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>

<ol>
	<li>Jacobberger, J. W., Horan, P. K., Hare, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+malaria+parasite-infected+blood+by+flow+cytometry.">Analysis of malaria parasite-infected blood by flow cytometry.</a> Cytometry. 4 (3), 228-237 (1983).</li><li>Bianco, A. E., Battye, F. L., Brown, G. V. <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:+rapid+quantification+of+parasitemia+in+fixed+malaria+cultures+by+flow+cytometry.">Plasmodium falciparum: rapid quantification of parasitemia in fixed malaria cultures by flow cytometry.</a> Exp Parasitol. 62 (2), 275-282 (1986).</li><li>Makler, M. T., Lee, L. G., Recktenwald, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiazole+orange:+a+new+dye+for+Plasmodium+species+analysis.">Thiazole orange: a new dye for Plasmodium species analysis.</a> Cytometry. 8 (6), 568-570 (1987).</li><li>Heyde, H. C., Elloso, M. M., van de Waa, J., Schell, K., Weidanz, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+hydroethidine+and+flow+cytometry+to+assess+the+effects+of+leukocytes+on+the+malarial+parasite+Plasmodium+falciparum.">Use of hydroethidine and flow cytometry to assess the effects of leukocytes on the malarial parasite Plasmodium falciparum.</a> Clin Diagn Lab Immunol. 2 (4), 417-425 (1995).</li><li>Pattanapanyasat, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+malaria+parasites+in+two+different+red+blood+cell+populations+using+biotin+and+flow+cytometry.">Culture of malaria parasites in two different red blood cell populations using biotin and flow cytometry.</a> Cytometry. 25 (3), 287-294 (1996).</li><li>Grimberg, B. T., Erickson, J. J., Sramkoski, R. M., Jacobberger, J. W., Zimmerman, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+Plasmodium+falciparum+growth+and+development+by+UV+flow+cytometry+using+an+optimized+Hoechst-thiazole+orange+staining+strategy.">Monitoring Plasmodium falciparum growth and development by UV flow cytometry using an optimized Hoechst-thiazole orange staining strategy.</a> Cytometry A. 73 (6), 546-554 (2008).</li><li>Theron, M., Hesketh, R. L., Subramanian, S., Rayner, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+adaptable+two-color+flow+cytometric+assay+to+quantitate+the+invasion+of+erythrocytes+by+Plasmodium+falciparum+parasites.">An adaptable two-color flow cytometric assay to quantitate the invasion of erythrocytes by Plasmodium falciparum parasites.</a> Cytometry A. 77 (11), 1067-1074 (2010).</li><li>Bei, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow+cytometry-based+assay+for+measuring+invasion+of+red+blood+cells+by+Plasmodium+falciparum.">A flow cytometry-based assay for measuring invasion of red blood cells by Plasmodium falciparum.</a> Am J Hematol. 85 (4), 234-237 (2010).</li><li>Clark, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RBC+barcoding+allows+for+the+study+of+erythrocyte+population+dynamics+and+P.+falciparum+merozoite+invasion.">RBC barcoding allows for the study of erythrocyte population dynamics and P. falciparum merozoite invasion.</a> PLoS One. 9 (7), e101041 (2014).</li><li>Malleret, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+robust+tri-color+flow+cytometry+assay+for+monitoring+malaria+parasite+development.">A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development.</a> Sci Rep. 1 (118), (2011).</li><li>Jimenez-Diaz, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+Plasmodium-infected+erythrocytes+in+murine+models+of+malaria+by+flow+cytometry+using+bidimensional+assessment+of+SYTO-16+fluorescence.">Quantitative measurement of Plasmodium-infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO-16 fluorescence.</a> Cytometry A. 75 (3), 225-235 (2009).</li><li>Jimenez-Diaz, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+detection+specificity+of+Plasmodium-infected+murine+erythrocytes+by+flow+cytometry+using+autofluorescence+and+YOYO-1.">Improvement of detection specificity of Plasmodium-infected murine erythrocytes by flow cytometry using autofluorescence and YOYO-1.</a> Cytometry A. 67 (1), 27-36 (2005).</li><li>Jun, G., Lee, J. S., Jung, Y. J., Park, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+determination+of+Plasmodium+parasitemia+by+flow+cytometry+and+microscopy.">Quantitative determination of Plasmodium parasitemia by flow cytometry and microscopy.</a> J Korean Med Sci. 27 (10), 1137-1142 (2012).</li><li>Lelliott, P. M., Lampkin, S., McMorran, B. J., Foote, S. J., Burgio, 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+flow+cytometric+assay+to+quantify+invasion+of+red+blood+cells+by+rodent+Plasmodium+parasites+in+vivo.">A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo.</a> Malar J. 13 (1), 100 (2014).</li><li>Morohashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+abnormalities+in+the+spleen+of+an+mFtz-F1+gene-disrupted+mouse.">Structural and functional abnormalities in the spleen of an mFtz-F1 gene-disrupted mouse.</a> Blood. 93 (5), 1586-1594 (1999).</li><li>Shet, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+functional+platelet+abnormalities+in+Berkeley+sickle+cell+mice.">Morphological and functional platelet abnormalities in Berkeley sickle cell mice.</a> Blood Cells Mol Dis. 41 (1), 109-118 (2008).</li><li>Duraisingh, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+variation+of+Plasmodium+falciparum+merozoite+proteins+directs+receptor+targeting+for+invasion+of+human+erythrocytes.">Phenotypic variation of Plasmodium falciparum merozoite proteins directs receptor targeting for invasion of human erythrocytes.</a> EMBO J. 22 (5), 1047-1057 (2003).</li><li>Okoyeh, J. N., Pillai, C. R., Chitnis, C. 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，?...

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>bisBenzimide H 33342 trihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>B2261</td>
			<td>Hoechst 33342. Store a 4mM stock solution at -20 &#176;C in distilled water</td>
		</tr>
		<tr>
			<td>Hoechst 34580</td>
			<td>Sigma-Aldrich</td>
			<td>63493</td>
			<td>Store a 2mM stock solution at -20 &#176;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 6mM stock solution at -20 &#176;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 2mg/ml stock solution at -20 &#176;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 25mg/ml stock solution at -20 &#176;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&#181;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 ...

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

Research

JoVE Journal

Immunology and Infection

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

9.5K 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。 