Exo-Flow 2.0 CD63 Analysis Kit (for Serum or Plasma)

See the performance that Exo-Flow 2.0 technology delivers

Exo-Flow 2.0 delivers undetectable background binding when analyzed by western blot

We isolated EVs from HEK293 cells and serum using ExoQuick^®-TC and ExoQuick^® Exosome Isolation Reagents, captured specific EV subpopulations using Exo-Flow 2.0 beads coated with either anti-CD63, anti-CD9, anti-CD81, or a no-antibody control, and analyzed the captured EVs by western blot probing for the exosome-specific marker TSG101 (Figure 3). The absence of detectable signal in the no-antibody control lane demonstrates the low background binding of Exo-Flow 2.0 technology. The varying signal intensity seen in Figure 3 also highlights the heterogeneity of EV subpopulations.

Exo-Flow 2.0 delivers undetectable background binding when analyzed using flow cytometry

We isolated EVs from HEK293 cells using ExoQuick-TC and captured the subpopulation of EVs expressing CD9 (Figure 4A, dark red trace) and the subpopulation of EVs bearing CD63 (Figure 4A orange trace). We also isolated EVs from serum using the SmartSEC HT EV Isolation system and captured the subpopulation of EVs bearing CD81 (Figure 4B).  After staining bead-bound EVs and washing away excess stain, we analyzed the EV subpopulations by flow cytometry. In both plots, distinct subpopulations of EVs can be seen—in Figure 4A, the peak seen on the red trace indicates a subpopulation of EVs bearing CD9 and the separate peak seen on the orange trace indicates a subpopulation of EVs bearing CD63. In Figure 4B, the peak seen on the green trace indicates a subpopulation of EVs bearing CD81.

In both plots, the overlap of signal in the beads-only and no-antibody traces demonstrates the low background binding of Exo-Flow 2.0 technology.

Understand surface marker differences between EV subpopulations with Exo-Flow 2.0

We isolated EVs from serum using SmartSEC HT, captured subpopulations of EVs using Exo-Flow 2.0 beads coupled with either biotinylated CD9 (Figure 5A) or CD63 (Figure 5B) antibodies, and analyzed these subpopulations for the presence of CD14, a myeloid lineage marker, using flow cytometry (Figure 5). The data show that EVs captured using anti-CD9 Exo-Flow 2.0 beads are more likely to also bear CD14 than EVs captured using anti-CD63- Exo-Flow 2.0 beads, demonstrating the effectiveness of Exo-Flow 2.0 technology for EV subpopulation analysis and enabling better biomarker screening.

Use Exo-Flow 2.0 to understand differences in cargo, such as miRNA levels, between EV subpopulations

We isolated EVs from HEK293-conditioned medium using ExoQuick-TC, captured subpopulations of EVs using Exo-Flow 2.0 beads coupled with either biotinylated CD63 or CD81 antibodies, and analyzed these subpopulations for the presence of miR-122 (Figure 6). The data show a statistically significant difference in the amount of miR-122 present in these EV subpopulations, with ~3.6-fold more miR-122 found in CD81 EVs than CD63 EVs, demonstrating the effectiveness of Exo-Flow 2.0 technology for EV subpopulation analysis.