Chapter 3 48 Figure 7 - Identification of single EVs on the basis of vesicular surface markers. a) Representative, fluorescence calibrated data obtained for buffer control (fPBS, left column) and PPP (right column) samples stained with anti-CD31-BV421 and anti-CD9-APC mAbs. Detergent treatment was performed by incubating the samples for 30 minutes with 20 µL 10% (v/v) TritonX-100 stock solution. Red gate: Single-positive CD9 events, purple gate: single-positive CD31 events, tan gate: double-positive events. I, double staining and II, double staining after detergent treatment. b) Visual interrogation of the gated populations in the representative PPP sample. c) Quantification of double-positive fluorescent events in 5 PPP samples and fPBS, stained with mAbs or isotypes, before and after detergent treatment. Approximately 93% of doublepositive events in PPP stained with mAbs represent PPP-derived single EV that were detected well above the fluorescent background. Red dots: means of sample spread. Symbols: individual PPP samples.
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