HCAECs were stained with PKH67 (Sigma-Aldrich; #MIDI67), according to the manufacturer’s instructions. PKH67-labeled sEVs were washed twice with PBS. 2 × 105 recipient ECs were co-incubated with PKH67-labeled sEVs (1.5 μg sEV, equivalent to sEVs collected from 8 × 106 donor ECs) for 0, 0.5, 6, or 24 h. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories; #H-1200). A Zeiss Axiovert 200M microscope and ZEN 2.3 Pro software was used to visualize the uptake of sEVs into the recipient HCAECs.
Pkh67
Manufactured by Vector Laboratories
Sourced in United States
PKH67 is a fluorescent cell linker used for general cell membrane labeling. It is a lipophilic tracer that inserts into the lipid regions of the cell membrane without affecting cell function.
Automatically generated - may contain errors
5 protocols using pkh67
1
Visualizing Endothelial Cell Uptake of sEVs
2
Exosome Uptake by Spermatogonium Cells
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Puri ed BMSC-Exo were labeled with 1 μM PKH67 (Invitrogen) as previously described. Brie y, BMSC-Exo were mixed with 1 μM PKH67, and the exosome-dye suspension was incubated for 5 min with regular mixing. Excess dye from the labeled exosomes was removed by ultracentrifugation at 100,000 g for 1 h at 4°C using a 70Ti rotor (Beckman Coulter), and the exosome pellets were washed three times by resuspending them in PBS. The nal pellets were resuspended in PBS. PKH67-labeled exosomes were cocultured with Spermatogonium cells(GC-1) for 6 h, then GC-1 were washed with PBS, and xed in 4% paraformaldehyde (PFA).The nucleus of cells were stained using medium containing 4,969-diamidino-2phenylindole (DAPI; Vector Laboratories, USA). The uptake was observed by uorescence microscopy.
3
Exosome Uptake by Spermatogonium Cells
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Puri ed BMSC-Exo were labeled with 1 μM PKH67 (Invitrogen) as previously described. Brie y, BMSC-Exo were mixed with 1 μM PKH67, and the exosome-dye suspension was incubated for 5 min with regular mixing. Excess dye from the labeled exosomes was removed by ultracentrifugation at 100,000 g for 1 h at 4°C using a 70Ti rotor (Beckman Coulter), and the exosome pellets were washed three times by resuspending them in PBS. The nal pellets were resuspended in PBS. PKH67-labeled exosomes were cocultured with Spermatogonium cells(GC-1) for 6 h, then GC-1 were washed with PBS, and xed in 4% paraformaldehyde (PFA).The nucleus of cells were stained using medium containing 4,969-diamidino-2phenylindole (DAPI; Vector Laboratories, USA). The uptake was observed by uorescence microscopy.
4
Exosome Uptake by Spermatogonium Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Puri ed BMSC-Exo were labeled with 1 μM PKH67 (Invitrogen) as previously described. Brie y, BMSC-Exo were mixed with 1 μM PKH67, and the exosome-dye suspension was incubated for 5 min with regular mixing. Excess dye from the labeled exosomes was removed by ultracentrifugation at 100,000 g for 1 h at 4°C using a 70Ti rotor (Beckman Coulter), and the exosome pellets were washed three times by resuspending them in PBS. The nal pellets were resuspended in PBS. PKH67-labeled exosomes were cocultured with Spermatogonium cells(GC-1) for 6 h, then GC-1 were washed with PBS, and xed in 4% paraformaldehyde (PFA).The nucleus of cells were stained using medium containing 4,969-diamidino-2phenylindole (DAPI; Vector Laboratories, USA). The uptake was observed by uorescence microscopy.
5
Exosome Uptake by Spermatogonium Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Puri ed BMSC-Exo were labeled with 1 μM PKH67 (Invitrogen) as previously described. Brie y, BMSC-Exo were mixed with 1 μM PKH67, and the exosome-dye suspension was incubated for 5 min with regular mixing. Excess dye from the labeled exosomes was removed by ultracentrifugation at 100,000 g for 1 h at 4°C using a 70Ti rotor (Beckman Coulter), and the exosome pellets were washed three times by resuspending them in PBS. The nal pellets were resuspended in PBS. PKH67-labeled exosomes were cocultured with Spermatogonium cells(GC-1) for 6 h, then GC-1 were washed with PBS, and xed in 4% paraformaldehyde (PFA).The nucleus of cells were stained using medium containing 4,969-diamidino-2phenylindole (DAPI; Vector Laboratories, USA). The uptake was observed by uorescence microscopy.
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