Figure 5

Effect of post-thaw processing on EVs using flow cytometry. (A) Schematic diagram of differentially processed plasma (S1, S2), respective freeze thaw samples (S1FR, S2FR), and secondary spin after post-freeze/thaw plasma S1FR (S1FRS2). (B) Representative flow cytometry dot plot of EV diameter (nm) versus fluorescent intensity in Quantum MESF units for CD9⁺ EVs, CD63⁺, and CD41⁺ EVs in S1FR, S2FR, and S1FRS2 conditions using FlowJo. Quantum Alexa Fluor 647 MESF is used for Alexa Fluor 647 conjugated CD9 stained plasma, Quantum Alexa Fluor 488 MESF is used for Alexa Fluor 488 conjugated CD63 stained plasma, and Quantum PE MESF is used for PE conjugated CD41 stained plasma. Events were gated from 150 to 1000 nm (green box) and from 1000 to 3000 nm (red box). (C) Box plot of CD9⁺, CD63⁺, CD41⁺ EV concentration from 1000–3000 nm (red) and 150–1000 nm (green) for S1FR, S2FR, and S1FRS2 using R. 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).