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Uptake Assay of Ram Seminal Plasma Extracellular Vesicles to Sperm
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DOI：10.21769/BioProtoc.5653.png)
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Abstract
En 中文
Extracellular vesicles (EVs) are critical mediators of cell-cell communication and play a key role in male reproductive biology by modulating sperm function. This protocol describes a robust and reproducible workflow for isolating EVs from ram seminal plasma using size-exclusion chromatography (SEC) and assessing their uptake by ram spermatozoa. In contrast to ultracentrifugation-based methods, SEC provides a gentle and more efficient isolation approach that preserves EV integrity and functionality. A central innovation of this protocol is the use of carboxyfluorescein succinimidyl ester (CFSE)labeled seminal plasma EVs (SP-EVs) to evaluate their incorporation into sperm cells through two complementary detection platforms: (i) flow cytometry with standard resolution and (ii) confocal microscopy, for spatial confirmation of EV-sperm interactions. By bridging the gap between EV isolation and functional analysis, this protocol provides a valuable tool for investigating the role of EV-cell interactions. Specifically, it offers potential applications in male fertility preservation, biomarker discovery, and the development of EV-based therapeutic strategies in reproductive medicine.
Keywords:
Extracellular vesicles
Seminal plasma
Sperm
Size-exclusion chromatography
CFSE
Flow cytometry
Journal
B
IF:
1.1
Papers:
191
Citations:
5.4K
Organization
Cited Papers