To determine the uptake of BMSC-derived exosomes by human podocytes, exosomes were labeled with red fluorescent dye PKH26 (Umibio, Shanghai, China) and incubated with podocytes in an incubator for 2 h, 6 h and 24 h. Following PBS rinsing, cells were fixed with 4% paraformaldehyde for 30 min, washed with PBS, and then stained with 4′,6-diamidino-2-phenylindole. The differential interference contrast (DIC) microscopy image and fluorescence microscopy image were taken by Leica SP8 confocal microscope (Leica Microsystems). And the exosome uptake efficiency was expressed by the proportion of PKH26-positive podocytes at 2 h, 6 h and 24 h.
Pkh26
Manufactured by Umibio
Sourced in China
PKH26 is a fluorescent cell linker dye used for long-term cell labeling. It is a lipophilic dye that incorporates into the cell membrane and is retained during cell division, allowing for the tracking of labeled cells.
Automatically generated - may contain errors
Lab products found in correlation
Pkh26, by Nikon (2 mentions)
Laser scanning confocal microscope, by Nikon (2 mentions)
Leica sp8 confocal microscope, by Leica Microsystems (1 mentions)
Huvecs, by Solarbio (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Huvecs, by iCell Bioscience (1 mentions)
Flow cytometry, by BD (1 mentions)
Confocal microscopy, by Zeiss (1 mentions)
Pkh26, by Beyotime (1 mentions)
Actin tracker green 488, by Beyotime (1 mentions)
Fluorescence microscope, by Leica camera (1 mentions)
Hoechst 33342, by Solarbio (1 mentions)
Total exosome isolation reagent, by Thermo Fisher Scientific (1 mentions)
12 protocols using pkh26
1
Quantifying Podocyte Exosome Uptake
2
Exosome Uptake by LPS-Induced H9C2 Cells
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The MSCs-Exo were labelled with the fluorescent dye PKH26 (Umibio, China) according to manufacturer's instructions. Then, the stained exosomes were coincubated with LPS-induced H9C2 cells. After 24 h incubation, the nuclei of each group of cells were stained with DAPI. Finally, the laser scanning confocal microscope (Nikon, Japan) was used to observe and record the red fluorescence of PKH26 in each group of cells.
3
Exosome Labeling and Internalization in DPSCs and HUVECs
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A total of 500 μg of M1-Exos and M2-Exos were labeled with the lipophilic dye PKH-26 (Umibio, Shanghai, China), respectively, followed by ultracentrifugation to remove excess dye. Then, the PKH-26-labeled exosomes were added to DPSCs or commercially obtained HUVECs (iCell, Shanghai, China) and incubated for 24 h. Afterward, the co-incubated cells were washed three times with PBS and subsequently fixed with PFA for 20 min. Next, fluorescent phalloidin (Abbkine, Wuhan, China) was employed to label the actin filament of the cytoskeleton in DPSCs and HUVECs for 30 min, and DAPI (Solarbio, Beijing, China) was utilized for staining cell nuclei for 5 min. Finally, the cells were rinsed three times with PBS and photographed under a laser confocal microscope.
4
PKH26-labeled Exosome Characterization
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PKH26 (UR52302, Umibio) was used to label exosomes according to the instructions and previous descriptions (Xu et al., 2017 (link)). After fluorescent labeling, the exosomes were collected by ultracentrifugation. Then, the exosome pellet was resuspended in pure water. Nanoflow was used to characterize the number of PKH26-labeled exosome particles, and the final concentration was adjusted to 1.1 × 108 Evts/µl (1 μg/μl).
5
Visualizing Exosome Uptake in A549 Cells
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Red fluorescent dye PKH26 (Umibio, UR52302, China) was used to mark the engineered exosomes. A549 cells were coincubated with stained Exo. 24 h later, 4′, 6-diamidino-2-phenylindole (DAPI) was used for marking the nucleus. We observed and recorded the PKH26 in A549 cells in each group by confocal microscope (Nikon, Japan).
6
Exosome Uptake and Surface Marker Analysis
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In order to evaluate the specific uptake ability of A549 for self-sourced exosomes, we first used the fluorescent dye PKH26 (Umibio, China) to label the extracted engineered exosomes according to the instructions. Then the stained exosomes were co-incubated with A549 cells, Hela cells and HepG2 cells. After 24-h incubation, the nuclei of each group of cells were stained with DAPI. Finally, the laser scanning confocal microscope (Nikon, Japan) was used to observe and record the green fluorescence of PKH26 in each group of cells.
We used flow cytometry to detect the surface marker CD63 of exosomes in each group of cells. In brief, the incubated A549, Hela and HepG2 cells were washed twice with PBS, then added with CD63-FITC monoclonal antibody (Abcam, UK) and incubated at room temperature for 30 min. The fluorescence intensity of CD63 was analyzed by flow cytometry (BD, USA).
We used flow cytometry to detect the surface marker CD63 of exosomes in each group of cells. In brief, the incubated A549, Hela and HepG2 cells were washed twice with PBS, then added with CD63-FITC monoclonal antibody (Abcam, UK) and incubated at room temperature for 30 min. The fluorescence intensity of CD63 was analyzed by flow cytometry (BD, USA).
7
Visualizing EV Uptake in SCAPs
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PKH26 (Umibio, Shanghai, China) was diluted in Diluent C and incubated with EVs for labeling and tracing EVs. After sequentially centrifuged as mentioned above, PKH26-labeled EVs were added to SCAPs and incubated for 48 h, cells were fixed with 4% paraformaldehyde and stained with Actin-Tracker Green-488 (Beyotime, Shanghai, China) for visualizing F-actin and 4′,6-diamidino-2-phenylindole (DAPI) for cell nucleus. The endocytosis of EVs by SCAPs was observed by confocal microscopy (Zeiss, Oberkochen, Germany).
8
Measuring Exosome Uptake in NPCs
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Exosome uptake was measured using PKH26 (Umibio) according to the manufacturer’s instructions. Briefly, the purified MSC-exosomes were incubated with PKH26 for 5 min at room temperature, and then the labelled exosomes were washed 2‒3 times with PBS, followed by centrifugation at 120,000
g for 90 min and resuspension in the culture medium. After suspension, the NPCs were incubated at 37°C for 12 h. The NPCs were washed twice with PBS. The nuclei were stained with Hoechst33342 (Solarbio, Beijing, China) for 10 min and finally observed under a fluorescence microscope (Leica, Wetzlar, Germany). In the follow-up experiments, NPCs were incubated with 20 μg of exosomes.
g for 90 min and resuspension in the culture medium. After suspension, the NPCs were incubated at 37°C for 12 h. The NPCs were washed twice with PBS. The nuclei were stained with Hoechst33342 (Solarbio, Beijing, China) for 10 min and finally observed under a fluorescence microscope (Leica, Wetzlar, Germany). In the follow-up experiments, NPCs were incubated with 20 μg of exosomes.
9
Nanoparticle Tracking with PKH26
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To track the nanoparticles, the fluorescent dye PKH26 (λex = 551 nm and λem = 567 nm, Umibio) was used to label An-BMP-TMZ according to the instructions.
10
In Vivo Kidney Exosome Tracking
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Briefly,40 μg of exosomes labelled with PKH26 (UR52302, Umibio, China) were locally injected into the rat kidneys (via methods described previously in the animal design section). Exosome accumulation and dynamics were tracked by in vivo fluorescence imaging on Day 1, 3, 7, 10and 14 via a Lago/Lago X Imager (Lago/Lago, Spectral Instruments Imaging, USA).
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