Pkh67 dye

sEVs were isolated from pooled plasma (approximately 480 µL/mouse; five mice per condition) by differential ultracentrifugation. Samples were centrifuged at 10,000× g for 10 min and then at 18,900× g for 30 min at 4 °C to remove cell debris and large vesicles. Supernatants were filtrated through a 0.22 µm membrane filter and then centrifuged twice at 100,000× g for 70 min at 4 °C using a Beckman Coulter Type 55Ti rotor (Beckman Coulter, Krefeld, Germany). sEVs were labeled with 2 µM PKH67 dye for 5 min (Sigma-Aldrich, Munich, Germany). The reaction was stopped by adding an equal volume of 1% BSA/NaCl 0.9%. Labeled sEVs were washed twice with saline solution by centrifugation at 100,000× g for 70 min. Pellet was resuspended in 100 µL saline solution and protein was quantified by Micro BCA^TMProtein assay (ThermoFisher Scientific GmbH, Dreieich, Germany). Naive recipient mice received 2 µg/100 µL of protein equivalents of sEV_PKH-67. This amount was assumed to distribute into 3 mL of blood and produce a blood level of 0.6 µg/mL, which represents about 1/75th the concentration of sEVs found in the mouse blood [15 (link)]. The injected amount of exosome protein represents about 2.6 × 10¹² sEVs, or 9.0 × 10¹¹ sEV particles per mL of blood [15 (link)].

The hADMSC-sEVs were labeled with a PKH67 dye (Sigma Aldrich, USA) for 4 min. Then, Bovine Serum Albumin (BSA) was used to terminate staining. The excess dye after labeling was removed by ultracentrifugation at 100,000g for 1 h at 4°. HUVECs (Cellcook, Guangzhou, China) and hADMSC-sEVs labeled with PKH67 were incubated in 37 °C with serum-free medium for 6 h and 12 h. Meanwhile, HUVECs and PKH67 dye were incubated in 37 °C for 6 h and 12 h as control. After fixation with 4% paraformaldehyde (PFA) for 30 min and staining with 4, 6-diamino-2-phenylindoles (DAPI, CST, USA), the samples were observed under a fluorescence microscope (Nikon, Japan).

Exosomes were labeled with PKH67 dye (Sigma) according to the manufacturer's instructions, and incubated with cells for 24 h. Then the cells were stained with phalloidin (Sigma) for intracellular cytoskeleton f-actin and observed under the confocal laser microscope.

ADSCs and L-ADSCs were stained with PKH67 dye (Sigma-Aldrich, USA) according to the manufacturer’s protocol. The cells were injected into the cavernosum of rats with CNI-induced ED. The PKH67 labelled cells in the penis, MPG, lung and spleen were detected using a fluorescence microscope at 3, 7 and 14 days after transplantation.

Exosomes were stained with PKH67 dye (sigma, USA) in room temperature for 2 minutes, then stopped with 1% BSA. Exosomes were stained with PBS as the negative control. The labeled exosomes were centrifuged at 100000g for 70 minutes. The HUVEC was co-incubated with PKH67-exosomes at 37°C for 4 hours, then stained with CM-Dil (KeyGEN, china) and Hoechst33342 (KeyGEN, china). Represent pictures were captured using the confocal microscope (Leica, Germany).

The PKH67 Fluorescent Cell Linker Kits (Sigma-Aldrich, United States) were used to label the oncolytic NDV AMHA1. A total of 1.5 × 10⁸ particles in PBS were labeled as follows: 1 µl of PKH67 dye (Sigma, St. Louis, MO) was dissolved in 2 ml of Diluent C before labeling as per the manufacturer's recommendations (Sigma, St. Louis, MO). Two volumes of diluted PKH67 were mixed with one volume of NDV suspension by pipetting. After 30 s, the labeling reaction was stopped by adding three volumes of the full medium and pipetting the suspension 5–6 times. The labeled virus was ready for exposure (Balogh et al., 2011 (link)).