Dil dye

Dil dye (Molecular Probes, USA) was incubated with exosomes at room temperature to label exosomes. Excess dye was removed by centrifugation (100,000 × g, 60 min, 4 °C), followed by PBS rinsing thrice. Dil-labeled exosomes were resuspended in DMEM medium containing 10% exosome-free FBS, and co-cultured with OS cells for 24 h. Cells were washed thrice with PBS, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.1% Triton X-100 for 5 min. The nuclei were stained with DAPI solution. Exosome internalization by MG63 and 143B cells was measured under the laser confocal microscope.

To evaluate the internalization of coincubated EVs in rCSCs by fluorescent microscopy, HLSC-EVs were labeled with 1 μM Dil dye (ThermoFisher, Waltham, MA, USA) as described previously [12 (link)]. Briefly, HLSC-EVs were resuspended in PBS supplemented with 1 μM Dil dye and ultracentrifuged at 100,000 g for 1 h at 4 °C. EVs were then washed with PBS by ultracentrifugation (100,000 g for 1 h at 4 °C). The EV pellet was resuspended in RPMI and processed for coincubation. Coincubated EVs were immediately used to treat previously plated rCSCs (50 × 10³ EVs/target cell) for 1 h, cells were then fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and processed for confocal microscopy.

The supernatant of HLSCs or MSCs was recovered and centrifuged for 20 min at 3000 g to remove cell debris and apoptotic bodies. An ultracentrifugation at 100,000 g for 2 hours at 4°C (Beckman Coulter Optima L-90 K, Fullerton, CA, USA) has followed the previous one. Both HLSC-EVs and MSC-EVs were resuspended in RPMI supplemented with 1% dimethyl sulfoxide (DMSO) and frozen at −80°C for later use. Concentration and size distribution of EVs were determined by the Nanosight LM10 system (NanoSight, Wiltshire, UK). Briefly, EV preparations were diluted (1:200) in sterile saline solution and analyzed by the Nanoparticle Analysis System using the NTA 1.4 Analytical Software (Figure 1A) [52 (link)]. To evaluate the internalization of EVs in G7 renal CSCs by fluorescent microscopy, EVs were labelled with 1 μM Dil dye (Thermo Fisher Scientific, Waltham, MA, USA) as described previously [52 (link)]. Briefly, purified EVs were resuspended in PBS supplemented with 1 μM Dil dye and ultracentrifuged at 100,000 g for 1 h at 4°C. Following labelling, the EVs were washed with PBS by ultracentrifugation as mentioned above. The pellet obtained was then resuspended in RPMI with 1% DMSO and frozen for subsequent studies.

Purified exosomes were fluorescently labeled with 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) dye (Thermofisher), a fluorescent dye that is incorporated into the cell membrane for 1 h at 37 °C, and excess dye was removed by ultracentrifugation at 100,000× g for 2 h. Exosomes were then re-suspended in PBS at the concentration of ~1 μg protein/μL. Fluorescent intensity (Ex 530, Em 590) was measured for the equal amount of the labeled exosomes from each sample (Hidex Plate Reader, Finland) and values were then corrected for differences in the total number of the viable cells for each condition.

EVs were labeled with Dil dye (Invitrogen) and coincubated with NFs for 24 hours. NFs were rinsed twice with PBS, fixed with 4% paraformaldehyde, counterstained with 4′,6-diamidino-2-phenylindole (Beyotime, Shanghai, China), and observed under the BX53 fluorescence microscope (×400) (Olympus, Japan).