An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 57 of incubation at room temperature in the dark. Control samples not stained with CFDA-SE were incubated with 100 µL fPBS instead. All samples were brought to a total volume of 380 µL using fPBS before IFCM measurements. Controls Assay controls were used in all experiments, as recommended by the MIFlowCytEV framework 18 (Supplementary Table 1 and 2). These controls consisted of fPBS, fPBS with reagents, unstained samples, single-stained samples, isotype controls (matched with their corresponding fluorophore-conjugated mAbs at the same concentrations) and samples subjected to detergent treatment. A 10% (v/v) Triton X-100 stock solution was made by dissolving 1 mL of TritonX-100 in 9 mL of fPBS. Detergent treatment was performed by the addition of 20 µL of the Triton X-100 stock solution (final concentration: 0.5% (v/v) per test), followed by 30 minutes of incubation at room temperature in the dark prior to acquisition. Note that samples were first acquired as described in the section “Data acquisition (Step VII)” before detergent treatment and corresponding re-acquisition was performed. Supplementary Table 3 gives an overview of these controls as well as the rationale behind their use. All controls contained 4% (v/v) 25x concentrated protease inhibitor cocktail solution (cOmplete Protease inhibitor cocktail tablets, Roche, Mannheim, Germany) in accordance with the PPP samples. Usage of polystyrene beads for calibration purposes A mix of commercial fluorescent polystyrene (PS) beads was used to calibrate fluorescence and light scattering signals. Megamix-Plus FSC (lot 203372) and Megamix-Plus SSC (lot 210812) beads (BioCytex) were mixed at a 1:1 ratio, resulting in a mix containing green fluorescent bead populations with sizes of 100, 300 and 900 nm from the Megamix-Plus FSC bead set, and 160, 200 and 240 nm from the Megamix-Plus SSC bead-set and 500 nm from both; this mix was termed Gigamix. Rainbow Calibration Particles (RCP-05-5, lot AL01, Spherotech) with known Equivalent number of Reference Fluorophores (ERF) values for C30/FITC/APC (as determined on a Beckman Coulter CytoFLEX) were used in the standardization of the fluorescent detection channels Ch01,Ch02 & Ch05, respectively. For each detection channel, the MFI of each peak from the four bead populations (1 blanc – 3 fluorescent) were measured, and a linear regression analysis was performed of
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