An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 79 Requirement Please Include Requested Information 4.4.1. Gate description The lower fluorescence threshold for Ch01 (BV421), Ch02 (CFSE), and Ch05 (APC) was set at 110, 170, and 170 a.u., respectively. These values were obtained by analyzing blanc-fluorescent Rainbow Calibration Particles (RCP-05-5, lot AL01, Spherotech), unstained PPP samples, and isotype control PPP samples. When scaled to ERF units, these values translated to 1397.171 / 38.40 / 28.03 ERF, respectively. Upper fluorescent limits (high-end gating cut-off) for Ch01 (BV421), Ch02 (CFSE), and Ch05 (APC) was set at 100.000, 50.553, and 10.302 a.u., respectively. These gating cut-offs were determined to encompass all obtained fluorescent events. When scaled to ERF units, these values translated to 89125 / 3656 / 133 ERF, respectively. 4.4.2. Gate statistics Median Fluorescent Intensity (MFI) – Count – Objects/mL 4.4.3. Gate boundaries See above Supplemental Table 2 – Checklist representing the Minimal Information about a Flow Cytometry (FC) experiment to allow standardized EV-FC-specific reporting (MIFlowCyt-EV checklist), as recommended by the Minimum Information for Studies of EVs (MISEV).
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