Isolation-Free Measurement of Single Urinary Extracellular Vesicles by Imaging Flow Cytometry 4 103 (M02, Ch02, Bright, 1) OR Peak (M05, Ch05, Bright, 1”. “Feature” is further applied upon Mask to analyze quantitative and positional information of selected pixels. Mask selection significantly influences feature characteristics because Mask determines the region of analysis for any given Feature.28 Hence, the combination of Feature and Mask is instrument/analysisspecific and independent of the sample. Intensity MC is designed to match Intensity Feature for quantifying fluorescence in Ch02, Ch03, Ch05, or SSC signals in Ch06. We chose Peak Masks (M02, Ch02, Bright, 1) and (M05, Ch05, Bright, 1) to be combined with “Spot Count Ch02” and “Spot Count Ch05” features, respectively, because Peak Mask can sensitively recognize fluorescent spot numbers in Ch02 and Ch05.22,28 Distance between spots presented in a single event but different channels can be analyzed after combining Masks in those channels. The feature “Spot Distance Min” was applied on the composite mask “Peak (M02, Ch02, Bright, 1) OR Peak (M05, Ch05, Bright, 1)” to measure the minimal distance between Alexa488+ and APC+ spots within an image.23 When this value of “Spot Distance (Ch02 & Ch05)” was 0, the positions of Alexa488+ and APC+ particles overlapped.23 An analysis template that summarizes all used features and accompanying masks can be found in Supplementary Table S2. Calibration of the IFCM Size-related calibration was performed by measuring SSC of beads of known diameter and refractive index (Gigamix, BioCytex, The Netherlands) followed by Mie theory using the scripts of Rosetta Calibration (v1.29, Exometry, The Netherlands).29 EVs were modeled as core-shell particles with a core refractive index of 1.38, a shell refractive index of 1.48 and a shell thickness of 6 nm. The calibration based on SSC using Gigamix and Rosetta Calibration has previously been reported by our team using the same machine and acquisition settings.23 Calibration of the fluorescence intensity was based on three QuantumTM MESF (molecules of equivalent soluble fluorochrome) kits containing four populations of beads with varying amounts of Alexa488, APC, or PE (Bangs Laboratories, USA).25 For each kit, the median fluorescence intensity (MFI) of beads was measured by IFCM and converted into MESF based on the instructions of the manufacturer (https://www.bangslabs.com/quickcal). Data was presented in Supplementary Table S3 & Figure S1. The regression coefficient values for each fluorochrome (R2-
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