Extracellular vesicles (EVs) are constantly secreted from both eukaryotic and prokaryotic cells. EVs, including those referred to as exosomes, may have an impact on cell signaling and an incidence in diseased cells. In this manuscript, a platform to capture, quantify, and phenotypically classify the EVs secreted from single cells is introduced. Microfluidic chambers of about 300 pL are employed to trap and isolate individual cells. The EVs secreted within these chambers are then captured by surface-immobilized monoclonal antibodies (mAbs), irrespective of their intracellular origin. Immunostaining against both plasma membrane and cytosolic proteins was combined with highly sensitive, multicolor total internal reflection fluorescence microscopy to characterize the immobilized vesicles. The data analysis of high-resolution images allowed the assignment of each detected EV to one of 15 unique populations and demonstrated the presence of highly heterogeneous phenotypes even at the single-cell level. The analysis also revealed that each mAb isolates phenotypically different EVs and that more vesicles were effectively immobilized when CD63 was targeted instead of CD81. Finally, we demonstrate how a heterogeneous suppression in the secreted vesicles is obtained when the enzyme neutral sphingomyelinase is inhibited.

细胞外囊泡 (EV) 不断从真核细胞和原核细胞中分泌出来。EV，包括被称为外泌体的那些，可能对细胞信号传导和患病细胞的发生率产生影响。在这份手稿中，介绍了一个捕获、量化和表型分类单细胞分泌的 EV 的平台。使用约 300 pL 的微流体室来捕获和分离单个细胞。这些腔室内分泌的 EV 然后被表面固定的单克隆抗体 (mAb) 捕获，而不管它们的细胞内来源。针对质膜和胞质蛋白的免疫染色与高度敏感的多色全内反射荧光显微镜相结合，以表征固定化囊泡。高分辨率图像的数据分析允许将每个检测到的 EV 分配给 15 个独特的群体之一，并证明即使在单细胞水平上也存在高度异质的表型。分析还表明，每种 mAb 分离出表型不同的 EV，并且当靶向 CD63 而不是 CD81 时，更多的囊泡被有效固定。最后，我们展示了当酶中性鞘磷脂酶被抑制时，如何在分泌的囊泡中获得异质抑制。