Chapter 4 90 permanently applied to distinguish non-A-F particles from A-F particles in all the following uEV-IFCM analyses (Supplementary Figure S5). Typical images of distinguished positive uEVs and excluded A-F particles were presented in Figure 3I. Figure 3 - Distinguish non-A-F particles/uEVs from A-F particles in the stained urine using IFCM. No compensation in A-C, E, F. Based on fDPBS (A), the gates“Negative Particle” and“Positive Particle” were established and applied to present A-F particles in the unprocessed urine (B). (C) A-F particles and positive uEVs could not be distinguished in urine with doubleCD63 staining. (D) Methods for removing A-F particles in unprocessed urine (n = 5).“Original urine”: unstained urine without any treatments. Compared to the control, the “Lower laser power” group used a quarter of the laser voltage of Ch02 and Ch05; “Detergent treatment” was incubated with 2.0% TritonX-100 at room temperature for 30 min; “Centrifugation” was centrifuged at 10.000 g for 10 min and removed the pellet. **p < 0.01; *p < 0.05; ns, no significance. (E) The absence of A-F particles in the urine supernatant after centrifugation (10.000 g 10 min). (F) Centrifuged urine was CD63-stained to present the distribution of non-A-F particles (uEVs). (G) A compensation matrix was applied in (F) to eliminate spillovers between Ch02 and Ch03, and the gate “Non-A-F Particles” was set up. (H) The compensation matrix and the gate “Non-A-F Particles” were applied to the CD63-stained urine (no centrifugation). (I) Typical images of positive uEVs and A-F particles in double-CD63-stained urine.
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