Emma Tordoff, Jillian Allen, Katya Elgart, Ahmed Elsherbini, Vrinda Kalia, Haotian Wu, Erden Eren, Dimitrios Kapogiannis, Olesia Gololobova, Kenneth Witwer, Olga Volpert, Erez Eitan
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引用次数: 0
Abstract
Small membranous extracellular vesicles (EV) incorporate proteins and nucleic acids from the parent cell. Proteins exposed on EV surface are dictated by cellular origin and biogenesis pathway. To better understand the EV origin and function, it is important to develop methods that reveal surface protein composition of heterogeneous EV populations in culture supernatants and in biofluids. Tetraspanins CD9, CD63, and CD81 are common and abundant EV markers. However, their relative enrichment (profile) on EVs of specific cellular origins is not fully elucidated. We introduce LuminEV, a novel version of the Luminex assay for the multiplexed analysis of EV surface proteins. Optimized LuminEV reagents enable direct, specific, and sensitive measurements of EV markers in biofluids and in culture supernatants, bypassing EV isolation step. LuminEV assay for CD9, CD63, and CD81 was validated by comparing simplex and multiplex measurements, establishing linearity, spike-in recovery, inter- and intra-assay precision, and reproducibility between operators. LuminEV measurements of CD9, CD63, and CD81 in conditioned media from 15 cell lines revealed strong variations between cell types and showed high sensitivity, which enabled EV detection without prior concentration. Using tetraspanin levels as a readout, we noted suppression and induction of EV release from the cultured cells by GW6869 and monensin. Measurement of EV CD9, CD63, and CD81 in blood plasma from 70 disease-free donors showed respective abundance of 72, 16, and 12%. CD63 displayed weak, albeit significant, negative correlation with age and was slightly lower in female samples. The assay was then used to detect cell type-specific EV surface markers, including CD235a (erythrocytes), GAP43 (neurons), and CD68 (macrophages), and to detect differences in tetraspanin profiles between healthy and diseased donors. In summary, LuminEV offers robust and sensitive approach for multiplexed assessment of EV surface proteins, to facilitate the research into EV biology, biomarker, and therapeutic applications.
小膜细胞外囊泡(EV)含有来自母细胞的蛋白质和核酸。暴露在 EV 表面的蛋白质由细胞来源和生物生成途径决定。为了更好地了解 EV 的起源和功能,开发揭示培养上清液和生物流体中异质 EV 群体表面蛋白质组成的方法非常重要。四蛋白 CD9、CD63 和 CD81 是常见且丰富的 EV 标记。然而,它们在特定细胞来源的 EV 上的相对富集(概况)尚未完全阐明。我们推出了 LuminEV,这是一种用于 EV 表面蛋白多重分析的新型 Luminex 检测方法。经过优化的 LuminEV 试剂可直接、特异、灵敏地测定生物流体和培养上清液中的 EV 标记物,从而绕过 EV 分离步骤。LuminEV 检测 CD9、CD63 和 CD81 的方法通过比较单倍和多倍测量进行了验证,确定了线性、尖峰回收率、检测间和检测内精度以及操作者之间的可重复性。LuminEV 对 15 种细胞系的条件培养基中 CD9、CD63 和 CD81 的测量结果表明,不同细胞类型之间的差异很大,而且灵敏度很高,无需事先浓缩即可检测到 EV。利用四泛素水平作为读数,我们注意到 GW6869 和莫能菌素抑制和诱导了培养细胞中的 EV 释放。对 70 名无病捐献者血浆中的 EV CD9、CD63 和 CD81 的测量显示,其丰度分别为 72%、16% 和 12%。CD63 与年龄呈微弱但显著的负相关,在女性样本中略低。然后,该测定法被用来检测细胞类型特异性的 EV 表面标记物,包括 CD235a(红细胞)、GAP43(神经元)和 CD68(巨噬细胞),并检测健康供体和患病供体之间四泛素谱的差异。总之,LuminEV 为 EV 表面蛋白的多重评估提供了稳健而灵敏的方法,有助于 EV 生物学、生物标记和治疗应用的研究。
期刊介绍:
The Journal of Extracellular Vesicles is an open access research publication that focuses on extracellular vesicles, including microvesicles, exosomes, ectosomes, and apoptotic bodies. It serves as the official journal of the International Society for Extracellular Vesicles and aims to facilitate the exchange of data, ideas, and information pertaining to the chemistry, biology, and applications of extracellular vesicles. The journal covers various aspects such as the cellular and molecular mechanisms of extracellular vesicles biogenesis, technological advancements in their isolation, quantification, and characterization, the role and function of extracellular vesicles in biology, stem cell-derived extracellular vesicles and their biology, as well as the application of extracellular vesicles for pharmacological, immunological, or genetic therapies.
The Journal of Extracellular Vesicles is widely recognized and indexed by numerous services, including Biological Abstracts, BIOSIS Previews, Chemical Abstracts Service (CAS), Current Contents/Life Sciences, Directory of Open Access Journals (DOAJ), Journal Citation Reports/Science Edition, Google Scholar, ProQuest Natural Science Collection, ProQuest SciTech Collection, SciTech Premium Collection, PubMed Central/PubMed, Science Citation Index Expanded, ScienceOpen, and Scopus.