Exosomes are among the most puzzling vehicles of intercellular communication, but several crucial aspects of their biogenesis remain elusive, primarily due to the difficulty in purifying vesicles with similar sizes and densities. Here we report an effective methodology for labelling small extracellular vesicles (sEV) using Bodipy FL C16, a fluorescent palmitic acid analogue. In this study, we present compelling evidence that the fluorescent sEV population derived from Bodipy C16-labelled cells represents a discrete subpopulation of small exosomes following an intracellular pathway. Rapid cellular uptake and metabolism of Bodipy C16 resulted in the incorporation of fluorescent phospholipids into intracellular organelles specifically excluding the plasma membrane and ultimately becoming part of the exosomal membrane. Importantly, our fluorescence labelling method facilitated accurate quantification and characterization of exosomes, overcoming the limitations of nonspecific dye incorporation into heterogeneous vesicle populations. The characterization of Bodipy-labelled exosomes reveals their enrichment in tetraspanin markers, particularly CD63 and CD81, and in minor proportion CD9. Moreover, we employed nanoFACS sorting and electron microscopy to confirm the exosomal nature of Bodipy-labelled vesicles. This innovative metabolic labelling approach, based on the fate of a fatty acid, offers new avenues for investigating exosome biogenesis and functional properties in various physiological and pathological contexts.

中文翻译：

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使用荧光棕榈酸类似物对小细胞外囊泡亚群进行代谢标记

外泌体是细胞间通讯最令人费解的载体之一，但其生物发生的几个关键方面仍然难以捉摸，这主要是由于难以纯化具有相似大小和密度的囊泡。在这里，我们报告了一种使用荧光棕榈酸类似物 Bodipy FL C16 标记小细胞外囊泡 (sEV) 的有效方法。在这项研究中，我们提出了令人信服的证据，证明源自 Bodipy C16 标记细胞的荧光 sEV 群体代表了遵循细胞内途径的小外泌体的离散亚群。Bodipy C16 的快速细胞摄取和代谢导致荧光磷脂掺入细胞内细胞器中，特别排除质膜，并最终成为外泌体膜的一部分。重要的是，我们的荧光标记方法促进了外泌体的准确定量和表征，克服了非特异性染料掺入异质囊泡群体的限制。Bodipy 标记的外泌体的表征揭示了它们富含四跨膜蛋白标记物，特别是 CD63 和 CD81，以及少量的 CD9。此外，我们采用 nanoFACS 分选和电子显微镜来确认 Bodipy 标记的囊泡的外泌体性质。这种基于脂肪酸命运的创新代谢标记方法为研究各种生理和病理背景下的外泌体生物发生和功能特性提供了新途径。