Extracellular Vesicles are Associated with Human Donor Kidney Characteristics 5 125 Figure 1 – Concentration and size distribution of particles released by a discarded kidney during (60, 180, 360 minutes - T60 / T180 / T360, respectively) Normothermic Machine Perfusion (NMP), as measured with Nanoparticle Tracking Analysis (NTA). T0 represents particle concentration and size distribution in baseline perfusate. A clear increase in particle concentration was observed during NMP and the majority of released particles were observed to be < 400 nm (red striped lines). Detergent treatment confirms the analysis of EVs Next, we stained the perfusate samples of 8 NMP kidneys with CFDA-SE and an anti-tetraspanin antibody mixture (anti-CD9/anti-CD63/anti-CD81) labeled with APC, and measured the samples with Imaging Flow Cytometry (IFCM). CFDASE is converted to CFSE (carboxyfluorescein succiminidyl ester) by intravesicular esterases and was used to discriminate EVs from contaminating agents such as lipoproteins. Identification and validation of single EV measurement by IFCM is presented in Supplemental Figure 1. For the stained samples, we observed CFSE and tetraspanin single positive – but very few double-positive (< 70 events) - fluorescent background events in perfusate samples drawn before exposure to the kidney (T0). Samples collected after 60 / 180 / 360 minutes of NMP showed increases in fluorescent events across all three populations (CFSE single-positive, Tetraspanin single-positive and CFSE and
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