Imaging Dendritic Cell Uptake of Parasite-Derived Extracellular Vesicles

Human monocyte derived dendritic cells were used to perform confocal microscopy assays. To generate dendritic cells, elutriated human monocytes were cultured at 37°C where recombinant human interleukin-4 and recombinant human granulocyte-macrophage colony-stimulating factor (Peprotech, Rocky Hill, NJ) were added at 50ng/ml on days 0, 3, and 6 of culture.
Mf-derived EV uptake by human dendritic cells was measured using confocal microscopy with Zeiss 780. Human monocyte-derived dendritic cells were labeled with PKH26 (red) and the mf-derived EVs were labeled with PKH67 (green) according to the manufacturer’s instructions (Millipore Sigma, Burlington, MA). Immediately following staining, the EVs were passed through Exo-spin columns to remove the dye. The EVs were then eluted using 1X PBS [35 (link)]. VECTASHIELD Hardset Antifade Mounting Media with DAPI was also used following the manufacturer’s instructions (Vector Laboratories, Burlingame, CA). Human dendritic cells were co-cultured with parasite EVs for 3 days. The co-culture was performed in an 8-well chambered coverglass (ThermoFisher) using 5000 cells/well and 2.2 x 10⁶ stained EVs. Settings for the acquisition of confocal images were as previously described [35 (link)].