Isolation, Characterization, and MicroRNA Analysis of Extracellular Vesicles from Bovine Oviduct and Uterine Fluids

Characterization and profiling analysis of bovine oviduct and uterine extracellular vesicles and their miRNA cargo through the estrous cycle

“…The isolation of OF‐EVs and UF‐EVs was performed as previously reported by our group, 23 based on size‐exclusion chromatography (SEC) PURE‐EV® (HansaBioMed Life Sciences); considered a very efficient technique for separating EVs from the circulating proteins and that does not affect the original shape and functionality of the vesicles 24 ; followed by ultracentrifugation. Briefly, after discarding the buffer provided within the SEC column, the column was washed with 30 ml of PBS − , then either OF (≃1 ml) or UF (≃2 ml) fluid sample was loaded onto the top of the SEC column, and when the sample was completely within the column, 11 ml of PBS − were loaded, avoiding the column to get dried.…”

Emerging micro-nanotechnologies for extracellular vesicles in immuno-oncology: from target specific isolations to immunomodulation

“…91 Immunoaffinity isolation reduces the processing time, in comparison to DGUC, and has high specificity for those intending to isolate subgroups of EVs. 92 2.1.3. Limitations of microtechnology based EV isolation and future of EV isolation.…”

“…Nanoparticle tracking analysis (NTA) is a powerful dark-field microscopy characterization technique that utilizes the properties of laser light scattering and the randomized Brownian motion of the particles, much like DLS. 92,101 Combining these two principles allows for the measurement of the sample size distribution and concentration in liquid suspension. 101 The NanoSight is the more common NTA capable instrument and, much like other instruments, operates by using a digital camera to capture the light scattered by the particles by laser illumination and measuring their Brownian motion through tracking.…”

“…91 Immunoaffinity isolation reduces the processing time, in comparison to DGUC, and has high specificity for those intending to isolate subgroups of EVs. 92…”

“…However, this size distribution is most accurate when the particles in suspension are monodispersed. 92 In polydisperse suspensions, larger particles influence the size distribution due to excessive light scattering. Therefore, the sample source for DLS is severely limited to non-biological sources or else some filtration step will be required to reduce the sample's heterogeneity.…”