Chapter 3 36 required to exclude false-positive particles from analysis. 0.20 µm filtered PBS (fPBS - Buffer Control) and platelet-poor plasma (PPP) samples from 5 healthy individuals was stained with CFDA-SE (carboxyfluorescein diacetate succiminidyl ester) or a mixture of tetraspanin-specific antibodies (anti-CD9/anti-CD63/anti-CD81) labeled with APC. CFDA-SE is a non-fluorescent molecule converted to fluorescent CFSE (carboxyfluorescein succiminidyl ester) by intravesicular esterases. This helps to discriminate EV from lipoproteins, as the latter do not contain esterase activity. PPP samples left unstained or singly stained with CFSE (Ch02) or the tetraspaninspecific antibody mixture (Ch05) were used to set the gating areas (Figure 2b) and compensation matrix. Following our gating strategy, analysis of unstained fPBS or unstained PPP or fPBS + CFSE resulted in ~E5 single-positive objects/mL within the CFSE gating area. In contrast, PPP samples single stained with CFSE showed an average of 4.23E7 ± 7.28E6 objects/mL (mean ± standard deviation), representing a 100-fold higher CFSE single-positive particle concentration compared to the unstained samples and fPBS (Figure 2c, left panel). Similarly, analysis of positive fluorescent events upon staining with the tetraspaninspecific antibody mixture showed that fPBS + anti-tetraspanin antibodies (fPBS Mix) yielded 5.98E6 objects/mL – a 3.6-fold increase over the concentrations of fPBS Unstained (1.65E6 objects/mL). Additionally, an isotype control was added to analyze the specificity of the antibodies in the tetraspanin mixture. Positive particle concentrations were obtained for both fPBS and PPP Isotypes, (6.16E5 and 1.97E5 ± 1.07E5 objects/mL, respectively). Analysis of PPP + anti-tetraspanin antibodies (PPP Mix) revealed an average of 1.69E8 ± 1.44E8 objects/mL – a 28-fold higher particle concentration than fPBS + anti-tetraspanin antibodies, a 350-fold higher particle concentration than PPP Unstained (4.86E5 ± 2.6E5 objects/mL), and an approximate 860-fold higher particle concentration than PPP Isotypes (Figure 2c, right panel). An approximate 4-fold higher concentration of fluorescent particles was observed in the PPP Mix vs CFSE after subtraction of background concentrations before comparison. Together, these findings show that positive fluorescently stained events can be successfully discriminated from background signals and that the anti-tetraspanin antibody binding in our protocol is specific. Moreover, as unstained samples and isotype controls yielded ~E5 (for CFSE) and fPBS with anti-tetraspanin antibodies yielded ~ E6 objects/mL in their respective fluorescent channels, we established the level of the background concentrations in our setup for single positive fluorescent events at E5 and E6 objects/mL, for CFSE ant anti-tetraspanin antibodies respectively.
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