The isolated urinary small EVs were labelled using two lipophilic dyes: PKH67 (Sigma) for the photooxidation experiment (due to the photostability of PKH) and BODIPY FL C16 (Thermo Fisher). The small EVs were incubated in 10 µM BODIPY in PBS for 30–40 min at 37 °C in 5% CO2. For the PKH67 labelling, the small EVs suspended in PBS were diluted 1:1 with 4 µM PKH67 that was prepared in dilution C buffer (provided in the PKH67 fluorescent cell linker kit, Sigma Aldrich, Buchs, Switzerland) and incubated at room temperature for 15 min. The reaction was stopped for both labelling reactions by adding an equal volume of 1% BSA (Sigma). Unincorporated dye was removed using Invitrogen™ spin columns (MW 3000, Thermo Fisher) according to the manufacturer’s instructions. Subsequently, the labelled small EVs were resuspended in ARPE-19 medium and tested for labelling efficiency or immediately transferred to the cell cultures for exosome uptake analysis. As a control, BODIPY alone was also column-purified and the eluate (after removal of unbound dye) was used to evaluate potentially residual unbound dye in the labelled experiment.
Pkh67
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Macao, China, Sao Tome and Principe, France, Switzerland, Japan
PKH67 is a lipophilic fluorescent dye that can be used to label cells. It is designed for long-term cell tracking and lineage analysis applications.
Automatically generated - may contain errors
Lab products found in correlation
Pkh26, by Merck Group (66 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (30 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (28 mentions)
Pkh67, by Thermo Fisher Scientific (18 mentions)
Lsm 780 confocal microscope, by Zeiss (14 mentions)
Diluent c, by Merck Group (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (9 mentions)
Lsm 700, by Zeiss (8 mentions)
Lsm 710, by Zeiss (8 mentions)
Lsm700b, by Zeiss (6 mentions)
Facscalibur, by BD (6 mentions)
Lsm 880, by Zeiss (6 mentions)
Exo red, by System Biosciences (6 mentions)
Axio observer z1, by Zeiss (6 mentions)
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
Fluorescence microscope, by Zeiss (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Lsm 780, by Zeiss (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
477 protocols using pkh67
1
Labeling and Purification of Small EVs
2
Tracking BMSC-Derived Exosome Uptake in 5637 Cells
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Extracted BMSC-Exo were incubated with the lipophilic dye PKH67 (10 μM, Sigma-Aldrich, USA) for 30 min at 37°C. After washed with PBS and removed the residual PKH67 by gel filtration chromatography, the 20 μg/mL PKH67-labeled exosomes were incubated with 5637 cells for 24 h. After washed with PBS, the 5637 cells were stained with DAPI (Sigma-Aldrich, USA) to label the cell nucleus. Finally, the internalization of BMSC-Exo into 5637 cells was observed under a confocal microscope (Olympus, Japan).
3
Labeling and Uptake of Exosomes in HUVECs
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NC-exos were labeled with PKH67 (Sigma-Aldrich), according to the manufacturer’s instructions. Briefly, 250 μg of NC-exos was mixed with PKH67 in 1 mL of diluent C (Sigma-Aldrich) with a final concentration of 2 × 10−6 M PKH67. Then, 2 mL of 5% BSA/PBS was added for neutralization after 5 min incubation. The NC-exos were then washed by PBS and incubated with HUVECs. After 6 h, HUVECs were observed by fluorescence confocal microscope (Nikon, Tokyo, Japan) and examined by flow cytometry. Additionally, NCs were transfected with Cy3-labeled miR-140-5p mimics and cultured in compressive loads with 0 MPa, 0.5 MPa, and 1.0 MPa. The NC-exos were labeled by PKH67 and incubated with HUVECs. 4′,6-diamidino-2-phenylindole (DAPI) was used for cellular nuclei staining. Imaging of exosome uptake was performed by fluorescence confocal microscope.
4
EV Labeling and Uptake in Cardiomyocytes
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Quantified EV suspension (100 μL, 20 µg) was mixed with 1 mL of diluted PKH67 (4 µL/mL, Sigma-Aldrich Chemical Company, St Louis, MO, USA) and incubated at room temperature for 4 min. Next, 1 mL of 0.5% bovine serum albumin (BSA) was added to the suspension EV re-extraction, which was stained with PKH67 (green) under a fluorescence microscope. The PKH67-labeled EVs were incubated with HL-1 cells stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 12 h. PKH67-labeled EVs were injected into the mouse atrium.
5
Fluorescence-based Uptake Analysis of Extracellular Vesicles
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For uptake study, the EVs collected from B16F1 and 3T3 Swiss Albino cells were collected with or without ET as described above, followed by fluorescence-labeling with PKH67 (Sigma-Aldrich, MO, USA) according to the manufacturer's protocol. The PKH67-labeled EVs were washed twice with PBS by using Amicon Ultra 10K (Merck Millipore). B16F1 and 3T3 Swiss Albino cells were seeded onto a 35-mm glass bottom dish at a density of 1 × 105 cells/dish. After 24-h incubation, PKH67-labeled EVs derived from B16F1 or 3T3 Swiss Albino were added to B16F1 or 3T3 Swiss Albino cells, respectively, to a final concentration of 5 μg protein/mL in serum free DMEM. After 6-h incubation, the media were removed and the cells were washed twice with PBS. Then, the cells were fixed with 4% paraformaldehyde for 20 min at 37 °C. After washing the cells with PBS three times, the cells were incubated with 1 μg/mL 4′, 6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) in PBS for 15 min at 37 °C to stain nuclei. After washing with PBS, the fluorescence was observed using a confocal laser scanning microscope (LSM700, Carl Zeiss). To evaluate relative uptake amount of each PKH-labeled EVs, fluorescence intensities of PKH67 were analyzed for 12 images per each group for one experiment using an image-analysis system Image J. The experiments were performed 3 times independently.
6
Uptake of Schistosoma japonicum Egg-Derived EVs
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Murine liver Hepa1-6 cells were obtained from the ATCC (CRL-1830) and grown according to the standard protocol in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) supplemented with 10 % fetal bovine serum (Life Technologies). Hepa1-6 cells were seeded in 12-well plates (1 × 105 cells/well) using advanced serum-free DMEM (Life Technologies) for 4 h. Purified EVs from S. japonicum eggs or Hepa1-6 cells were labeled with the green fluorescent dye PKH67 (Sigma-Aldrich, St. Louis., MO, USA) as described by Hazan-Halevy et al. [37 (link)] with minor modifications. Briefly, 10 μg of the PKH67-stained EVs were washed three times using a 300-kDa Amicon (Merck Millipore, Merck KGaA, Darmstadt, Germany) to remove excess dye, EVs were then added to the cells and incubated for 1 h at 37 °C. As a control for non-specific labeling of cells, PBS was stained with PKH67, washed, and added to the cells. Following 1 h incubation, the medium was aspirated, cells were washed twice with PBS, fixed with 4 % formaldehyde solution for 15 min, and washed twice more with PBS; nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies). Finally, the cells were observed using confocal fluorescence microscopy (Leica TCS SP5 II, Heidelberg, Germany).
7
Tracking Extracellular Vesicle Internalization
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To track the internalization of EVs, EVs were labelled with PKH67 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) as previously described [33 (link), 34 (link)]: EVs resuspended in a buffer provided in the kits were mixed with the PKH67 dyes and were incubated for 5 min at room temperature. Next, the samples were added to PBS supplemented with 5% bovine serum albumin and were ultracentrifuged at 100,000 g for 1 h to remove free dyes. The labelled EVs were co-cultured with HUVECs for 6 h, which were then fixed with 4% paraformaldehyde at 25 °C for 20 min. The internalization of labelled EVs by the HUVECs was analysed using a fluorescence confocal microscope. All the fluorescence images were captured.
8
Tracking EV Internalization in HUVECs
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To track the internalization of EVs, EVs were labelled with PKH67 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) as previously described [33] [34] : EVs resuspended in a buffer provided in the kits were mixed with the PKH67 dyes and were incubated for 5 min at room temperature. Next, the samples were added to PBS supplemented with 5% bovine serum albumin and were ultracentrifuged at 100,000 g for 1 h to remove free dyes. The labelled EVs were cocultured with HUVECs for 6 h, which were then xed with 4% paraformaldehyde at 25˚C for 20 mins. The internalization of labelled EVs by the HUVECs was analysed using a uorescence confocal microscope. All the uorescence images were captured.
9
Exosome Uptake Assay in Osteoblasts
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The uptake assay was performed as previous described [69 (link), 70 (link)], the exosomes were labeled with 10 μM PKH67 (a green fluorescent dye, Sigma Aldrich), and incubated in the dark at room temperature for 12 min, and the excess dye was removed using a 100kD ultrafiltration device (Millipore, USA), then the exosomes resuspended in culture medium. Subsequently, the PKH67-labelled exosomes were added to MC3T3-E1 cells and incubated for 24 h at 37◦C. After the cells were fixed with 4% formaldehyde, the nuclei of the cells were stained with 4', 6-diamidino-2-phenylindole (DAPI, Sigma Aldrich), and the cells were visualized by fluorescence under a laser-scanning confocal microscope (LSM710 Carl Zeiss AG).
10
Fluorescent Labeling of Glioblastoma-Derived Nanoparticles
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For DiR labelling, GDNs (50 mg in 1 mL PBS) were mixed with 1 µL near-infrared lipophilic carbocyanine dye (1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indotricarbocyanine-iodide, DiR; Invitrogen, Carlsbad, CA, 5 mM in DMSO) and incubated at 22°C for 20 min.
For PKH67 labelling, GDNs (50 mg in 1 mL dilute C) were mixed with 2 µL PKH67 (Sigma, 1 mM in ethanol) and incubated at 37°C for 5 min. Labelling was stopped by adding 1 mL of exosomes-depleted FBS (supernatant of centrifuging at 150,000g for overnight).
For IRDye-700DX labelling, GDNs (5 mg in 1 mL PBS) were mixed with 1 µL IRDye-700DX NHS Ester (5 mg/mL in DMSO) and incubated at 37°C for 2 h.
All unlabelled dye was washed away by centrifugation at 150,000g for 90 min, and labelled GDN pellets were re-suspended in PBS.
For PKH67 labelling, GDNs (50 mg in 1 mL dilute C) were mixed with 2 µL PKH67 (Sigma, 1 mM in ethanol) and incubated at 37°C for 5 min. Labelling was stopped by adding 1 mL of exosomes-depleted FBS (supernatant of centrifuging at 150,000g for overnight).
For IRDye-700DX labelling, GDNs (5 mg in 1 mL PBS) were mixed with 1 µL IRDye-700DX NHS Ester (5 mg/mL in DMSO) and incubated at 37°C for 2 h.
All unlabelled dye was washed away by centrifugation at 150,000g for 90 min, and labelled GDN pellets were re-suspended in PBS.
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