Figure 4

Effect of a freeze thaw cycle on EVs using flow cytometry. (A) Schematic diagram of differentially processed plasma (S1, S2) and respective freeze thaw processes (S1FR, S2FR). (B) Box plot of CD9⁺, CD63⁺, and CD41⁺ of gated events from 1000–3000 nm (red) and 150–1000 nm (green) for S1, S1FR, S2, and S2FR using R. CD9⁺, CD63⁺, CD41⁺ events were converted to concentrations using calibrated flow rate in a given acquisition time. EV concentration defined as the number of EVs per μl was determined by number of EVs detected in a given sample volume multiplied by the dilution factor. The sample volume was calculated by the product of measured flow rate and acquisition time. Statistical significance were obtained from three healthy volunteers for each freeze thaw processing condition using Tukey’s multiple comparisons (ns = not significant, P > 0.05; *P < 0.05, ***P < 0.001, ****P < 0.0001).