，opsonizing反应进行关联从临床疾病和高密度的寄生虫血症10保护对于不同的队列研究，可能有必要调节血浆稀释，以保证吞噬细胞引起的THP-1细胞中不饱和或低于检测水平。 流式细胞仪可以快速和可量化的吞噬作用与提高精度超过显微镜。这个协议是一种高通量，基于板和自动采集的96孔板来研究自然获得的体液免疫。该方法需要裂殖子用溴化乙锭染色，但是替代的DNA污渍如SYBRgreen，D​​API和碘化丙啶，膜的污渍或蛋白质的污渍可以被利用。此法是服从PRIMARÝ单核细胞或嗜中性粒细胞，并可以适应如吞噬细胞或生物的FcR是感兴趣的。 体外分化用PMA的原代细胞或THP-1细胞可用于研究吞噬作用于疟疾和其他病原体12，32-34。然而，使用原代细胞可以是具有挑战性的，因为这些细胞是贴壁，并显示非抗体介导的吞噬作用35。此外，变异纯度，活力和功能的一些主要限制的使用主要吞噬细胞。参与裂殖子的吞噬Fc受体依然未知，因此采用封闭抗体对特定的Fc受体可以阐明各自的Fc受体，以裂殖子吞噬的贡献。由于THP-1细胞吞噬功能的Fc受体依赖，这使得观察到的吞噬简单的解释。该测定也适合于寻址opsonizing可被util的实现抗体的抗原特异性定义敲除寄生于裂殖子表面抗原，或用亲和纯化的人抗体的裂殖子表面蛋白。此外，在深度细胞因子应答的下列裂殖子的吞噬作用研究缺乏，并且可以使用该测定法来实现。 这两项技术构成，以功能antimerozoite抗体的研究相当大的进展。高品质的裂殖子的净化是有利的，使用冷冻裂殖子，或涂裂殖子抗原荧光微球。虽然该试验中已被用作用于评价自然获得性免疫的一个工具，它可能被证明为处理采集的免疫响应于免疫接种的重要工具。而最近已表明，吞噬作用或opsonised裂殖子被保护免于临床疟疾相关联，这是不可能得出结论直接从体外的THP-1细胞吞噬到体内 phagocyto裂殖子吞噬作用在这个实验的SIS静态条件下，并在没有竞争的红血细胞的发生。因此，这个实验的潜在适应可提供一个多功能的工具，进一步了解疟疾和裂殖子吞噬。

抗体的抗恶性疟原虫疟疾的重要性，表明40年前，从超免疫的成年人，当免疫球蛋白被动地被转移到儿童造成病情缓解1重症疟疾。因此，相当大的努力一直试图确定的保护性疟疾免疫的目标，主要是通过测定抗体滴度，以用ELISA肽或细菌表达的蛋白质。基于ELISA的血清学检查也证明研究之间充满变数，并且不涉及抗体的功能2。许多疟疾抗原诱导嗜细胞IgG1和IgG3抗体形象，特别是裂殖子表面抗原3。本小类偏压表明与吞噬细胞的抗体的Fc受体（FcR的）相互作用对于opsonizing antimerozoite抗体4的效应子功能重要的。正在开发中的几个裂殖子抗原疫苗被设计引起巨噬细胞的效应功能5，6，虽然对抗体Fc受体的相互作用在啮齿类动物疟疾模型的重要性显著证据7-9，和最近的一些研究支持功能的抗体和吞噬细胞效应功能的免疫力疟疾在人类的重要性10，11，这方面的研究仍然很差。研究成裂殖子的具体opsonizing抗体已经由两个因素受到限制;难度在隔离良好的品质裂殖子;从原代细胞和吞噬变量的反应。 直到最近，高速离心或Percoll密度梯度被用于从裂殖体破裂文化的培养上清液分离的裂殖子。这些裂殖子很少是可行的，而且往往是由密度梯度离心进一步处理和多次洗涤步骤12，或者冷冻保存11试验后方可使用。这些PROCS弯潜在的分离从裂殖子表面许多周边相关的蛋白质，称为是疟疾的免疫力13抗原的目标蛋白质。最近的半胱氨酸蛋白酶抑制剂反式Epoxysuccinyl-L-leucylamido（4 - 胍基）丁烷（E64）已被用来产生可行的裂殖子。 E64阻止裂殖体破裂，产生膜封闭的裂殖子14，它可以通过过滤打乱解放可行的裂殖子15，16。这种技术已导致许多蛋白质的红细胞浸润15，17-19在空间分辨率和澄清的几个抗疟药16,20的阶段特异性作用。然而，可行的裂殖子的生成保持在技术上具有挑战性。为了帮助这个技术和应用，以免疫功能分析，对可行的裂殖子净化和其使用的一个详细的协议的传播抗体的标准化功能测定：在调理作用和吞噬细胞的合作在这里描述。 这种技术表明了显著提前比以前的体外测定裂殖子调理作用，如嗜中性粒细胞呼吸爆发，抗体依赖性细胞抑制（ADCI），和替代裂殖子的吞噬作用测定。这些测定是由于变异寄生虫的输入和主吞噬细胞11，21的活性重复性差。污染疟原虫色素也可能会深刻地影响吞噬细胞功能22。最近报告的稳健和可重复的裂殖子的吞噬功能试验23采用了前单核细胞株THP-1 24。这是一种理想的细胞类型用于高通量流式细胞仪检测，因为它是无粘性的具体执行的Fc-受体介导的吞噬作用25,26。一个额外的复杂性，同时不变拟tigating吞噬作用是，吞噬作用的程度取决于相对于THP-1细胞和血浆浓度的裂殖子的数量。为了确保实验之间的重现性，裂殖子应列举和规定浓度使用。由于其体积小，流式细胞仪定量分析是必要的。 这里所描述的过程将删除疟原虫色素生成可行的裂殖子，并介绍了这些裂殖子的裂殖子后调理作用和吞噬作用的流式细胞计数的应用程序。虽然技术要求高，所描述的技术可在阐明的裂殖子表面特异性抗体应答的贡献，以自然获得与疫苗诱导的免疫证明是有用的。

<table><tbody><tr><td>THP-1 cell line</td><td>American Type Culture Collection</td><td>TIB-202</td><td></td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>31800-089</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Invitrogen</td><td>10099-141</td><td></td></tr><tr><td>2-mercapthoethanol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M6250-100ML</td><td>Use in Fume Hood</td></tr><tr><td>Pen/Strep Solution (100x)</td><td>Sigma</td><td>P0781</td><td></td></tr><tr><td>HEPES</td><td>SAFC</td><td>90909C</td><td>Cell culture grade</td></tr><tr><td>Hypoxanthine</td><td>Calbiochem</td><td>4010</td><td></td></tr><tr><td>Human Serum&amp;nbsp;</td><td>Donation from Australian Red Cross</td><td></td><td>Available commercially (i.e. Invitrogen)</td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Merck</td><td>1.06329.0500</td><td></td></tr><tr><td>Gentamycin</td><td>Pfizer</td><td>61022027</td><td>Injection Quality</td></tr><tr><td>Heparin Sodium BP (5000 IU/ml)</td><td>Pfizer</td><td></td><td>Porcine origin</td></tr><tr><td>Blasticidin S-hydrochloride</td><td>Sigma-Aldrich</td><td>15205</td><td></td></tr><tr><td>D-Sorbitol</td><td>Sigma-Aldrich</td><td>50-70-4</td><td></td></tr><tr><td>E64 Protease Inhibitor</td><td>Sigma-Aldrich</td><td>E3132-10MG</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>98281-100G</td><td>Use for phosphate buffer</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4.&lt;/sub&gt;2H&lt;sub&gt;2&lt;/sub&gt;0</td><td>Merck</td><td>10383.4G</td><td>Use for phosphate buffer</td></tr><tr><td>Giemsa&#039;s azur eosin methylene blue solution</td><td>Merck</td><td>1.09204.0500</td><td>1:10 dilution in 6.7 mN Phosphate buffer (make fresh each stain)</td></tr><tr><td>EDTA disodium salt</td><td>Merck</td><td>10093.5V</td><td>0.1 M, pH 7.2</td></tr><tr><td>Acrodisc Syringe Filters 1.2-&amp;mu;m/32-mm</td><td>Pall Life Sciences</td><td>4656</td><td></td></tr><tr><td>QuadroMACS Separator (for small column)</td><td>MACS Miltenyi BioTec</td><td>130-091-051</td><td>Interchangeable with MidiMACS&amp;nbsp;</td></tr><tr><td>VarioMACS Separator (for large columns)</td><td>MACS Miltenyi BioTec</td><td>130-090-282</td><td></td></tr><tr><td>MACS MultiStand</td><td>MACS Miltenyi BioTec</td><td>130-042-303</td><td></td></tr><tr><td>LS Column (small magnetic column)</td><td>MACS Miltenyi BioTec</td><td>130-042-401</td><td></td></tr><tr><td>Large magnetic column</td><td>MACS Miltenyi BioTec</td><td>130-041-305</td><td></td></tr><tr><td>Ethidium Bromide</td><td>Bio-Rad</td><td>1510433</td><td>Cytotoxic</td></tr><tr><td>CountBright Absolute Counting Beads</td><td>Invitogen</td><td>C36950</td><td></td></tr><tr><td>BD FACSCalibur Flow Cytometer with HTS plate reader</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>EDTA disodium salt</td><td>Merck</td><td>10093.5V</td><td>0.1 M, pH 7.2</td></tr><tr><td>FlowJo cytometry analysis software</td><td>Tree Star</td><td></td><td></td></tr></tbody></table>

high yield purification of plasmodium falciparum merozoites for use in opsonizing antibody assays

恶性疟原虫裂殖子抗原正在开发作为潜在的疟疾疫苗。免疫抵抗疟疾的一个方面是消除游离裂殖子从血液吞噬细胞。然而评估裂殖子特异性opsonizing抗体的功能性功效是由于裂殖子的短半衰期和主吞噬细胞的变异性的挑战。本文详细描述的是这样一种方法，用于产生可行的裂殖子使用E64蛋白酶抑制剂和裂殖子的使用亲单核细胞系THP-1的测定调理素依赖性吞噬作用。 E64阻止裂殖体破裂，同时允许它们由处理裂殖体的过滤释放的裂殖子的发展。溴化乙啶标记的裂殖子被调理的与人的血浆样品，并添加到THP-1细胞。吞噬作用是通过一个标准化的高通量协议进行评估。可行的裂殖子是评估NUMER的宝贵资源P的项方面恶性生物学，包括免疫功能的评估。此法检测抗体水平与临床免疫疟疾的自然暴露的人有关。该法也可用于评价疫苗诱导的抗体。

之前E64治疗寄生虫的成熟阶段是用于产生膜封闭的裂殖子的关键。 图1AI显示可用于添加E64裂殖体的适当成熟阶段。寄生虫要大，几乎填满红细胞。姬姆萨染色的斑驳的外观表示裂殖生殖已经开始，和E64应该加入以产生膜封闭的裂殖子（ 图1Aii）。如果E64是较早加入滋养体阶段的寄生虫，即使经过12小时的E64不会产生膜封闭的裂殖子。相反寄生虫承担形态异常，如消化液泡（ 图1BI和1Bii）的扩大，和裂殖子都没有形成。如果E64是后来添加到裂殖体，不会产生膜封闭裂殖子的裂殖体破裂是不羁（ 图1CI和1Cii）。寄生虫同步高水平的要求，其他明智的一系列描述的所有三种结果都将E64治疗后可以看到。 去除疟原虫色素是用于净化裂殖子的另一个关键步骤。如果裂殖子不会从疟原虫色素纯化，然后裂殖子，疟原虫色素簇形成不能用移液被抛下。这些聚集体的流式细胞仪上运行的单个事件，虽然比单一的裂殖子不同的前向散射和侧散射特性，从 ​​而导致不准确的裂殖子的计数通过流式细胞术（ 图2A）。如THP-1细胞也可以吞噬疟原虫色素，这些裂殖子，疟原虫色素簇也可以吞噬（ 图2B）。这些总量是高度溴化乙锭荧光，因为它们包含多个裂殖子。吞噬作用的聚集体的结果等效于所观察到的多个单独的裂殖子的吞噬一个THP-1的EtBr荧光分布。因此，如果疟原虫色素不会被删除，THP-1细胞的溴化乙锭荧光将邻verestimate的opsonizing潜力被测试的血浆。疟原虫色素也被报告给显著改变巨噬细胞功能。 GFP阳性裂殖子被计数器沾上的EtBr增加裂殖子由THP-1细胞吞噬的可视化。使用本协议所述的条件，所有的GFP阳性裂殖子被counterstained用溴化乙锭其中有一个光明的荧光强度（ 图3A）。通过溴化乙锭荧光吞噬评估使裂殖子的吞噬作用比GFP荧光（ 图3B）优异的解析度。 加入到测定裂殖子的数目将调制吞噬观察到的量。出于这个原因，精确计数通过流式细胞术是至关重要的。增加裂殖子的比率：THP-1，将导致裂殖子的增加粘附到THP-1细胞在无等离子体的（ 图4A）。增加裂殖子：THP-1的比率也导致增加的pH值agocytosis反应时裂殖子被调理（ 图4B）。 4:1裂殖子：THP-1的比例，建议稳健的吞噬反应。使用这种比例，血浆指示的稀释，该测定是能够解决opsonizing抗体的低，中和高的水平。使用PNG血浆样本和说明的其他条件介于0和78％的吞噬反应已被观察到。例如响应近来显示出与天然获得的临床免疫疟疾10。 图5示出的4个四分位数从PNG个体的吞噬反应的实例相关联。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51590/51590fig1highres.jpg" width="500" /> 图1的时序E64加入是用于产生膜封闭的裂殖子的关键。 （一）适当maturatio不生成的n个级i）之前，E64治疗寄生虫，以及ii）6小时E64治疗后产生的膜封闭裂殖子（B）的膜封闭的裂殖子，如果E64加到未成熟的寄生虫。; ⅰ）晚期滋养体，以及ii）12小时E64的后（C）的裂殖破裂不如果E64被添加到末分段裂殖抑制。; I）晚期裂殖体，以及ii）后6小时E64的。标尺为10微米。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51590/51590fig2highres.jpg" width="500" /> ，裂殖子，疟原虫色素聚集体形式以下的膜封闭裂殖子过滤，除疟原虫色素被磁力从裂殖子中分离如图2所示。疟原虫色素去除是重要的，以产生单细胞裂殖子悬浮液（A）前向散射和侧向散射图裂殖子带疟原虫色素移除和疟原虫色素保留（B）疟原虫色素-裂殖子聚集体可以通过THP-1细胞进行吞噬。 THP-1细胞培养与PNG等离子的差异，快速染色的细胞质自旋幻灯片调理裂殖子准备带或不带疟原虫色素。标尺为10微米。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51590/51590fig3highres.jpg" width="500" /> 图3。溴化乙锭染色允许裂殖子的吞噬作用的优良的分辨率。D10-GFP纯化的裂殖子被counterstained用溴化乙锭，以提高荧光强度（A）流式细胞术纯化的裂殖子histrogram与浇口对GFP阳性的裂殖子，和斑点印迹示出的EtBr这门GFP阳性人群中荧光（B）前向散射与GFP和前向散射与THP-1细胞的下面的GFP和溴化乙锭双荧光裂殖子吞噬溴化乙锭。盖茨是ð<html> rawn基于THP-1细胞与裂殖子和没有等离子体控制。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51590/51590fig4highres.jpg" width="500" /> 图4中的裂殖子：THP-1比值影响吞噬作用的观察到的程度。 （一）五裂殖子：THP-1的比例被描绘; 50:1，20:1，10:1，4:1，1:1。细胞只控制表示背景荧光是由于THP-1细胞。 THP-1的EtBr荧光每个比表示为裂殖子调理与PNG血浆池或与非免疫澳大利亚等离子（B）的平均数荧光THP-1细胞进行不同的裂殖子的强度：将THP-1的比例，并调理带游泳池PNG等离子或非免疫澳大利亚等离子（平均值±SEM）的。 <a href="https://www.jove.com/files/ftp_upload/51590/51590fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ove_content“FO：保持together.within页=”总是“&gt; <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51590/51590fig5highres.jpg" width="500" /> 图5。可测大范围opsonizing抗体。裂殖子的调理与来自巴布亚新几内亚的个人血浆和孵育THP-1细胞。显示四个有代表性的吞噬反应。的基础上，THP-1细胞盖茨被画与裂殖子，并没有等离子控制和数字表示的THP-1细胞与非免疫等离子控制的减法后的％吞噬。 <a href="https://www.jove.com/files/ftp_upload/51590/51590fig5highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays at JoVE.com

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

测量抗体功能的关键是了解免疫力恶性疟原虫疟疾。这个方法描述了可行的裂殖子的纯化和调理作用的依赖性吞噬作用通过流式细胞术测量。

高产净化<em&gt;恶性疟原虫</em&gt;裂殖子适用于Opsonizing抗体检测

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

high-yield-purification-plasmodium-falciparum-merozoites-for-use

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

23.0K 次观看.  Walter and Eliza Hall Institute of Medical Research. 测量抗体功能的关键是了解免疫力恶性疟原虫疟疾。这个方法描述了可行的裂殖子的纯化和调理作用的依赖性吞噬作用通过流式细胞术测量。 

视频: High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

一个简单的协议介导的血小板聚集<em&gt;恶性疟原虫</em&gt;受感染的红细胞在一个资源匮乏的设置

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

科研

免疫与感染

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

分离<em&gt;恶性疟原虫</em&gt;晚期感染的红细胞由磁性装置

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectrometer in conjunction with Multivariate Data Analysis (MVDA). 3D7 P. falciparum were cultured to 10% parasitemia ring stage parasites and used to spike fresh donor isolated RBCs to create a dilution series between 0&#8211;1%. 10 &#181;L of each sample were placed onto the center of the ATR diamond window to acquire the spectrum. The sample data was treated to improve the signal to noise ratio and to remove the contribution of water, and then the second derivative was applied to resolve spectral features. The data were then analyzed using two types of MVDA: first Principal Component Analysis (PCA) to determine any outliers and then Partial Least Squares Regression (PLS-R) to build the quantification model.

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

Here, we present a protocol for the detection and quantification of Plasmodium falciparum in infected aqueous red blood cells using an attenuated total reflection infrared spectrometer and multivariate data analysis.

衰减全反射红外光谱和多变量数据分析检测和定量水红血球中恶性疟原虫

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total ...

化学

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

方法调查小RNA和核糖体占用的调控作用<em&gt;恶性疟原虫</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

通过流细胞学测定，测量自然获得的噬菌体诱导恶性疟Plasmodium falciparum原虫抗体

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

用于检测按蚊媒介中恶性疟原虫感染的标准膜喂养测定

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8+ and gamma delta T cells (&#947;&#948; T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8+ T, &#947;&#948; T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes.&#160;

体外 细胞毒性淋巴细胞杀伤疟 原虫感染红细胞的测定

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

基于海拉细胞自由表达系统中的表达<em&gt;疟原虫</em&gt;棒状体蛋白

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

生物学

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.

使用酶联免疫吸附测定法检测按蚊中的疟原虫孢子

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female ...

<meta charset="utf8"></head><body>鉴定疟疾寄生虫中的激酶重新布线:一种化学遗传学方法

Understanding the Development of Compensatory Pathways in a Mutant Malaria Parasite Harbouring Hypomorphic Allele of Plant-Like Kinases

One of the mechanisms for subverting the effect of drugs by&#160;the malaria parasite is through rewiring of its transcriptome. The effect is more pronounced for target genes belonging to the multigene family. Plasmodium falciparum protein kinases belonging to the CDPK family are essential for blood stage development. As such, CDPKs are considered good targets for the development of anti-malarial compounds. The chemical genetics approach has been historically used to elucidate the function of protein kinases in higher eukaryotes. It requires the substitution of gatekeeper residue for another amino acid with a different side chain through genetic manipulation. Amino acid substitution at the gatekeeper position modulates the activity of a protein kinase and changes its susceptibility to a specific class of compounds known as bumped kinase inhibitors (BKIs) that help in the functional identification of the target gene. Here, we have exploited the chemical genetics approach to understand compensatory mechanisms evolved by a mutant parasite harboring a hypomorphic allele of cdpk1. Overall, our approach helps in identifying compensatory pathways that may be simultaneously targeted to prevent the development of drug resistance against individual kinases.

<meta charset="utf8"></head><body>作者聚焦:鉴定含有必需蛋白激酶亚型等位基因的疟疾寄生虫中的代偿途径

Chemical genetics involves the substitution of a gatekeeper residue with an amino acid containing a different side chain at the target locus. Here, we have generated a mutant parasite containing a hypomorphic allele of cdpk1 and identified compensatory pathways adopted by the parasite in the mutant background.

高产净化

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一个简单的协议介导的血小板聚集

分离

衰减全反射红外光谱和多变量数据分析检测和定量水红血球中恶性疟原虫

方法调查小RNA和核糖体占用的调控作用

通过流细胞学测定，测量自然获得的噬菌体诱导恶性疟Plasmodium falciparum原虫抗体

用于检测按蚊媒介中恶性疟原虫感染的标准膜喂养测定

体外细胞毒性淋巴细胞杀伤疟原虫感染红细胞的测定

基于海拉细胞自由表达系统中的表达

使用酶联免疫吸附测定法检测按蚊中的疟原虫孢子

了解携带植物样激酶亚型等位基因的突变疟疾寄生虫中补偿途径的发展

高产净化

摘要

探索更多视频

此视频中的章节

一个简单的协议介导的血小板聚集

分离

衰减全反射红外光谱和多变量数据分析检测和定量水红血球中恶性疟原虫

方法调查小RNA和核糖体占用的调控作用

通过流细胞学测定，测量自然获得的噬菌体诱导恶性疟Plasmodium falciparum原虫抗体

用于检测按蚊媒介中恶性疟原虫感染的标准膜喂养测定

体外 细胞毒性淋巴细胞杀伤疟 原虫感染红细胞的测定

基于海拉细胞自由表达系统中的表达

使用酶联免疫吸附测定法检测按蚊中的疟原虫孢子

了解携带植物样激酶亚型等位基因的突变疟疾寄生虫中补偿途径的发展

体外细胞毒性淋巴细胞杀伤疟原虫感染红细胞的测定