Pkh26 red fluorescent membrane linker dye

Purified exosomes isolated from the culture medium were collected and labeled with PKH26 red fluorescent membrane linker dye (Sigma, USA) according to the manufacturer’s instructions. The labeled exosome pellets were then resuspended and added to unstained macrophage cells for exosome uptake studies. They were incubated for 30 minutes, 2 hours or 12 hours at 37°C. After this, the cells were washed twice with PBS and fixed. The nuclei were stained with 4 ‘, 6-diamidino-2-phenylindole (DAPI). Finally, the cell samples were measured by fluorescence microscope.

Busulfan (Sigma-Aldrich, St Louis, MO, USA, B2635; 40 mg/kg) was used in order to eliminate endogenous testicular germ cells in 4-week-old recipient BALB/c nude mice (n = 4) (ORIENTBIO INC., Seongnam, South Korea). GDC cells formed from cryopreserved testicular cell culture were isolated and labelled with 2 μM PKH26 Red Fluorescent Membrane Linker Dye (Sigma-Aldrich, St Louis, MO, USA, P9691) for 5 min. Afterward, PKH26-stained GDC cells were washed 3 times with DMEM and resuspended in the same medium containing 10% FBS. Trypan blue (0.001%) was used as an indicator of the injection into recipient seminiferous tubules. An aliquot of 1 × 10⁵cells/10 μl PKH26-labelled donor cells was injected into each recipient testis, which resulted in up to 80% filling of the recipient seminiferous tubules. Recipient testes injected with PKH26-labeled GDC cells were collected 8 weeks after the transplantation, and PKH26-positive cells and the localization of GDC cells in seminiferous tubules were detected by fluorescence microscopy, using an excitation filter of 450–560 nm (Nikon, Tokyo, Japan). Additionally, immunohistochemical analyses were performed, and seminiferous tubules were stained with anti-PGP9.5 antibody.

The busulfan-treated recipient mouse model has been described in our previous studies^{38 (link),39 (link)}. Briefly, 10-week-old BALB/c immunodeficient mice (n = 4; Orient Bio, Inc., Seongnam, South Korea) were used as recipient animals because we chose these recipients to avoid immunological rejection of donor cells. The mice were injected with 40 mg/kg busulfan (Sigma–Aldrich) at least 5 weeks before CD14⁺cell transplantation to deplete germ cells in the testis of recipients^{7 (link)}.
CD14⁺ cells membranes were labelled with 2 μM PKH26 red fluorescent membrane linker dye (Sigma–Aldrich) for 3 min after sorting, then washed five times with medium (Dulbecco’s Modified Eagles Medium) and resuspended in 10% foetal bovine serum/DMEM. An aliquot 10 μL (1 × 10⁵ cells) of L PKH26-labelled CD14⁺ cells was injected into each recipient testis. Eight weeks later, the recipient mice were sacrificed and testes were harvested for analysis. The image of localisation of PKH26-labelled CD14⁺ cells in the seminiferous tubules was collected by fluorescence microscopy (Nikon).

EV preparations were labeled with PKH26 red fluorescent membrane linker-dye (Sigma) as previously described55 (link) and according to the manufacturer’s protocol with minor modifications. In brief, purified EVs (200 μL) corresponding to 100 μg of protein were labeled by adding 800 μL diluent C (Sigma linker kit) and 1 ml diluted PKH26 dye. After 5 min of incubation, reactions were interrupted with 2 mL of 1% BSA. Labeled EVs were washed in PBS, pelleted by ultracentrifugation at 100,000 g for 1 h, suspended in a final volume of 400 μL and stored at −20 °C. EV membranes were fluorescently labeled with PKH26 and EV binding to the immobilized lectins were recorded as fluorescence intensity.