A simple flow cytometry method improves the detection of phosphatidylserine-exposing extracellular vesicles.
A simple flow cytometry method improves the detection of phosphatidylserine-exposing extracellular vesicles.
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DOI：
10.1111/jth.12767
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发表时间：
2015-02
期刊：
影响因子：
--
通讯作者：
Brisson AR
中科院分区：
文献类型：
--
作者：
Arraud N;Gounou C;Linares R;Brisson AR
血浆中含有细胞来源的细胞外囊泡，参与机体的生理病理过程，作为疾病的生物标志物具有潜在的应用价值。然而，由于EV的亚微米尺寸和表征方法（主要是流式细胞术（FCM））的固有限制，EV的计数面临主要问题。我们的目标是通过以磷脂酰丝氨酸（PS）暴露EV的人群为原型，对血浆中的EV进行计数，这些EV构成了主要的EV人群之一，并导致血栓性疾病。通过FCM使用光散射或荧光作为触发剂和荧光膜联蛋白-5（Anx 5）作为特异性标记来测量健康受试者的无血小板血浆（PFP）中暴露于PS的EV的浓度。此外，PS-暴露的EV在用Anx 5金纳米颗粒标记并在EM网格上沉降后通过电子显微镜（EM）计数。我们表明，与光散射相比，当检测触发荧光时，FCM检测到约50倍以上的Anx 5阳性EV。通过荧光触发，使用两种不同的流式细胞仪测定每μL PFP中22 000-30 000个Anx 5阳性EV的浓度。荧光触发方法的检测限估计为约1000-2500个Anx 5分子。来自EM的结果表明，通过荧光触发检测到直径低至100-150 nm的EV。这项研究提出了一种简单的方法来计数电动汽车。我们相信，这种方法适用于一般情况下，并将提高我们的理解EV的作用，在病理生理情况下，这将开辟道路EV为基础的诊断检测的发展。
Plasma contains cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications as disease biomarker. However, the enumeration of EVs faces major problems, due to their sub-micrometer size and to intrinsic limitations in methods of characterization, mainly flow cytometry (FCM). Our objective is to enumerate EVs in plasma, by taking as the prototype the population of phosphatidylserine (PS)-exposing EVs, which constitute one of the major EV populations and are responsible for thrombotic disorders. The concentration of PS-exposing EVs in platelet-free plasma (PFP) of healthy subjects was measured by FCM using either light scattering or fluorescence as the trigger and fluorescent Annexin-5 (Anx5) as the specific label. In addition, PS-exposing EVs were enumerated by electron microscopy (EM) after labeling with Anx5 gold nanoparticles and sedimentation on EM grids. We show that about 50× more Anx5-positive EVs are detected by FCM when detection is triggered on fluorescence as compared with light scattering. By fluorescence triggering, concentrations of 22 000–30 000 Anx5-positive EVs per μL PFP were determined, using two different flow cytometers. The limit of detection of the fluorescence triggering method was estimated at about 1000–2500 Anx5 molecules. Results from EM suggest that EVs down to 100–150 nm diameter are detected by fluorescence triggering. This study presents a simple method for enumerating EVs. We believe that this method is applicable in a general context and will improve our understanding of the roles of EVs in pathophysiological situations, which will open avenues for the development of EV-based diagnosis assays.
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影响因子：
7.5
作者：
Manly DA;Wang J;Glover SL;Kasthuri R;Liebman HA;Key NS;Mackman N
通讯作者：
Mackman N
影响因子：
3.2
作者：
HERCHER, M;MUELLER, W;SHAPIRO, HM
通讯作者：
SHAPIRO, HM
影响因子：
16.6
作者：
Bouter A;Gounou C;Bérat R;Tan S;Gallois B;Granier T;d'Estaintot BL;Pöschl E;Brachvogel B;Brisson AR
通讯作者：
Brisson AR
DOI：
10.1126/science.1181928
发表时间：
2010-01-29
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Boilard E;Nigrovic PA;Larabee K;Watts GF;Coblyn JS;Weinblatt ME;Massarotti EM;Remold-O'Donnell E;Farndale RW;Ware J;Lee DM
通讯作者：
Lee DM
影响因子：
20.3
作者：
BEVERS, EM;WIEDMER, T;SIMS, PJ
通讯作者：
SIMS, PJ