Name: in vivo assessment of rodent plasmodium parasitemia and merozoite invasion by flow cytometry
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我们承认，从国家健康与医学研究委员会的资金支持（授予APP605524，490037和1047082），澳大利亚研究理事会（批DP12010061），澳大利亚从创新系国家合作研究基础设施战略和教育投资基金，产业，科学和研究。 PML是澳大利亚研究生奖的获得者。

所有的程序均按照麦考瑞大学的政策进行，符合国家卫生和医学研究委员会（NHMRC）实践澳洲代码。根据协议道德没有ARA 2012/017批准，并从动物伦理委员会在麦考瑞大学获得进行这项工作。所有实验均在SJL / J小鼠进行，除非另有说明。 1.鼠标和实验疟疾感染<ol><li>家鼠控制温度（21°C）具有12:12小时明暗周期下。 </li><li>解冻冷冻保存P的等分试样chabaudi阿达米 DS用5％寄生红细胞（pRBCs）注入200微升进入腹腔的C57BL / 6小鼠供体腔。 </li><li>使用流式细胞仪在第3节中描述2天 - - 本协议的4监察捐助鼠标原虫每1。一旦捐助者达到5 - 15％原虫，通过心脏穿刺采血如下: <ol><li>吸100微升肝素溶液（300 U / ml肝素在小鼠林格液（MTR）（154毫米氯化钠，5.6毫米氯化钾，1毫米氯化镁2，2.2毫米氯化钙2，20毫米的HEPES，10毫米的葡萄糖，30 U / ml的肝素，pH值7.4，0.22微米过滤灭菌））到一个1ml注射器用26ģ针。 </li><li>麻醉小鼠通过吸入，用5％异氟烷，由缺乏踏板戒断反应及角膜反射确认麻醉的所需深度。 </li><li>放置鼠标在其一侧并垂直插入针的正下方的弯头，通过肋，并进入心脏。慢慢拉出注射器活塞和0.5，直到旋转针 - 1毫升血液中获得。 </li><li>通过颈脱位麻醉下执行人道的安乐死。 </li></ol></li><li>计算每个供体血容量pRBCs假设的9×10 6 RBC /μL（血液计数，即，如果捐助者是在10％原虫牺牲时，血液将包含9×10 5猪红细胞/微升）。稀释血液寄生于Krebs缓冲盐水（126 mM氯化钠，2.5mM的氯化钾，25mM的碳酸氢钠 ，1.2毫的NaH 2 PO 4，1.2毫的MgCl 2，2.5mM的氯化钙 ，0.2％葡萄糖，pH为7.2，灭菌0.22μm的过滤器）使浓度为每200微升1×10 4个pRBCs并注入200μl的进腹腔所需红细胞标签和输血实验小鼠的空腔。 </li><li>使用流式细胞仪在第3描述2天 - - 4本协议的监控原虫每1。 </li></ol> 2.标签红细胞和输血<ol><li>如在步骤1.3中描述收集来自小鼠的肝素化血液通过心脏穿刺，并放置在冰上。保持血液在4℃下在任何时候。收集每只小鼠约200微升的血液被注入。如果需要的话，分离血成两个样品，并根据需要处理一个样本。 注:以这种方式，侵入经处理的SAMP的乐可以比作对照，未处理的样品（ 见图1的原理图）。 </li><li>制备各荧光红细胞标签的2倍的溶液。 <ol><li>稀阿托633-N-羟基（阿托633-NHS）在地铁的2毫克/毫升的储备溶液，使其浓度为20微克/毫升。 </li><li>稀释生物素-N-羟基琥珀酰亚胺（生物素NHS）在地铁的25毫克/毫升的储备溶液，使其浓度为250微克/毫升。 </li></ol></li><li>拆分血样成两个管和2倍阿托633-NHS溶液等体积添加到一个管中，与2倍生物素 - NHS等体积的其他管。立即混合。孵育血液在4℃下1小时以恒定缓慢混合，或可替代地，混合样品每10 - 15分钟。 </li><li>与地铁（MTR + 0.5％牛血清白蛋白（BSA），灭菌0.22μm滤器）洗涤标记的血液的3倍，如下所示: <ol><li>至少添加2体积地铁，离心机在750×g离心5分钟，去除上清，并resuspend。在至少2卷地铁沉淀。重复2次以上。 </li><li>在最后的洗涤，离心，在750×g离心5分钟，并结合后以不同的组合的沉淀血液（ 即，未处理的阿托633 /治疗生物素，未处理的生物素/处理阿托633）。加入地铁弥补对每只小鼠200微升体积被注入。 </li></ol></li><li>注入标记的血液进入血管内灭鼠疟疾感染的小鼠或未受感染的控制如下: <ol><li>在寄生虫裂殖生殖高峰15％原虫 - 执行输血2。 注:本约一半是通过暗周期（ 即在12 -上午02时在一个正常的光照周期，或12 -下午2点在反向光周期）。 </li><li>用热灯5-10分钟暖小鼠增加血流量，但须小心不要过热的小鼠。小鼠放置在约束装置和血管内注入200微升标记的血液（约1 - 2×10 9 RBCS）通过尾静脉。 </li></ol></li><li>如在第3节中所述采集血样，在注射后不同的时间点。 注:侵入率可以定量小于10分钟注射后，根据不同的受体小鼠的寄生虫血症。 </li></ol> 3.收集血液样品，并准备流式细胞仪<ol><li>制备50微升的每个样品染色溶液，再加额外，在实验当天如下: <ol><li>除霜6毫JC-1储存在-20℃，在​​二甲基亚砜（DMSO）的等分试样。 </li><li>温暖的50微升地铁每个样品，再加上额外的，到37℃。 </li><li>而连续地涡旋预热地铁，加入JC-1，使最终浓度为12μM。这样做是为了减少JC-1聚集体的形成;如果不聚集形成，不离心管，因为这将防止染色。 注:少量聚集不会影响结果。 </li><li>添加抗CD45 APC eFluor 780和抗CD71 PerCP eFluor 710，以使最终浓度为1微克/毫升。如果执行所述标记的RBC实验中，添加链霉抗PE-Cy7的，以使最终浓度为1微克/毫升。 </li></ol></li><li>通过尾部内的血管的尾部或裂伤的尖端的截肢执行尾出血。将一滴鲜血尾巴上的小重量船或搏命。采血后，确保出血已停止。吸管3微升尾巴血成50微升染色溶液预加热到37℃，并孵育样品在37℃下20分钟。 </li><li>除霜的4mM的赫斯特33342储存在-20℃下在蒸馏水中（如果使用355纳米的激光）或2mM的赫斯特34580储存在-20℃下在蒸馏水中（如果使用405nm的激光）的等分试样。准备500微升每个样品，再加上额外的，对赫斯特的地铁4微米或2微米的解决方案，分别为。 </li><li>加入500μl的4μM赫斯特33342或2微米的Hoechst 34580到样品和在室温下孵育20分钟。 </li><li>离心机细胞在750 XG为3分钟，在4°C，弃上清，加入700μl地铁和流式细胞流动分析。 </li></ol> 4.流式细胞仪<ol><li>用355/488/633 nm激光或405/488/633 nm激光仪器分析样品。 </li><li>通过一个35微米的细胞分析之前，立即滤网过滤器样品。冲洗细胞过滤的样品，并再利用之间蒸馏水。 </li><li>记录足够的事件，以便人口最少包含至少500事件（通常是100 - 10,000,000每个样品总的事件）。 </li><li>激发的Hoechst 33342使用355纳米的激光，并通过一个五十分之四百六十〇滤波器检测事件。激发的Hoechst 34580使用405nm的激光，并通过一个五十分之四百六十〇滤波器检测事件。激发JC-1，抗CD71 PerCP eFluor 710和链霉PE-Cy7的使用488nm的激光，并通过一个四十〇分之五百三检测事件，40分之692和750LP过滤各自LY。激发阿托633和抗CD45 APC eFluor 780使用633纳米激光分别检测通过三十零分之六百七十或750LP过滤事件。 </li><li>使用适当的流式细胞仪分析软件如下进行分析和补偿: <ol><li>选择全细胞和排除噪声，碎屑，并且基于FSC / SSC特性（ 图2A）的血小板。选择基于任一触发脉冲宽度（ 图2B）或通过使用FSC峰面积与高度之比（ 图2C）的单细胞。 </li><li>选择成熟红细胞，并排除红细胞祖细胞和白细胞，基于在PerCP eFluor 710和APC eFluor 780的通道（ 图2D）负荧光。 </li><li>如果执行标记RBC实验，选择基于PE-Cy7的和阿托633荧光标记的红细胞的每个人口和分析这些分开（ 图3A）。 </li><li>基于积极的荧光为JC-1和赫斯特选择pRBCs（ <str翁&gt;图2E-H）。 </li></ol></li></ol> 5.计算和统计<ol><li>除以pRBCs的数目由红细胞的总数目计算原虫（ 即在栅极Q2由事件数在闸门G4划分事件的数量）。当进行两个标记的种群的侵袭测定寄生虫血症可以通过将标记pRBCs的数目由标记的RBCs的数目中，人口（计算即数量的事件，因为他们位于两个栅极L1和Q2，由事件的数量除以在门的L1）。 注意:标记原虫而变化很大，取决于多种因素，例如内源性原虫和循环裂殖子的数量。出于这个原因，它是有帮助的报告原虫比率，其计算公式为经处理的标记的人口由控制标记的人口的寄生虫在每个单独小鼠划分的寄生虫血症。为了纠正可能的染料的影响，报告结果正穿过两个染料组合的平均原虫比例。 </li><li>确定使用一个样本t检验与1为假想的平均比率的统计显着性。 </li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum+field+isolates+commonly+use+erythrocyte+invasion+pathways+that+are+independent+of+sialic+acid+residues+of+glycophorin+A.">Plasmodium falciparum field isolates commonly use erythrocyte invasion pathways that are independent of sialic acid residues of glycophorin A.</a> Infect Immun. 67 (11), 5784-5791 (1999).</li><li>Dolan, S. A., Miller, L. H., Wellems, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+switching+mechanism+in+the+invasion+of+erythrocytes+by+Plasmodium+falciparum.">Evidence for a switching mechanism in the invasion of erythrocytes by Plasmodium falciparum.</a> J Clin Invest. 86 (2), 618-624 (1990).</li></ol>

作者什么都没有透露。

我们已经描述了两种原虫和裂殖子侵入体内的样品的测定的方法。在寄生虫血症的测量而言，这种方法提供了比以前的方法10-13在于HJ-红细胞的优点可以从pRBCs区分，从而降低假阳性事件的数目。而HJ-红细胞通常在人类中很少见，一些研究报告高水平小鼠15,16使这些细胞和pRBCs为啮齿动物寄生虫血症的精确测量重要的区别。采用这种方法检测原虫的极限是约0.007％14，虽然这是可能的，以减少在适当的条件下，本低至0.0005％。值得注意的是，此限制是由流式细胞仪中使用，并取得了大量的事件在一个合理的时间量的能力的清洁度决定。与使用这种技术的相关的最常见的问题是染色机智不足ħJC-1。 JC-1的荧光取决于该寄生虫的线粒体膜电位，和寄生虫必须是健康，以获得最佳的染色。应该染色前因此，必须避免寄生虫或固定长时间存放。 分析寄生虫已受到药物干预时还应注意行使，因为这也可能影响与JC-1染色的程度。在这些情况下，相比寄生虫血症在添加的JC-1染料可能是分析的基础上的Hoechst荧光单独，原虫很有帮助。实际上，这可以提供的量度寄生虫计数，以及寄生虫健康的整体指示。最后，将JC-1染料可以从一个峰在529纳米的在590nm处通过形成J-聚集改变其荧光的峰。这些聚集体的形成取决于染料的浓度，而这又取决于细胞的线粒体膜电位。线粒体膜势的损失人引起荧光的移位朝529纳米，其可以指示细胞凋亡。在我们沾有JC-1的经验pRBCs总是表现出这是在两个四十分之五百三十〇纳米通道和四十二分之五百八十五纳米通道约等于强度类似的荧光图案。然而，在一些研究中，如那些使用药物干预，它可能是值得监测变化四十分之五百三纳米通道和四十二分之五百八十五纳米通道的线粒体膜电位丧失的任何指示之间的荧光比。同样重要的是要注意，JC-1特别容易分解，应该从所述冷冻等分新鲜配制，同时应避免冷冻/解冻循环。染色后，JC-1荧光是具有很少或没有减少在荧光，至少在几个小时的相对稳定。 用于体内侵袭测定中，两个最重要的因素是在无线闭塞中心标记PR一致性ocess和注射标记的血液的时间。为红细胞标记，染料的最小量应使用，以防止与寄生虫侵入RBC，已用表面标签7时提出这样一种可能性的能力的任何干扰。根据这里所描述的标记条件的入侵不抑制14，但是仔细注意应给予维持一致的标签的浓度，和染料应被切换，以防止由于侵入抑制由红细胞标签的任何不精确性。作为寄生虫经历一个同步周期的入侵，这是非常重要的标记的细胞在适当的时间，在寄生虫生命周期的注入。这应该以最大限度的入侵事件的数量，因此，测定的精确度来实现。根据我们的经验入侵的峰值大约一半是通过黑暗的周期，使得反向光周期室有利的这个实验。 经tWO标签侵袭实验提供了一个准确的方法，以确定裂殖子侵入效率成不同类型的红细胞在体内，这种方法因此允许处理的红细胞， 即胰蛋白酶处理，并控制红细胞之间的比较。或者，从转基因小鼠中的红细胞可以比那些对照小鼠，或不同的生理参数或不同年龄红细胞不能比拟的。而类似的测定法已在体外 9被描述为侵袭的测定，该测定在此提供了只占本体内作用，包括免疫系统，这可能改变寄生虫入侵机制17-19的影响的可能性。最后，该测定也有可能提供深入了解疾病，如pRBCs的免疫清除，和寄生虫的生长，如果被标记的种群内的pRBCs监测在延长的时期。 OveraLL，对于寄生虫血症的体内样本中的精确测量的详细协议被描述，它具有比以前的试验在于HJ-红细胞的优点可以从pRBCs区别开来。此外，一个测定在体内描述的侵入效率量化成不同的RBC类型。

The clinical symptoms associated with malaria occur during the Plasmodium parasite&#8217;s asexual replicative cycle within red blood cells (RBCs). Merozoites, released during the liver stage of infection, quickly attach to and invade RBCs. After gaining entry into the cell, the parasite grows and matures, eventually undergoing schizogony, splitting open the cell, and releasing a cluster of newly formed merozoites which go on to repeat this cycle. As such, an assessment of malaria infection often involves monitoring both parasitemia, which is the percentage of RBCs appropriated by one or more parasites, and the rate of merozoite invasion into uninfected RBCs. 
Flow cytometry is a powerful tool which can be used to record the properties of vast numbers of cells in a short period of time. This technique has clear applicability for the measurement of malaria parasitemia and invasion, and offers several advantages over traditional microscopy techniques. These include the accurate measurement of very low parasitemia, which would be prohibitively time consuming by microscopy, the unbiased nature of the measurement, and the ability to measure multiple cell parameters simultaneously. Flow cytometry is widely used to determine both parasitemia and merozoite invasion in in vitro culture1-9, however, techniques for measuring these parameters in vivo are less well developed, and can be complicated by the presence of additional cell types which interfere with analysis. No assays have been described for measurement of in vivo invasion, and while some assays exist for the analysis of in vivo parasitemia, these lack the ability to distinguish between parasitized RBCs (pRBCs) and RBCs containing Howell-Jolly bodies (HJ-RBCs)10-13. The later issue is particularly important as in mice HJ-RBCs may account for up to 0.9% of mature RBCs14-16, thereby preventing the accurate measurement of low parasitemia. 
We have previously demonstrated an approach for the measurement of parasitemia and merozoite invasion in a rodent model of malaria infection14. Here, we provide a more detailed protocol and accompanying video. This approach builds on previous methodologies and allows for the accurate identification of parasitized RBCs, as distinct from leukocytes, RBC progenitors, and HJ-RBCs. Additionally, this assay allows the simultaneous measurement of merozoite invasion into two labeled RBC populations, a treated, or target, population, and a control population, thereby providing a robust platform for the assessment of invasion into different cell types.

<table><tbody><tr><td>bisBenzimide H 33342 trihydrochloride</td><td>Sigma-Aldrich</td><td>B2261</td><td>Hoechst 33342. Store a 4 mM stock solution at -20 &amp;deg;C in distilled water</td></tr><tr><td>Hoechst 34580</td><td>Sigma-Aldrich</td><td>63493</td><td>Store a 2 mM stock solution at -20 &amp;deg;C in distilled water</td></tr><tr><td>JC-1 Dye</td><td>Life Technologies</td><td>T-3168</td><td>Store small aliquots of 6 mM stock solution at -20 &amp;deg;C in DMSO</td></tr><tr><td>Anti-Mouse CD45 APC-eFluor 780</td><td>eBioscience</td><td>47-0451-80</td><td>Clone 30-F11</td></tr><tr><td>Anti-Mouse CD71 PerCP-eFluor 710</td><td>eBioscience</td><td>46-0711-80</td><td>Clone R17217</td></tr><tr><td>Atto 633 NHS ester</td><td>Sigma-Aldrich</td><td>1464</td><td>Atto 633-NHS. Store a 2 mg/ml stock solution at -20 &amp;deg;C in DMF</td></tr><tr><td>EZ-Link Sulfo-NHS-LC-Biotin</td><td>Thermo Fisher Scientific</td><td>21335</td><td>Biotin-NHS. Store a 25 mg/ml stock solution at -20 &amp;deg;C in DMF</td></tr><tr><td>Streptavidin PE-Cyanine7</td><td>eBioscience</td><td>25-4317-82</td><td>Streptavidin PE-Cy7</td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H478</td><td></td></tr><tr><td>35 &amp;micro;M filter cap tubes</td><td>Becton Dickinson</td><td>352235</td><td></td></tr><tr><td>Flow cytometer: BD LSRFortessa</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Flow cytometer: BD FACSAria II</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Flow cytometer: BD Influx</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Flow cytometer: CyAn ADP Analyzer</td><td>Beckman Coulter</td><td></td><td></td></tr></tbody></table>

in vivo assessment of rodent plasmodium parasitemia and merozoite invasion by flow cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

测量原虫的。 对于寄生虫血症的测定，血细胞应首先被选择，和噪音，碎片和血小板中排除，基于FSC / SSC特性（ 图2A）。根据所使用的流式细胞仪，然后应根据任一触发脉冲宽度（ 图2B），或FSC峰高度以面积比（ 图2C）中选出的单细胞。其余的事件应包括白细胞，染色呈阳性的APC eFluor 780，红细胞祖细胞（包括网织红细胞），染色阳性PerCP eFluor 710和成熟红细胞，负两种这些污渍（ 图2D）。选择成熟RBC人口后，事件应该分成四个群体，当JC-1荧光暗算赫斯特33342（ 图2E，F）和赫斯特34580（ 图2G，H）的荧光。双阳性人群表示寄生的红细胞，而第remaining种群要么未感染RBC（双阴性），HJ-红细胞（Hoechst的正，JC-1阴性），或未知的细胞（JC-1阳性，Hoechst的负数）。值得注意的是，JC-1荧光可能会移动从最大在529纳米到一个宽的发射峰值在590nm处，这取决于细胞的线粒体膜电位。无论这种转变的JC-1将在四十〇分之五百三纳米通道强烈荧光，使这一渠道的分析最适合的。然而，在某些情况下，可能值得在42分之585纳米通道，其可以提供细胞的线粒体膜电位的指示附加记录荧光。在未感染的样本事件小于0.007％应是双重阳性（ 图2E，G）。这一结果将取决于流式细胞仪的清洁度，并且可以有很大的不同，在分析前彻底清洗可能改进结果。 HJ-红细胞占0.3 - 成熟红细胞的0.9％，使得加入的JC-1染料临界用于ACC低原虫的尿酸盐测量。 裂殖子入侵评估。 注射标记的RBCs成感染小鼠后的相对侵袭率为两个标记的群体可以被确定。血样应截取的并且描述的用于测定寄生虫血症通过加入链霉抗PE Cy7的制备。在该样品所用的时间取决于实验条件和分析的期望结果。在一般情况下，一个较早的时间点会最好地反映了入侵的表型，虽然它可能是有益的，收集多个时间点，以增加精确度。用于流式细胞术数据的分析，成熟的红细胞应该被选择为在图2中，从这些细胞中，阿托633和生物素标记的RBCs可以基于分别荧光在三十○分之六百七十和750LP通道（ 图3A）来鉴定。从这里的三个红细胞种群（阿托633，生物素，以及未标记的）应分别进行分析。在每个这些种群原虫可以基于染色用Hoechst和JC-1（ 图3B-D）来确定。 <img alt="图1" src="/files/ftp_upload/52736/52736fig1.jpg" /> 示图1示意图的体内寄生虫侵袭测定的。如何使用此测定法可用于确定相对侵袭率红细胞根据用户的生物处理过的质询的一个例子。血液从感染小鼠收集被分成两管。一个管被视为所希望的，而其他的样品放未处理的（A）中。管被再次分割与一个标记的NHS-生物素和其他与NHS-阿托633（B）中 。然后样品被结合在两个组合;生物素标记处理的红细胞与阿托633标记未经处理的红细胞和阿托633标记的处理的红细胞与生物锡标记未处理的RBC（℃）。 15％原虫（D） -这两个组合在裂殖2中分别注入两批感染小鼠。总共6受体小鼠建议以获得统计意义的结果，虽然或多或少如果需要的话也可以使用。从Lelliott 等 14改编，最初是由生物医学中心出版。 <img alt="图2" src="/files/ftp_upload/52736/52736fig2highres.jpg" width="430" /> 图通过流式细胞2.测量寄生物血症的流式细胞仪。基于FSC / SSC特性（A） 的碎片，噪声和血小板从分析中删除。单个细胞，然后从基于任一触发脉冲宽度（B）或FSC峰面积比（C）中剩余的细胞中选择。细胞类型然后可以基于正染色的CD45 APC eFluor尊贵780（白细胞），CD71 PerCP eFluor 710（红细胞祖细胞），或负染色（成熟红细胞）（D）中 。选择成熟红细胞之后，将细胞或者是:Hoechst公司和JC-1阳性（寄生的红细胞），Hoechst的正和JC-1阴性（含有霍维尔Jolly小体的红细胞），JC-1阳性和阴性赫斯特（未知的细胞），或双阴性（未感染RBC）。未感染和P. chabaudi用Hoechst 33342（E，F），或赫斯特34580（G，H）感染的样本，显示， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52736/52736fig2highres.jpg" target="_blank">请点击这里查看此图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52736/52736fig3highres.jpg" width="430" /> 图裂殖子入侵3.评估分为两个标记RBC人群体内 。情节是成熟红细胞，门控如在图2中。可以根据它们的标签（A）中来选择这两个标记的RBC群体。阿托633标记细胞荧光在三十分之六百七十信道（L1）中，生物素标记的细胞结合的链霉亲和荧光中的PE-Cy7的通道（L3），和未标记的细胞是阴性的这些污渍（L4）。在这些人群红细胞寄生可以根据赫斯特和JC-1阳性（Q2）（BD）来识别的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52736/52736fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry at JoVE.com

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

疟原虫侵入和重复内红细胞。裂殖子入侵和原虫的准确评估，因此在评估疟疾感染的过程中是至关重要的。在这里，我们描述了一种流式细胞仪基础的协议用于疟疾的小鼠模型中，这些参数的测量。

在啮齿动物疟原虫原虫的体内评估和裂殖子入侵流式细胞仪

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

in-vivo-assessment-rodent-plasmodium-parasitemia-merozoite-invasion

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

9.9K 次观看.  Macquarie University. 疟原虫侵入和重复内红细胞。裂殖子入侵和原虫的准确评估，因此在评估疟疾感染的过程中是至关重要的。在这里，我们描述了一种流式细胞仪基础的协议用于疟疾的小鼠模型中，这些参数的测量。 

视频: In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

科研

免疫与感染

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

活体鼠脑微循环显微镜使用一个封闭的颅窗口

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

神经科学

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

脑幽静的白细胞的分离与分析<em&gt;伯氏疟原虫</emANKA感染的小鼠

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

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

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

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

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

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 ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

一个<em&gt;体外</em&gt;模型在HIV合并感染的背景下测量免疫应答疟疾

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

使用磁共振成像实验性脑疟疾模型中的水肿发育​​和微血管病理学的体内追踪

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

医学

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 ...

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 ...

在啮齿动物疟原虫原虫的体内评估和裂殖子入侵流式细胞仪

摘要

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此视频中的章节

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

活体鼠脑微循环显微镜使用一个封闭的颅窗口

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

脑幽静的白细胞的分离与分析

高产净化

一个

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

使用磁共振成像实验性脑疟疾模型中的水肿发育和微血管病理学的体内追踪

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

体外细胞毒性淋巴细胞杀伤疟原虫感染红细胞的测定

在啮齿动物疟原虫原虫的体内评估和裂殖子入侵流式细胞仪

摘要

探索更多视频

此视频中的章节

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

活体鼠脑微循环显微镜使用一个封闭的颅窗口

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

脑幽静的白细胞的分离与分析

高产净化

一个

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

使用磁共振成像实验性脑疟疾模型中的水肿发育​​和微血管病理学的体内追踪

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

体外 细胞毒性淋巴细胞杀伤疟 原虫感染红细胞的测定

使用磁共振成像实验性脑疟疾模型中的水肿发育和微血管病理学的体内追踪

体外细胞毒性淋巴细胞杀伤疟原虫感染红细胞的测定