Exoglow protein ev labeling kit green

The cells were mechanically dissociated and washed with PBS. After one wash with staining buffer (0.1% BSA/PBS, Sigma), 100000 cells were incubated with PE-conjugated antibodies against CD105 (5:100, Biolegend), CD90 (5:100, Biolegend), CD73 (5:100, Biolegend), CD45 (5:100, Biolegend) and Isotype control PE-mouse IgG1 (5:100, Biolegend). For the exosomes, we used the ExoGlow-protein EV labeling kit green (System Biosciences) according to manufacturer instructions. Then, we analyzed the freshly stained samples with the FACS CitoFlexS flow cytometer (Beckman). We detected autofluorescence background signals from the same samples without labeling and subtracted it from the data. The flow cytometry data were analyzed using FloJo (FloJo LLC).

Exosomes were labeled by using an ExoGlow-Protein EV Labeling Kit (Green, System Biosciences; Palo Alto, CA) according to the manufacturer’s instructions. Briefly, the exosomes were incubated with the green fluorescent dye at 37 ℃ for 20 min and then incubated at 4 ℃ overnight. The next day, the labeled exosomes were isolated and resuspended in the macrophage growth medium. Macrophages were incubated in the medium that contained 50 µg/ml exosomes for various times (3 h, 6 h, 12 h, or 24 h). The macrophages were harvested and incubated with a 1:10,000 diluted Hoechst 33,342 (Thermo Fisher Scientific) to label the nucleus. The fluorescent pictures were taken under fluorescent microscopy (Olympus DP70, Japan). For flow cytometry analysis, the cells were collected and analyzed by using a Guava easyCyte flow cytometer (Millipore Sigma). The results were further analyzed by using FlowJo software.