Characterization of exosomes secreted by breast cancer cells and exosome uptake by HMECs.

<p>Exosomes were isolated from conditioned media of 3 different breast cancer cell lines, T47DA18, MCF7 and MDA-MB-231 and characterized by (A) detection of exosome specific proteins by western blotting and (B) electron microscopy. (A) Western blotting for endoplasmic reticulum specific protein calnexin and exosome marker proteins Alix and CD63 in total cellular lysates (lanes 1, 3 and 5) and exosome preparations (2, 4 and 6). 10 µg of protein was analyzed for each sample. (B) Characterization of exosomes from MDA-MB-231 cells by transmission electron microscopy (TEM). Exosomes isolated by multi-step centrifugation were fixed, negatively stained using phosphotungstic acid and observed by TEM. (C) Exosomes isolated from conditioned media of MDA-MB-231 cells were labeled with fluorescent dye PHK67. 10 µg protein equivalent of labeled exosomes were incubated with 5×10⁴ HMECs for 24 h. HMECs were washed extensively with PBS, fixed in PFA and observed using an epifluorescence microscope. Green fluorescent “specs” represent PKH-67 labeled exosomes taken up by the HMECs. (D) Flow cytometric analysis of HMECs exposed to PKH-67 labeled exosomes as described in (C).