PKH26 labeling of extracellular vesicles: Characterization and cellular internalization of contaminating PKH26 nanoparticles
PKH26 labeling of extracellular vesicles: Characterization and cellular internalization of contaminating PKH26 nanoparticles
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DOI：
10.1016/j.bbamem.2018.03.013
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发表时间：
2018-06-01
影响因子：
3.4
通讯作者：
Lenassi, Metka
中科院分区：
文献类型：
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作者：
Dominkus, Pia Puzar;Stenovec, Matjaz;Lenassi, Metka
PKH亲脂性染料通过将其脂肪部分插入暴露的脂双层中而成为高度荧光和染色的膜。它们已被用于标记和追踪体内和体外的细胞。尽管PKH标记的胞外小泡(EV)在细胞靶向和功能研究中得到了广泛的应用，但非EV相关的荧光结构从未被系统地研究过，它们也没有被细胞内化。在这里，我们使用共聚焦成像和不对称流动分离与多角度光散射检测器分析相结合的方法，通过超速离心、过滤、蔗糖垫和蔗糖梯度法染色，表征了淋巴母细胞B外切体样本和无外切体对照中的PKH26阳性颗粒。我们首次发现，在基于超速离心的外切体染色过程中形成了大量的PKH26纳米颗粒(十个PKH26阳性颗粒中有九个)，它们在大小、表面积和荧光强度方面与PKH26标记的外切体几乎没有区别。当PKH26标记的外切体通过蔗糖纯化时，PKH26纳米颗粒与PKH26标记的外切体的区别在于它们的尺寸减小。然而，只有当在蔗糖梯度上分离时，PKH26纳米颗粒才能物理地从PKH26标记的外切体中移除，并且以低PKH26标记的外切体回收率为代价。总体而言，低PKH26阳性颗粒回收率是基于滤过的外切体染色的特征。重要的是，PKH26纳米颗粒被原代星形胶质细胞内化到与PKH26标记的外切体相似的亚细胞室中。总之，PKH26纳米颗粒可以导致染色EV的假阳性信号，这可能会损害EV内化的解释。因此，为了用于EV摄取和功能研究，基于蔗糖梯度的分离方法应该是获得不含PKH26纳米颗粒的PKH26标记外切体的首选方法。
PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent structures have never been examined systematically, nor was their internalization by cells. Here, we have characterized PKH26-positive particles in lymphoblastoid B exosome samples and exosome-free controls stained by ultracentrifugation, filtration, and sucrose-cushion-based and sucrose-gradient-based procedures, using confocal imaging and asymmetric-flow field-flow fractionation coupled to multi-angle light-scattering detector analysis. We show for the first time that numerous PKH26 nanoparticles (nine out of ten PKH26-positive particles) are formed during ultracentrifugation-based exosome staining, which are almost indistinguishable from PKH26-labeled exosomes in terms of size, surface area, and fluorescence intensity. When PKH26-labeled exosomes were purified through sucrose, PKH26 nanoparticles were differentiated from PKH26-labeled exosomes based on their reduced size. However, PKH26 nanoparticles were only physically removed from PKH26-labeled exosomes when separated on a sucrose gradient, and at the expense of low PKH26-labeled exosome recovery. Overall, low PKH26-positive particle recovery is characteristic of filtration-based exosome staining. Importantly, PKH26 nanoparticles are internalized by primary astrocytes into similar subcellular compartments as PKH26-labeled exosomes. Altogether, PKH26 nanoparticles can result in false-positive signals for stained EVs that can compromise the interpretation of EV internalization. Thus, for use in EV uptake and functional studies, sucrose-gradient-based isolation should be the method of choice to obtain PKH26-labeled exosomes devoid of PKH26 nanoparticles.