Cellular differentiation induced by microvesicles. (A) THP-1 cells and (B) monocytes were treated with microvesicles from PMA- or GM-CSF–stimulated cells, respectively. After 48 hours, adherent cells possessing macrophage-like phenotype were stained with crystal violet and dye uptake was measured at 550 nm. As controls, THP-1 cells were treated with vehicle (DMSO), PMA, or microvesicles from untreated cells, while monocytes were left untreated (media only) or treated with GM-CSF. The absorbance was measured and shown as the average optical density ± SEM (n = 3). (C-D) CD206, CD16, and CCR5 surface expression by flow cytometry. Quantification of surface marker expression is presented as average fluorescence ± SEM (n = 3). Significance was determined comparing to vehicle or freshly isolated monocytes as indicated. (E) Monocytes cells were treated with GM-CSF or microvesicles from GM-CSF–stimulated cells for 48 hours. qRT-PCR was performed using primers for CD16, CD206, or CCR5. Data are normalized over the average C_t value of housekeeping genes GAPDH and CAP-1 and expressed as relative fold increase of in gene expression compared with freshly isolated monocytes. Data represents the average fold change in expression ± SEM for 3 independent experiments. (F) Freshly isolated monocytes were treated with GM-CSF or microvesicles from GM-CSF–stimulated cells for 48 hours. Phagocytic SRBC were counted from 100 cells under fluorescent microscope. Total number of phagocytes and total number of SRBC within 100 phagocytes is presented as phagocytic index. Data shown are the average fold change ± SEM from independent recipient blood donors treated with microvesicles generated from 3 different donors.