Direct Detection of Circulating Donor-Derived Extracellular Vesicles in Kidney Transplant Recipients 6 167 speed of 43.59 ± 0.07 mm/sec (mean ± standard deviation). Core size was set at 6 µm, autofocus was activated and the ‘Remove Speedbead’ option was checked. BV421 fluorescence signals were collected in channel 1 (435–505-nm filter), APC signals in channel 5 (642–745 nm filter), and SSC signals in channel 6 (745–785 nm filter). Particle enumeration was achieved through the advanced fluidic control of the ISx coupled with continuously running speed beads, resulting in the “objects/ mL” feature within the ISx Data Exploration and Analysis Software (IDEAS). Data Analysis Data analysis was performed using Amnis IDEAS software (version 6.2). Image display mapping was linearly adjusted for all fluorescent events for each channel and then applied to all files of the respective experiment. 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 number of Reference Fluorophores (ERF) values based on previously published calibration data 23. Lower and upper gating area cut-offs were defined as 677 – 112,201 ERF for BV421, and 6.40 – 123 ERF for APC. Statistical Analysis Statistical analysis was performed using R version 4.0.2 and RStudio (RStudio Team (2016). RStudio: Integrated Development for R. RStudio, Inc., Boston, MA URL http:// www.rstudio.com/.) version 1.1.463. All concentrations reported in this work were corrected for sample dilution (before acquisition – 380 µL total volume per test containing 30 µL sample = ~12.33-fold dilution factor) and are shown as the mean ± standard deviation unless specified otherwise. Statistical significance between EV concentrations and groups was determined through two-sided t-tests, 95% CI with unpaired data.
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