Direct and high-throughput assays for human cell killing through trogocytosis by Entamoeba histolytica.
Direct and high-throughput assays for human cell killing through trogocytosis by Entamoeba histolytica.
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DOI：
10.1016/j.molbiopara.2020.111301
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发表时间：
2020-09
影响因子：
1.5
通讯作者：
Ralston KS
中科院分区：
文献类型：
--
作者：
Bettadapur A;Ralston KS
溶组织内阿米巴是阿米巴病的病原体。发病机制与人体组织的严重损伤有关。我们之前的研究表明，阿米巴通过巨噬细胞增多杀死人类细胞。巨噬细胞增多症很可能是感染过程中组织损伤的基础，尽管其机制尚不清楚。巨噬细胞增多症很难定量测定，这给研究带来了困难。在这里，我们开发了两种新的、互补的检测方法，通过量化人类细胞死亡来测量滋养细胞增多症。一种检测方法是使用CellTiterGlo，一种ATP的发光读数，作为细胞死亡的代用品。我们发现，CellTiterGlo可以用来检测与阿米巴共同孵育后的人类细胞的死亡，并且它对肌动蛋白或阿米巴表面Gal/GalNAc凝集素的抑制很敏感，这两种条件都能抑制阿米巴促红细胞增多症。另一种方法使用两种荧光核染色法在显微镜下直接区分活的和死亡的人类细胞，并且通过干扰肌动蛋白对阿米巴促红细胞增多症的抑制也很敏感。这两种检测方法都简单且廉价，可用于悬浮和贴壁的人类细胞类型，并适用于高通量方法。这些新的检测方法是提高对巨噬细胞增多症和阿米巴病发病机制的了解的工具。溶组织内阿米巴通过巨噬细胞增多症杀死人类细胞。我们开发了新的巨噬细胞增多症检测方法。一种方法是使用荧光核染色法来区分活的(蓝色)和死亡的(绿松石色)人类细胞。
Entamoeba histolytica is the causative agent of amoebiasis. Pathogenesis is associated with profound damage to human tissues. We previously showed that amoebae kill human cells through trogocytosis. Trogocytosis is likely to underlie tissue damage during infection, although the mechanism is still unknown. Trogocytosis is difficult to assay quantitatively, which makes it difficult to study. Here, we developed two new, complementary assays to measure trogocytosis by quantifying human cell death. One assay uses CellTiterGlo, a luminescent readout for ATP, as a proxy for cell death. We found that the CellTiterGlo could be used to detect death of human cells after co-incubation with amoebae, and that it was sensitive to inhibition of actin or the amoeba surface Gal/GalNAc lectin, two conditions that are known to inhibit amoebic trogocytosis. The other assay uses two fluorescent nuclear stains to directly differentiate live and dead human cells by microscopy, and is also sensitive to inhibition of amoebic trogocytosis through interference with actin. Both assays are simple and inexpensive, can be used with suspension and adherent human cell types, and are amenable to high-throughput approaches. These new assays are tools to improve understanding of trogocytosis and amoebiasis pathogenesis. Entamoeba histolytica kills human cells by performing trogocytosis. We developed new trogocytosis assays. One assay uses fluorescent nuclear stains to differentiate live (blue) and dead (turquoise) human cells.
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影响因子：
15.3
作者：
Bracha, R;Mirelman, D
通讯作者：
Mirelman, D
DOI：
10.1084/jem.152.2.377
发表时间：
1980-08-01
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Ravdin JI;Croft BY;Guerrant RL
通讯作者：
Guerrant RL
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
6.4
作者：
Miller, Hannah W.;Suleiman, Rene L.;Ralston, Katherine S.
通讯作者：
Ralston, Katherine S.
影响因子：
3.6
作者：
Bracha, R;Nuchamowitz, Y;Mirelman, D
通讯作者：
Mirelman, D