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.
4 969 diamidino 2phenylindole dapi
Manufactured by Vector Laboratories
Sourced in United States
4,969-diamidino-2phenylindole (DAPI) is a fluorescent dye commonly used in molecular biology and cell biology applications. It binds strongly to DNA, allowing it to be used for staining and visualizing nucleic acids in fixed cells or tissues.
Automatically generated - may contain errors
5 protocols using 4 969 diamidino 2phenylindole dapi
1
Exosome Uptake by Spermatogonium Cells
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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+ 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.
4
Exosome Uptake by Spermatogonium Cells
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+ 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
Fluorescent Labeling of MSC Exosomes
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MSCs were labeled with a fluorescent dye CM-DiI (Molecular Probes, US) by incubating them in the CM-DiI working solution (1 µM) for 15 min at 37°C, followed by washing with PBS and centrifuging at 100,000 g for 2 h at 4°C. The excess dye was removed by precipitation of exosomes. HUVECs were previously cultured to 80% confluency and incubated with DMEM containing CM-DiI-labeled exosomes for 12 h at 37°C with 5% CO 2 . After incubation, cells were washed with PBS and fixed in 4% paraformaldehyde at room temperature. The nucleus of cells were stained using medium containing 4,969-diamidino-2-phenylindole (DAPI; Vector Laboratories, USA). Cellular uptake of MSC-derived exosomes by HUVECs was observed using an inverted fluorescent microscope (IX71-A12FL/PH, Olympus, Japan).
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