Two-color, laser-induced fluorometry for evaluating the efficiency of click chemistry-based labeling of extracellular vesicles

Extracellular vesicles (EVs), including exosomes, have attracted considerable attention as promising biomarkers for the early diagnosis and prognosis of cancer. In this study we evaluated the efficiency of a click chemistry-based EV labeling method using a two-color laser-induced fluorometry (LIF) system. During cell culture, cellular membranes were metabolically functionalized with azide groups, and the released EVs were subsequently labeled with Cy5 via click chemistry. The collected EVs were then stained with a phycoerythrin-conjugated anti-CD63 antibody (PE-anti-CD63) to achieve dual labeling. A two-color LIF system equipped with 488- and 635-nm lasers was used to distinguish Cy5-labeled, CD63-positive EVs from Cy5-unlabeled, CD63-positive EVs. Because PE and Cy5 are selectively excited at 488 and 635 nm, respectively, Cy5-labeled, CD63-positive EVs generated coincident dual-color fluorescence, whereas Cy5-unlabeled, CD63-positive EVs emitted fluorescence only upon 488-nm excitation. Using this platform, we investigated the effect that serum concentration in the culture medium exerts on click-chemistry labeling efficiency for CD63-positive EVs. The highest labeling efficiency for both HeLa and A549 cells was obtained in medium supplemented with 10% fetal bovine serum. These findings demonstrate the utility of the proposed two-color LIF system for quantitatively evaluating the click-chemistry labeling efficiency of CD63-positive EVs under different cell-culture conditions.