Unambiguous determination of Plasmodium vivax reticulocyte invasion by flow cytometry
Unambiguous determination of Plasmodium vivax reticulocyte invasion by flow cytometry
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DOI：
10.1016/j.ijpara.2015.08.003
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发表时间：
2016-01-01
影响因子：
4
通讯作者：
Malleret, Benoit
中科院分区：
文献类型：
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作者：
Cho, Jee-Sun;Russell, Bruce;Malleret, Benoit
间日疟原虫对CD71+网织红细胞的入侵是一个关键的事件，但其特征尚不明确。将流式细胞术应用于间日疟原虫网织红细胞体外侵袭实验，有助于对间日疟原虫网织红细胞侵袭的定量分析。然而，由于缺乏针对间日疟原虫细胞嗜性的特定设计，目前的方案存在灵敏度低的问题。重要的是，裂殖子从裂殖体接种物侵入受污染的红细胞(自体侵入)可能会扰乱分析。在这里，我们提出了一种稳定的双色流式细胞术，用于准确定量间日疟原虫裂殖子对细胞内标记的CD71+网织红细胞的侵袭。各种酶处理、抗体和侵袭抑制分子被成功地证明了该方法的实用性。红细胞荧光标记不影响间日疟原虫的入侵和早期红细胞内发育。重要的是，这种便携式现场检测允许经济地使用有限的生物材料(寄生虫和网织红细胞)，并且目标细胞的细胞内标记减少了对高度纯化的裂殖体接种的需求。本试验将有助于间日疟原虫裂殖子生物学的研究和间日疟候选疫苗的测试。(C)2015年提交人。爱思唯尔有限公司出版。
The invasion of CD71+ reticulocytes by Plasmodium vivax is a crucial yet poorly characterised event. The application of flow cytometry to ex vivo invasion assays promises to facilitate the quantitative analysis of P. vivax reticulocyte invasion. However, current protocols suffer from a low level of sensitivity due to the absence of a particular design for P. vivax cell tropism. Importantly, merozoite invasion into contaminating red blood cells from the schizont inoculum (auto-invasion) may confound the analysis. Here we present a stable two-color flow cytometry assay for the accurate quantification of P. vivax merozoite invasion into intracellularly labelled CD71+ reticulocytes. Various enzymatic treatments, antibodies and invasion inhibitory molecules were used to successfully demonstrate the utility of this method. Fluorescent labelling of red blood cells did not affect the invasion and early intra-erythrocytic development of P. vivax. Importantly, this portable field assay allows for the economic usage of limited biological material (parasites and reticulocytes) and the intracellular labeling of the target cells reduces the need for highly purified schizont inoculums. This assay will facilitate the study of P. vivax merozoite biology and the testing of vaccine candidates against vivax malaria. (C) 2015 The Authors. Published by Elsevier Ltd.