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首页> 外文期刊>Clinical and diagnostic laboratory immunology >Early Events in Macrophage Killing ofAspergillus fumigatus Conidia: New Flow Cytometric Viability Assay
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Early Events in Macrophage Killing ofAspergillus fumigatus Conidia: New Flow Cytometric Viability Assay

机译：烟曲霉分生孢子巨噬细胞杀伤的早期事件：新的流式细胞仪活力测定

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Detailed investigations of macrophage phagocytosis and killing ofAspergillus fumigatus conidia have been limited by technical difficulties in quantifying fungal uptake and viability. In order to study early events in cell pathogen ingestion and killing, we developed a new flow cytometry assay that utilizes the fungus-specific viability dye FUN-1. Metabolically active A. fumigatusconidia accumulate orange fluorescence in vacuoles, while dormant or dead conidia stain green. After incubation within THP-1 cells, recovered conidia are costained with propidium iodide (PI) to discriminate between dormant and dead cells. Flow cytometric measurements of FUN-1 metabolism and PI uptake provide indicators of conidial viability, dormancy, and death. Conidial phagocytosis and killing are also assessed by measurement of green and orange FUN-1 fluorescence within the THP-1 cell population. Compared to previously described methods, this assay has less error introduced by membrane permeability changes and serial dilution of filamentous fungal forms. Results suggest that the THP-1 cells kill conidia rapidly (within 6 h) after exposure. Conidia that are preexposed to human serum are ingested and killed more quickly than are nonopsonized conidia.
机译：巨噬细胞吞噬作用和烟曲霉分生孢子杀死的详细研究受到定量真菌吸收和生存能力的技术难题的限制。为了研究细胞病原体摄入和杀灭的早期事件，我们开发了一种新的流式细胞术测定法，该方法利用了真菌特异性生存力染料FUN-1。代谢活跃的 A。烟的分生孢子在液泡中积累橙色荧光，而休眠或死的分生孢子则染成绿色。在THP-1细胞中孵育后，将回收的分生孢子与碘化丙啶（PI）染色，以区分休眠细胞和死细胞。 FUN-1代谢和PI摄取的流式细胞仪测量可提供分生孢子活力，休眠和死亡的指标。分生孢子的吞噬作用和杀灭作用还可以通过测量THP-1细胞群体中的绿色和橙色FUN-1荧光来评估。与先前描述的方法相比，该测定法具有较小的误差，该误差是由膜渗透性变化和丝状真菌形式的连续稀释引起的。结果表明，THP-1细胞在暴露后迅速（6小时内）杀死分生孢子。与未调理过的分生孢子相比，预先暴露于人血清中的分生孢子的摄取和杀死速度更快。

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