Fig. 2.

Extracellular vesicles mediated cell-cell communication from NIH/3T3 to MC3T3-E1. (a) Fluorescence protein labeling of cells via virus transfection. MC3T3-E1 was labeled with GFP, and NIH/3T3 was labeled with RFP. (b) IF images of labeled coculture cells (MC/NIH) under a fluorescence microscope. Green cells were MC3T3-E1 (MC), and red cells were NIH/3T3 (NIH). (c) MC/NIH cocultures were tripsinized into single cells, and were imaged under a fluorescence microscope. Yellow arrows indicated double positive cells. (d–e) Flow cytometry analysis of GFP-MC3T3-E1, RFP-NIH/3T3 and GFP-MC/RFP-NIH cocultures on day 1 and 10. GFP-MC3T3-E1 cells in cocultures on day 10 were smeared, and shifted into double positive area (d), the number of which was ~20% (e). (f–g) GFP-MC3T3-E1 and RFP-NIH/3T3 were cocultured indirectly via transwell system: RFP-NIH/3T3 cells cultured in transwells and GFP-MC3T3-E1 cells cultured on tissue culture plates (f); or GFP-MC3T3-E1 cells cultured in transwells and RFP-NIH/3T3 cells cultured on tissue culture plates (g). Cells on tissue culture plates were imaged. Images and data are representative of n = 3 individual experiments, and bar heights and error bars represent means ± SD. Scale bars: 100 μm.