Generation and Application of a Reporter Cell Line for the Quantitative Screen of Extracellular Vesicle Release.
Generation and Application of a Reporter Cell Line for the Quantitative Screen of Extracellular Vesicle Release.
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DOI：
10.3389/fphar.2021.668609
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发表时间：
2021
影响因子：
5.6
通讯作者：
Hayashi T
中科院分区：
文献类型：
--
作者：
Shpigelman J;Lao FS;Yao S;Li C;Saito T;Sato-Kaneko F;Nolan JP;Shukla NM;Pu M;Messer K;Cottam HB;Carson DA;Corr M;Hayashi T
细胞外囊泡（EVs）被认为是细胞间通讯和细胞调节的介质。在免疫系统中，EV作为细胞通讯的一部分在抗原呈递中发挥作用。为了使药物发现和表征的化合物，影响EV生物发生，功能，并在免疫细胞中释放，我们开发和表征了一种报告细胞系，允许定量的EV脱落到培养基中的表型高通量筛选（HTS）格式。四跨膜蛋白CD 63和CD 9先前被报道在EV中富集;因此，工程化了具有由CD 63-Turbo-荧光素酶（Tluc）和CD 9-Emerald绿色荧光蛋白（EmGFP）组成的双报告子的构建体。将该构建体转导到人单核细胞白血病细胞系THP-1中。通过流式细胞术将表达最高EmGFP的细胞分选为单细胞，并在抗生素选择压力下扩增克隆池。传代4次后，绿色荧光变暗，然后通过培养上清液中的荧光素酶活性追踪EV生物发生。培养上清液中从CD 63 Tluc-CD 9 EmGFP报告细胞脱落的EV的Tluc活性与通过纳米颗粒跟踪分析测量的释放的EV的浓度正相关。为了检查在HTS中使用的潜力，我们首先将测定小型化到机器人384孔板格式中。然后在不同的日子筛选2210商业化合物文库（Maybridge）两次，用于诱导细胞外荧光素酶活性。筛选数据显示第1天和第2天的重现性高（78.6%），信号窗宽，Z′因子极佳（2天筛选的平均值为0.54）。在第1天和第2天筛选中，187种化合物显示出高于阴性对照3SD的响应比，并被视为命中候选物（约10%）。40种重新测试的化合物中有22种得到验证。这些结果表明，CD 63 Tluc-CD 9 EmGFP报告细胞的性能对于调节免疫细胞EV释放的化合物的HTS是可靠的、可再现的、稳健的和可行的。
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 four 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, 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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影响因子：
7.3
作者：
Andreu Z;Yáñez-Mó M
通讯作者：
Yáñez-Mó M
DOI：
10.1073/pnas.1521230113
发表时间：
2016-02-23
影响因子：
11.1
作者：
Kowal, Joanna;Arras, Guillaume;Thery, Clotilde
通讯作者：
Thery, Clotilde
影响因子：
4.6
作者：
Brennan, K.;Martin, K.;Mc Gee, M. M.
通讯作者：
Mc Gee, M. M.
影响因子：
7.4
作者：
Garcia-Romero, N.;Madurga, R.;Ayuso-Sacido, A.
通讯作者：
Ayuso-Sacido, A.
影响因子：
9.7
作者：
Datta A;Kim H;Lal M;McGee L;Johnson A;Moustafa AA;Jones JC;Mondal D;Ferrer M;Abdel-Mageed AB
通讯作者：
Abdel-Mageed AB