Isolation-Free Measurement of Single Urinary Extracellular Vesicles by Imaging Flow Cytometry 4 91 Detect fluorescently labelled uEV singlets Next, we aimed to distinguish between single-positive and double-positive uEVs. The cutoff fluorescent intensity of APC -/+ was established by staining samples with CD63-Alexa488 and isotype (IgG1)-APC. Based on the APC threshold, we set up the green gate in Supplementary Figure S6B to include the Alexa488-single-positive uEVs. Likewise, the Alexa488 -/+ cutoff value was set by staining urine with IgG1Alxea488 and CD63-APC and then obtaining the red gate in Supplementary Figure S6C for APC-single-positive uEVs. Doing so allowed us to establish a compensation matrix to minimize fluorescent spillover between Ch02 and Ch05. Fluorescent thresholds were set at 22 MESF-Alexa488, and 1463 MESF-APC. Using these fluorescent thresholds, we established a double-positive fluorescent region which allowed identification of double-positive uEVs (blue region in Supplementary Figure S6D) from single-positive uEVs. These thresholds were established based on multiple urine samples without gating differences observed, indicating that these gates can be repeatedly applied. Here, we summarized the logic of the whole IFCM gating strategy (Figure 4). After excluding A-F particles, the analysis of double-CD63-stained HC urine samples demonstrated three uEV populations as the final readout: CD63-Alexa488 singlepositive, CD63-APC single-positive, and double-positive uEV singlets.
RkJQdWJsaXNoZXIy MTk4NDMw