Cst 4412

Co-labeled Evs were prepared from co-transfection of the plasmids as described above. A total of 2 × 10⁴ cells each of A431 and MCF-7 cells were mixed and seeded to an 8-well chamber slide (Nunc Lab-Tek, Thremo Scientific, Waltham, MA, USA) 24 h before EV treatment. These co-cultured cells were incubated with 2.0 × 10⁷ E626-mCherry or RDG-mCherry co-labeled EVs for 10 min, followed by 3× PBS washes to remove unbound EVs. The cells were fixed with 4% PFA at room temperature for 10 min, washed with PBS three times, and blocked with blocking buffer (1% BSA in PBST) for 60 min. Then, the cells were incubated with the primary antibody (CST D38B1, Cell Signaling Technology, Danvers, MA, USA) in the humidified chamber overnight at 4 °C. After three 5 min PBS washes, they were incubated in the diluted secondary antibody (CST 4412, Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature in the dark. The slide received a coverslip following applying mounting medium (P36930 Life Technologies, Carlsbad, CA, USA) containing DAPI. Fluorescence images were taken at 60× objective magnification by confocal laser scanning microscopy (FluoView FC1000, Olympus, Shinjuku City, Tokyo, Japan).