Figure 4. Effect on cell detachment of the crude MVs and SB purification fractions.

Cell detachment activity of samples fractionated using SB-Epoxy was investigated using Ca9.22 cell monolayers. (A) The cell monolayers were treated with PBS only (PBS), crude MV preparation (Crude), first unbound fraction (Unbound, 1^st), first urea fraction (Urea, 1^st), and CHAPS fraction (CHAPS). In order to confirm complete removal of urea and CHAPS, the monolayers were also treated with dialyzed urea buffer [Urea (vehicle only)], and dialyzed CHAPS buffer [CHAPS (vehicle only)]. Bars: 50 nm. (B) The amount of detached cells was determined by crystal violet staining and measuring absorbance at 595 nm (mean ± SD), assayed in triplicate. *P<0.05, compared to PBS-treated cells. ^†P<0.05, compared to cells treated with the crude MV preparation (Crude). (C) Cell appearances after treatment with PBS control (PBS) and PBS containing MVs at concentrations of 1, 5, 10, and 20 µg/ml are shown. After treatment with different concentrations of MVs, micrographs of the treated cells were taken. Bar: 50 µm. (D) Time-lapse images of Ca9.22 cells in the presence of the SB-purified MVs. Morphological changes of Ca9.22 cells are shown. Loss of focal adhesion and cell-cell adhesion resulted in cell rounding, followed by detachment from the culture wells. The images were acquired at intervals of 30 sec for 30 min immediately after addition of MVs [See Video S1 with S2 (movie of cells in the absence of MVs)]. Bar: 50 µm.