GFP Reporter Cell Line - BV2

Mounting evidence suggests that storage affects the properties of extracellular vesicles (EVs). Here, researchers found that storage at -80°C reduces EV concentration and sample purity in a time-dependent manner. Furthermore, it increases particle size and size variability, and alters the zeta potential of EVs, causing a shift in the size-charge diagram. None of the testing conditions prevented these observed effects. Freeze-thaw cycling resulted in a decrease in EVs after the first cycle and an increase in particle size with increasing cycle number. Flow cytometry revealed a large number of double-positive EVs (GFP+-mCherry+) after storage. This observation may indicate fusion during storage. These results suggest that storage has a significant impact on EV samples, including particle loss, reduced purity, and fusion leading to artificial particles. Based on subsequent analysis and experimental setup, in most cases, EV processing should likely utilize fresh, non-archived samples.

Western blot analysis was performed on extracellular vesicle (FE) samples derived from fresh plasma (three samples from three different healthy donors) and BV2-derived FE samples (three different samples prepared for each of the following three scenarios: BV2 WT, BV2 fGFP, and BV2 mCherry-f). BV2 fGFP and mCherry-f cells served as controls. Western blot analysis confirmed the presence of extracellular vesicle markers, such as LAMP1, ALIX, Flotillin-1, and Annexin A1 (ANXA-1), in both FE and BV2-derived FE samples (Figure 1a). Except for histone H3, which was detectable in the two BV2 WT-derived FE samples (Figure 1a), other non-extracellular vesicle markers, such as histone H3, GM130, and apolipoprotein E, were not detected in the FE samples. As expected, ANXA-1 was highly expressed in BV2 cells. The analysis also confirmed the expression of GFP and mCherry in engineered BV2 cells and their corresponding extracellular vesicles (EVs). The researchers performed transmission electron microscopy (TEM) analysis on the flow cytometry (FE) samples. TEM analysis confirmed the presence of particles with a typical cup-shaped morphology, consistent in size with EVs (Figure 1b). Immunogold labeling showed that the observed particles were positively stained by the tetraspan membrane protein CD63 (a typical marker of EVs) (Figure 1c).

Figure 1. EVs characterization: Western Blotting and Transmission Electron Microscopy. (Gelibter S, et al., 2022)