Extracellular Vesicles are Associated with Human Donor Kidney Characteristics 5 139 Nanoparticle Tracking Analysis Size distribution and concentration of nanoparticles within the perfusates were measured with the Malvern Panalytical NanoSight NS300 and analyzed with Nanoparticle Tracking Analysis (NTA) software version NTA 3.4 Build 3.4.003. Samples were diluted in 0.2 µm filtered PBS (fPBS) until 20 – 60 particles were in the field of focus during acquisition and 10 videos of 15 seconds were recorded with camera level 11 and analyzed with detection threshold 5. Sample Labelling and Controls Perfusates were stained with monoclonal antibodies (mAbs) and Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) as extensively described in our previous work 23 and detailed in SDC, Materials and Methods; Sample Labelling. To ascertain EV measurements the following controls were applied, as recommended by the MIFlowCyt-EV framework 25: buffer only, buffer with reagents, unstained samples, isotype controls, and detergent treatment, which aims to disrupt the membranous structure of EVs thereby allowing discrimination between biological and artificial events. Detergent treatment was performed by adding 20 µL of a 10% (V/V) TritonX-100 detergent to the samples followed by 30 minutes of incubation at room temperature prior to acquisition. Data acquisition and analysis All samples were acquired on an ImageStreamX MkII instrument (ISx; Luminex). Settings as extensively described in our previous work 23 and detailed in SDC, Materials and Methods; Acquisition were used. Data analysis was performed using Amnis IDEAS software (version 6.2). To ensure the analysis of EVs we 1) selected all particles with SSC intensities ≤ 900 a.u., and 2) identified and excluded coincidence detection by counting the number of fluorescent spots within the pixel grid for each event acquired; events showing multiple spots were excluded from analysis 23. This gating strategy ensures the selection and analysis of single spot fluorescent particles ≤400 nm. Gating areas and cut-offs were established through identification of (fluorescent) populations in unstained and single stained samples, and arbitrary fluorescent intensities were converted into Equivalent Molecules of Fluorescence (ERF) values based on
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