Comparison of Generic Fluorescent Markers for Detection of Extracellular Vesicles by Flow Cytometry
Comparison of Generic Fluorescent Markers for Detection of Extracellular Vesicles by Flow Cytometry
复制标题
DOI：
10.1373/clinchem.2017.278978
复制
发表时间：
2018-04-01
影响因子：
9.3
通讯作者：
Coumans, Frank A. W.
中科院分区：
文献类型：
--
作者：
de Rond, Leonie;van der Pol, Edwin;Coumans, Frank A. W.
背景：生物体液中的细胞外囊泡（EV）是潜在的疾病生物标志物。为了探索 EV 的临床相关性，一种特定的通用 EV 标记物将很有用，该标记物不需要抗体并与所有 EV 结合。这里我们评估了5种常用的流式细胞术通用标记物。 方法：使用流式细胞术（A60-Micro，Apogee）评估通用EV标记物钙黄绿素乙酰氧基甲酯、钙黄绿素乙酰氧基甲酯紫、羧基荧光素琥珀酰亚胺酯（CFSE）、4-(2-[6-(二辛氨基)-2-萘基]的能力。乙烯基)1-(3-磺丙基)吡啶 (di-8-ANEPPS) 和乳粘素，对来自 MCF7 人乳腺癌细胞系条件培养基 [上皮细胞粘附分子阳性 (EpCAM(+))] 的 EV 或来自人血浆的血小板 EV [整合素 β 3 阳性 (CD61(+))] 进行染色。以侧向散射触发作为参考，评估非 EV 成分（蛋白质和脂蛋白）的影响。结果：Di-8-ANEPPS、乳粘素和侧向散射检测到 100% 的 EpCAM(+) MCF7 EV。乳粘素和侧向散射分别检测到 33% 和 61% 的 CD61(+) EV。仅当首先去除可溶性蛋白时，Di-8-ANEPPS 才能检测到血小板 EV。由于所有通用标记都会对蛋白质进行染色，因此最多检测到血浆中 33% 的血小板 EV。钙黄绿素标记物和 CFSE 要么对两个样本中的 EV 不敏感，要么与群体检测相关。结论：没有一种通用标记物检测到血浆中的所有且仅检测到的 EV。侧向散射触发在我们的 A60-Micro 上检测到血浆 EV 浓度最高，其次是乳粘素。散射或乳粘素之间的选择主要取决于所用流式细胞仪的分析灵敏度。 (c) 2018 年美国临床化学协会
BACKGROUND: Extracellular vesicles (EVs) in biofluids are potential biomarkers of disease. To explore the clinical relevance of EVs, a specific generic EV marker would be useful, one that does not require antibodies and binds to all EVs. Here we evaluated 5 commonly used generic markers for flow cytometry.METHODS: Flow cytometry (A60-Micro, Apogee) was used to evaluate the ability of the generic EV markers calcein acetoxymethyl ester, calcein acetoxymethyl ester violet, carboxyfluorescein succinimidyl ester (CFSE), 4-(2-[6-(dioctylamino)-2-naphthalenyl] ethenyl)1-(3-sulfopropyl) pyridinium (di-8-ANEPPS), and lactadherin to stain EVs from MCF7 human breast adenocarcinoma cell line-conditioned culture medium [epithelial cell adhesion molecule positive (EpCAM(+))] or platelet EVs from human plasma [integrin beta 3 positive (CD61(+))]. Side scatter triggering was applied as a reference, and the influence of non-EV components (proteins and lipoproteins) was evaluated.RESULTS: Di-8-ANEPPS, lactadherin, and side scatter detected 100% of EpCAM(+) MCF7 EVs. Lactadherin and side scatter detected 33% and 61% of CD61(+) EVs, respectively. Di-8-ANEPPS detected platelet EVs only if soluble protein was first removed. Because all generic markers stained proteins, at best 33% of platelet EVs in plasma were detected. The calcein markers and CFSE were either insensitive to EVs in both samples or associated with swarm detection.CONCLUSIONS: None of the generic markers detected all and only EVs in plasma. Side scatter triggering detected the highest concentration of plasma EVs on our A60-Micro, followed by lactadherin. The choice between scatter or lactadherin primarily depends on the analytical sensitivity of the flow cytometer used. (c) 2018 American Association for Clinical Chemistry