Chapter 3 38 Human plasma single EV can be discriminated from artifact signals through detergent treatment After optimizing the protocol to identify single fluorescent sub-micron sized particles above background in PPP of healthy individuals, we tested the protocols’ ability to discriminate legitimate EV signals from artefact signals. We hypothesized that single EV could be identified as double-positive events after staining with both CFDA-SE and the anti-tetraspanin mixture, as these events would represent structurally intact, esterase containing sub-micron sized particles bearing common EV antigens. To test this hypothesis, we examined the fluorescent populations of particles ≤400 nm in diameter in 1 fPBS and the same 5 PPP samples by combining both fluorescent stains. Following our gating strategy, gating areas were reestablished on the basis of unstained and single-stained fPBS and PPP samples, as well as isotype controls. Gating cut-offs were determined to encompass all obtained fluorescent events for all PPP samples. Visual interrogation of the events in the identified fluorescent gates confirmed that the events analyzed met the criteria imposed by the gating strategy: (co-localized) single-spot fluorescence (Figure 3a). After acquisition of double-stained PPP (Figure 3b - I), we used detergent treatment (30 minutes incubation with 20 µL 10% (v/v) TritonX-100) to disrupt the lipid bilayer of EV and thereby remove EV signals from the measurement (Figure 3b – II). Fluorescent particles such as free antibodies or disrupted membrane fragments bearing antigens-antibodies remaining after detergent treatment were measured to allow the identification of artifact events, and the number of fluorescent events still present after detergent treatment were compared with the number of total fluorescent events before detergent treatment on a gate-by-gate basis to identify false positive signals (Figure 3c-e). Analysis of CFSE single-positive events before detergent treatment showed a total of 3.25E7 ± 1.16E6 objects/mL acquired for PPP samples, and a 31% reduction was observed after detergent treatment resulting in 2.25E7 ± 1.03E6 objects/mL (~69% of total CFSE-single positive fluorescent events) (Figure 3c). Analysis of antibody mixture single-positive events showed a total of 1.47E8 ± 9.35E7 objects/mL events acquired for PPP samples, and 5.31E7 ± 6.88E7 objects/mL after detergent treatment (~36% of total events (Figure 3d).
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