Chapter 4 94 Figure 5 - Quantification of background signals and presence of CD63+ uEVs with IFCM. Concentrations of uEVs included in the double-positive gate (++; A), CD63-Alexa488 singlepositive gate (B), and CD63-APC single-positive gate (C). Negative controls compared with the double-stained healthy urine samples (n = 5).“CD63”: staining samples with CD63Alexa488 and CD63-APC;“Isotype” labeling urine with IgG1-Alexa488 and IgG1-APC;“+T”: urine incubated with 0.5% (v/v) TritonX-100 at room temperature for 30 min. “ns”: no significance; **p < 0.01. Serial dilutions confirm the single-particle analysis Serial dilutions were performed to verify the detection of single uEVs and the selection of singlets in the IFCM gating strategy.33 We serially diluted HC urine samples 3- and 9-fold in fDPBS and observed a linear decrease in CD63+ uEV concentration (R2 = 0.9992; Figure 6A). This decrease corresponded with the dilution factor, indicating the analysis of single that CD63+ uEVs.33 Moreover, diluted samples maintained consistent MESF-Alexa488 and MESF-APC signals of the CD63+ uEVs (Figures 6B & 6C). These findings confirmed that our gating strategy correctly identifies and selects single uEVs.
RkJQdWJsaXNoZXIy MTk4NDMw