Chapter 5 146 BV421 fluorescence signals were collected in channel 1 (435–505-nm filter), CFSE signals were collected in channel 2 (505–560 nm filter), PE fluorescence signals were collected in channel 3 (560–595-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®). Supplementary Figure 1 – Identification and validation of single EV measurements using Imaging Flow Cytometry (IFCM). A) From left to right: fluorescence intensity scatterplots representative for unstained, stained (CFDA-SE & anti-tetraspanin antibody mixture (antiCD9/anti-CD63/anti-CD81)), and isotype control end-point NMP perfusate samples (T = 360 minutes). Fluorescent populations were established on the basis of unstained and single-stained end-point NMP samples (data not shown). B) Visual examination of events representative for each fluorescent population demonstrated the selection and subsequent analysis of particles showing single spot fluorescence (no coincidence events), indicating the selection and analysis of single EVs. C) Quantification of fluorescent events in each gate for unstained, stained and isotype control (N=2), showing the specificity of our staining protocol. In unstained samples, no fluorescent events were observed indicating that no auto-fluorescent events were detected. In stained samples, we observed concentrations of fluorescent events (>E8 objects/mL) well above unstained and isotype levels (<E5 Objects/mL), indicating that the fluorescent events acquired in samples stained with the anti-tetraspanin antibody mixture represent specific fluorescent positive events.
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