Large Extracellular Vesicles Can be Characterised by Multiplex Labelling Using Imaging Flow Cytometry.
Large Extracellular Vesicles Can be Characterised by Multiplex Labelling Using Imaging Flow Cytometry.
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DOI：
10.3390/ijms21228723
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发表时间：
2020-11-18
影响因子：
5.6
通讯作者：
McCabe MG
中科院分区：
文献类型：
--
作者：
Johnson SM;Banyard A;Smith C;Mironov A;McCabe MG
细胞外囊泡（EVs）在大小（30 nm-10µm）、含量（脂质、RNA、DNA、蛋白质）和潜在功能上是异质性的。许多分离技术通常在小EV或外泌体分离方案的早期阶段放弃大EV。我们在此描述了一种从成神经管细胞瘤细胞中分离大型EV的标准化方法，并使用成像流式细胞术检测EV标记物的表达和直径。我们的方法允许将每个大型电动汽车表征为单个事件，并用多个荧光共轭标记进行装饰，具有可视化每个事件的额外优势，以确保应用稳健的门控策略。方法：我们描述了从成神经管细胞瘤细胞系UW228-2中逐步分离和表征的一个大ev亚群，通过荧光显微镜、透射电子显微镜（TEM）和可调电阻脉冲传感（TRPS）进行评估。用流式细胞术检测Annexin V暴露测定亲本细胞活力。成像流式细胞术（Imagestream Mark II）通过细胞掩膜橙（CMO）与EV标记物直接荧光膜标记来鉴定EV。采用基于侧散射和荧光强度的严格门控算法，评估EV标记CD63、CD9和LAMP 1的表达。结果：UW228-2细胞可增殖释放最大6µm的ev。我们表明，Imagestream Mark II成像流式细胞仪可以对大型ev进行稳健且可重复的分析，包括直径评估。我们还证明了EV大小的增加与筛选的标记物的共表达之间的相关性。结论：我们已经开发了一种标记和严格的门控策略，能够在广泛异质群体中的单个EV上探索EV标记（CD63， CD9和LAMP1）的表达。综上所述，本文提供的数据有力地支持了在临床样本中探索大型ev的潜在生物标志物的价值，这些生物标志物可用于诊断筛查和疾病监测。
Extracellular vesicles (EVs) are heterogeneous in size (30 nm–10 µm), content (lipid, RNA, DNA, protein), and potential function(s). Many isolation techniques routinely discard the large EVs at the early stages of small EV or exosome isolation protocols. We describe here a standardised method to isolate large EVs from medulloblastoma cells and examine EV marker expression and diameter using imaging flow cytometry. Our approach permits the characterisation of each large EVs as an individual event, decorated with multiple fluorescently conjugated markers with the added advantage of visualising each event to ensure robust gating strategies are applied. Methods: We describe step-wise isolation and characterisation of a subset of large EVs from the medulloblastoma cell line UW228-2 assessed by fluorescent light microscopy, transmission electron microscopy (TEM) and tunable resistance pulse sensing (TRPS). Viability of parent cells was assessed by Annexin V exposure by flow cytometry. Imaging flow cytometry (Imagestream Mark II) identified EVs by direct fluorescent membrane labelling with Cell Mask Orange (CMO) in conjunction with EV markers. A stringent gating algorithm based on side scatter and fluorescence intensity was applied and expression of EV markers CD63, CD9 and LAMP 1 assessed. Results: UW228-2 cells prolifically release EVs of up to 6 µm. We show that the Imagestream Mark II imaging flow cytometer allows robust and reproducible analysis of large EVs, including assessment of diameter. We also demonstrate a correlation between increasing EV size and co-expression of markers screened. Conclusions: We have developed a labelling and stringent gating strategy which is able to explore EV marker expression (CD63, CD9, and LAMP1) on individual EVs within a widely heterogeneous population. Taken together, data presented here strongly support the value of exploring large EVs in clinical samples for potential biomarkers, useful in diagnostic screening and disease monitoring.
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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：
16
作者：
Menck K;Bleckmann A;Wachter A;Hennies B;Ries L;Schulz M;Balkenhol M;Pukrop T;Schatlo B;Rost U;Wenzel D;Klemm F;Binder C
通讯作者：
Binder C
影响因子：
16
作者：
Crescitelli R;Lässer C;Szabó TG;Kittel A;Eldh M;Dianzani I;Buzás EI;Lötvall J
通讯作者：
Lötvall J
影响因子：
4.6
作者：
Headland SE;Jones HR;D'Sa AS;Perretti M;Norling LV
通讯作者：
Norling LV
影响因子：
16
作者：
Simonsen, Jens B.
通讯作者：
Simonsen, Jens B.