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Challenges in counting of target cell-derived exosome : A protocol for minimized the influence of exosome derived from fetal bovine serum

  • 이기쁨 (Ki-ppeum Lee, 서강대학교 일반대학원)

초록

Exosomes are nano-sized (30–150 nm) membrane vesicles secreted by various types of cells and are thought to play an important role in cell-to-cell communication. Since exosomes reflect the characteristics of the blast cell of origin, tumor-derived exosomes contain various biomarkers such as nucleic acids and proteins that characterize the tumor, and diagnosis using exosomes has been extensively studied. The issue to consider in exosome research is that the amount of exosomes secreted is not fixed, so counting and normalization are essential for accurate analysis. However, there are some challenges in counting or analyzing cell-derived exosomes. The reason is that FBS (Fetal Bovine Serum) used for cell growth has a significant effect on exosome analysis. Even if the exosome- depleted serum is used, FBS-derived exosomes are not completely removed, and a large number (x10^7) of bovine-derived exosomes are present in the isolated exosomes. This causes errors in biomarker analysis. Although many studies overlook this issue, only cell- derived exosomes need to be differentiated for accurate analysis. Therefore, here we propose a protocol to minimize the influence of exosomes derived from FBS for accurate exosome analysis from target cells.

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목차

1. Introduction 2
2. Materials and Methods 5
2.1 Cell culture 5
2.2 Transmission electron microscopy (TEM) 5
2.3 Exosome Isolation and fluorescent labeling 6
2.4 Nanoparticle Tracking Analysis (NTA) 6
2.5 RNA extraction and cDNA synthesis 7
2.6 Quantitative real-time Polymerase Chain Reaction (qRT-PCR) 8
3. Results and Discussion 9
3.1 Exosome Characterization 9
3.2 . Influence of FBS on exosome counting 11
3.3 Count only target cell-derived exosome 13
3.4 Normalization of miRNA expression level by exosome number 15
4. Conclusion 17
5. Reference 18

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