An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 59 We adjusted the default sample core size of 7 µm to 6 µm using the “Defaults Override” option within INSPIRE software (version 200.1.681.0), as recommended by the manufacturer. Data were acquired over 180 seconds for standardization among samples with the autofocus setting activated and the “Remove Speedbead” option unchecked. These settings are shown in Supplementary Table 4 for quick reference. BV421 fluorescence signals were collected in channel 1 (435–505-nm filter), CFSE signals in channel 2 (505–560-nm filter) and APC signals in channel 5 (642–745-nm filter). Channel 4 was used as the brightfield channel, and channel 6 (745–785-nm filter) was used for SSC detection. Particle enumeration was achieved through the advanced fluidic control of the ISx coupled with continuously running SBs (used by the IFCM to measure sample velocity for camera synchronization during acquisition, and enables particle enumeration during analysis), and application of 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). The image display mapping was linearly adjusted for all fluorescent events for each channel and then applied to all files from their respective experiments. The IDEAS software utilizes ‘masks’ – defined as the algorithm which selects pixels within an image based on their intensity and localization – to define the analysis area of each event within the pixel grid. The “masks combined” (MC) standard setting was used to quantify all fluorescence intensities in the channels used during acquisition corresponding to the fluorochromes used (Ch01, Ch02 & Ch05). Fluorescent events from singly stained PPP samples were used in the setting of compensation matrices (to compensate for spectral overlap between fluorochromes) such that straight fluorescent populations were obtained when depicted in scatterplots. Singlepositive gating areas were established based on these single-positive fluorescent populations, and double-positive gates were set based on the boundaries of the single-positive gates. Unstained samples were used in the definition of the lowend of the various gates. Fluorescent thresholds were verified using cut-off values from the blanc fluorescent bead populations in the Rainbow Calibration Particles.
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