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. 2021 Oct 19;22(20):11269. doi: 10.3390/ijms222011269

Figure 1.

Figure 1

Characterization of MSC-EVs and their uptake by different leukocyte subpopulations. (A) Representative images of EVs assessed by nanoparticle tracking analysis; (B) representative Western blots of Hsp70, TSG101 and CD9 proteins in 30 µg of loaded of EVs; absence of calnexin signifies a pure EV preparation; (C) representative transmission electron microscopy images of isolated EVs. Scale bar: 100 nm. EVs showed positive immunogold-labeling of the transmembrane extracellular protein CD63, a canonical EV marker. (D) Peripheral blood mononuclear cells and neutrophils were incubated with carboxyfluorescein succinimidyl ester (CFSE)-labeled EVs for 3 h at 37 °C and EV internalization was assessed by flow cytometry. As a negative control, PBS was mixed with CFSE and added to immune cells in parallel. Cell types were analyzed by specific surface markers for T-cells (CD4+ and CD8+), B-cells (CD19+), monocytes (CD14+), natural killer cells (CD56+) and neutrophils (CD15+). EV internalization was measured by fluorescence intensity and is represented as the percentage of EVs internalized by each cell population. Graphs represent mean ± SD of three independent experiments.