Extracellular vesicles (EVs) are identified as mediators of intercellular communication and cellular regulation. In the immune system, EVs play a role in antigen presentation as a part of cellular communication. To enable drug discovery and characterization of compounds that affect EV biogenesis, function, and release in immune cells, we developed and characterized a reporter cell line that allows the quantitation of EVs shed into culture media in phenotypic high-throughput screen (HTS) format. Tetraspanins CD63 and CD9 were previously reported to be enriched in EVs; hence, a construct with dual reporters consisting of CD63-Turbo-luciferase (Tluc) and CD9-Emerald green fluorescent protein (EmGFP) was engineered. This construct was transduced into the human monocytic leukemia cell line, THP-1. Cells expressing the highest EmGFP were sorted by flow cytometry as single cell, and clonal pools were expanded under antibiotic selection pressure. After 4 passages, the green fluorescence dimmed, and EV biogenesis was then tracked by luciferase activity in culture supernatants. The Tluc activities of EVs shed from CD63Tluc-CD9EmGFP reporter cells in the culture supernatant positively correlated with the concentrations of released EVs measured by nanoparticle tracking analysis. To examine the potential for use in HTS, we first miniaturized the assay into a robotic 384-well plate format. A 2210 commercial compound library (Maybridge) was then screened twice on separate days, for the induction of extracellular luciferase activity. The screening data showed high reproducibility on days 1 and 2 (78.6%), a wide signal window, and an excellent Z' factor (average of 2-day screen, and 0.54). One hundred eighty-seven compounds showed a response ratio that was 3SD above the negative controls in both day 1 and 2 screens and were considered as hit candidates (approximately 10%). Twenty-two out of 40 re-tested compounds were validated. These results indicate that the performance of CD63Tluc-CD9EmGFP reporter cells is reliable, reproducible, robust, and feasible for HTS of compounds that regulate EV release by the immune cells.

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用于细胞外囊泡释放的定量筛选的报告细胞系的产生和应用

细胞外囊泡（EVs）被确定为细胞间通讯和细胞调节的介质。在免疫系统中，电动汽车在抗原呈递中起着细胞通信的作用。为了能够发现和鉴定影响EV生物发生，功能和在免疫细胞中释放的化合物的药物，我们开发并鉴定了一种报告细胞系，该细胞可以定量分析以表型高通量筛选（HTS）格式注入到培养基中的EV。以前曾报道四跨膜蛋白CD63和CD9富含EV。因此，设计了具有由CD63-涡轮荧光素酶（Tluc）和CD9-翡翠绿色荧光蛋白（EmGFP）组成的双重报告基因的构建体。该构建体被转导至人单核细胞白血病细胞系THP-1。通过流式细胞术将表达最高EmGFP的细胞分类为单细胞，并在抗生素选择压力下扩增克隆库。4次传代后，绿色荧光变暗，然后通过培养物上清液中的萤光素酶活性追踪EV生物发生。从培养物上清液中的CD63Tluc-CD9EmGFP报告基因细胞释放出的EV的Tluc活性与通过纳米颗粒跟踪分析测量的释放的EV的浓度呈正相关。为了检查在HTS中使用的潜力，我们首先将测定法微型化为自动384孔板形式。然后在分开的日子中两次筛选2210商业化合物库（Maybridge），以诱导细胞外萤光素酶活性。筛选数据显示第1天和第2天的重现性高（78.6％），宽信号窗口和出色的Z' 系数（2天屏幕的平均值和0.54）。在第1天和第2天的筛选中，一百八十七种化合物的反应率比阴性对照高3SD，被认为是命中候选物（约10％）。验证了40种重新测试的化合物中的22种。这些结果表明，CD63Tluc-CD9EmGFP报告基因细胞对于调节免疫细胞释放EV的化合物的HTS而言是可靠，可重现，强大且可行的。