Isolation-Free Measurement of Single Urinary Extracellular Vesicles by Imaging Flow Cytometry 4 99 uEVs.24 In contrast, in our protocol without filtration, we found around 107 objects/ mL of CD63+ uEVs. These findings suggest not using filtration or centrifugation when aiming to investigate the full (detectable) spectrum of uEVs. We chose two CD63 antibodies to demonstrate our methodology. Researchers can easily replace the labeling for detecting other markers but should be cautious in the antibody selection. The single staining with CD63-Alexa488 detected around a 5-fold uEV concentration compared to CD63-APC (Compare Supplementary Figure S5B with S5C). This finding might be attributed to the fluorophore to protein (F/P) ratio of CD63-Alexa488 (5.20) being much higher than CD63-APC (1.22). A Higher F/P value means brighter fluorescence of each antibody-epitope complex and hence higher sensitivity in the EV detection by FCM.41 In this study, we developed our protocol for application in patient samples. During kidney injury, the kidney excretes more THP than in healthy conditions,34 leading to more entrapment of uEVs.15 In the patient samples, we found the recovery of uEVs using DTT (10%) is not as significant as previous reports (20%),16,42 which might result from a lower concentration of DTT (25 mg/mL) we used than previous studies (200 mg/mL), because considering the detrimental effects of DTT on antibodies.36,43 The limitation of this study is the absence of other quantitative techniques available to compare our IFCM results. However, we do see a correlation between TR-FIA and IFCM (Supplementary Figure S9A). TR-FIA, independent of isolation, revealed that uEV concentration, not the epitope density on single uEVs, might determine the total uEV protein numbers. Our study met all the requirements of accurate IFCM measurement in the MISEV2018 guideline and MIFlowCyt-EV framework.1,33 As a method paper, we demonstrate that IFCM is a feasible tool to quantitate and characterize uEVs without bias-concomitant isolation. With our protocol (Figure 8), uEV researchers can measure targeted subpopulations of uEVs from 500 µL of urine by simply changing the antibody to other markers. More patient urine samples should be enrolled to make the results more robust for clinical application. Most EV studies relying on isolation used disunified isolation methods, which results in non-comparable data among them. Promoting standardized and isolation-free detection equals diminishing the research heterogeneity and integrating the data from single-center clinical studies. In conclusion, IFCM provides insight into the differences between single uEVs, A-F particles, and uEV multiplets, allowing for characterizing single uEVs without purification.
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