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Fig. 2 | Biomarker Research

Fig. 2

From: Selective isolation of extracellular vesicles from minimally processed human plasma as a translational strategy for liquid biopsies

Fig. 2

Intact EV subpopulations spiked in plasma can be completely recovered using antibody-conjugated beads. (A) Single-particle analysis of fluorescent 22RV1-NIR EVs by nFCM. The dot plot on the left shows that the majority of 22RV1 EVs incorporated the NIR fluorophore, whereas the one on the right evidences the CD9-positive subpopulation of these NIR-EVs, determined after staining with CD9-PE. Percentages of fluorescent particles were background-corrected (BC) with buffer (PBS). (B) IP of 22RV1-NIR EVs spiked in plasma (donor 6). Recovery (% of input) was appreciated indirectly, through the NIR signal of IP flow-throughs (FT) and directly, by measuring the fluorescence of NIR EVs captured on beads. 22RV1-NIR EVs were used to plot a calibration curve correlating particle numbers with their fluorescent signal, which allowed to present percentages of input based on the actual number of particles recovered vs. input particles. Specificity (S) was calculated for both readouts. (C) IP of 22RV1-NIR-CD9-PE-stained EVs spiked in plasma (donor 6). Recovery, plotted as % of input, was assessed by indirect and direct fluorescence readouts. The NIR signal of 22RV1-NIR-CD9-PE EVs was used to plot a calibration curve, correlating particle numbers with their fluorescent signal, which allowed to present percentages of input based on the actual number of particles recovered vs. input particles. Recovery with each bead source was equivalent, as suggested by both readouts. (D) Cryo-TEM images of EVs captured from plasma (1) and plasma spiked with HEK293 EVs (2), using triple-coated beads. Respective scale bars are shown on the top right corner of each image

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