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.
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DOI：
10.1002/cyto.a.20972
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发表时间：
2010-11
期刊：
影响因子：
3.7
通讯作者：
Rayner, Julian C.
中科院分区：
文献类型：
--
作者：
Theron, Michel;Hesketh, Richard L.;Subramanian, Sathish;Rayner, Julian C.
关键词：
恶性疟原虫基因分型最近经历了一场革命，目前正在收集大量寄生虫分离株的全基因组基因型数据集。相比之下，表型分析技术已经落后，几乎没有高通量表型分析平台可用。恶性疟原虫对人红细胞的侵入是一种特别感兴趣的表型，因为它在寄生虫发育中起着重要作用。入侵是一种受寄生虫和宿主的自然遗传变异影响的可变表型，并受多种重叠和在某些情况下冗余的寄生虫-红细胞相互作用的影响。为了促进红细胞侵袭表型的扩大，我们开发了一种基于双色流式细胞术的新平台，该平台区分寄生虫侵袭和寄生虫生长。用细胞内染料CFDA-SE或DDAO-SE预标记用酶处理去除了一种或多种受体的靶细胞，与恶性疟原虫寄生虫一起孵育，用荧光DNA嵌入染料Hoechst 33342或SYBR绿色I检测侵入标记或未标记细胞的寄生虫。两种细胞标记均不干扰红细胞侵袭，并且细胞和寄生虫染料的组合概括了三种标准实验室菌株的已知侵袭表型。已经验证了具有最小重叠的三种不同染料组合，这意味着相同的测定可以适用于具有几种不同激光线组合的仪器。该测定是敏感的，在96孔格式中操作，并且可用于定量天然或实验遗传变异对红细胞侵袭效率的影响。© 2010国际细胞计数促进学会
Plasmodium falciparum genotyping has recently undergone a revolution, and genome-wide genotype datasets are now being collected for large numbers of parasite isolates. By contrast, phenotyping technologies have lagged behind, with few high throughput phenotyping platforms available. Invasion of human erythrocytes by Plasmodium falciparum is a phenotype of particular interest because of its central role in parasite development. Invasion is a variable phenotype influenced by natural genetic variation in both the parasite and host and is governed by multiple overlapping and in some instances redundant parasite–erythrocyte interactions. To facilitate the scale-up of erythrocyte invasion phenotyping, we have developed a novel platform based on two-color flow cytometry that distinguishes parasite invasion from parasite growth. Target cells that had one or more receptors removed using enzymatic treatment were prelabeled with intracellular dyes CFDA-SE or DDAO-SE, incubated with P. falciparum parasites, and parasites that had invaded either labeled or unlabeled cells were detected with fluorescent DNA-intercalating dyes Hoechst 33342 or SYBR Green I. Neither cell label interfered with erythrocyte invasion, and the combination of cell and parasite dyes recapitulated known invasion phenotypes for three standard laboratory strains. Three different dye combinations with minimal overlap have been validated, meaning the same assay can be adapted to instruments harboring several different combinations of laser lines. The assay is sensitive, operates in a 96-well format, and can be used to quantitate the impact of natural or experimental genetic variation on erythrocyte invasion efficiency. © 2010 International Society for Advancement of Cytometry
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影响因子：
12.3
作者：
Dharia NV;Sidhu AB;Cassera MB;Westenberger SJ;Bopp SE;Eastman RT;Plouffe D;Batalov S;Park DJ;Volkman SK;Wirth DF;Zhou Y;Fidock DA;Winzeler EA
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影响因子：
3.1
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通讯作者：
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影响因子：
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