Sung Bong H, Weaver Alissa M
Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA.
Methods Mol Biol. 2023;2608:83-96. doi: 10.1007/978-1-0716-2887-4_6.
Exosome secretion and uptake regulate cell migration through autocrine and paracrine mechanisms. Monitoring exosome secretion and uptake during cell migration is critical for investigation of these mechanisms. Exosomes can be visualized by direct labeling with fluorescent dyes or by tagging intrinsic markers with fluorescent proteins for live imaging. Due to several limitations of fluorescent dye-labeled exosomes, we created two bright genetically encoded reporters of exosome secretion, pHluorin_M153R-CD63 and pHluorin_M153R-CD63-mScarlet. Here, we describe how to visualize secretion and uptake of exosomes labeled with these pH-sensitive and pH-insensitive fluorescent protein-tagged exosomal markers during cell migration using time-lapse fluorescent microscopy.
外泌体的分泌和摄取通过自分泌和旁分泌机制调节细胞迁移。在细胞迁移过程中监测外泌体的分泌和摄取对于研究这些机制至关重要。外泌体可以通过用荧光染料直接标记或用荧光蛋白标记内在标志物以进行实时成像来可视化。由于荧光染料标记的外泌体存在若干局限性，我们创建了两种用于外泌体分泌的明亮的基因编码报告分子，即pHluorin_M153R-CD63和pHluorin_M153R-CD63-mScarlet。在此，我们描述了如何使用延时荧光显微镜在细胞迁移过程中可视化用这些对pH敏感和对pH不敏感的荧光蛋白标记的外泌体标志物标记的外泌体的分泌和摄取。
