Purified exosomes were labelled with the membrane-labelling dye PKH67 (Invitrogen, USA) according to the manufacturer's instructions and were then washed and resuspended in serum-free medium. Next, VSMCs were seeded into glass bottom dishes (Cellvis, Mountain View, CA, US) for single layer and then cocultured with PKH67-labelled vesicles for 30 min, 60 min and 120 min; washed with PBS three times; fixed in 4% paraformaldehyde; stained with DiI (Invitrogen); washed with PBS; and imaged by confocal microscopy (Leica TCS SP8).
Pkh67
Manufactured by Thermo Fisher Scientific
Sourced in United States
PKH67 is a fluorescent cell linker kit designed for general cell membrane labeling. The kit provides a simple and reproducible method for fluorescently labeling cells without affecting cell viability or function.
Automatically generated - may contain errors
Lab products found in correlation
Pkh67, by Merck Group (18 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (7 mentions)
Pkh67, by Vector Laboratories (4 mentions)
4 969 diamidino 2phenylindole dapi, by Vector Laboratories (4 mentions)
70ti rotor, by Beckman Coulter (4 mentions)
Tcs sp8, by Leica camera (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Facscanto 2, by BD (3 mentions)
8 well chamber slide, by Thermo Fisher Scientific (3 mentions)
Glass bottom dishes, by Cellvis (2 mentions)
Axio imager lsm 800, by Zeiss (2 mentions)
Rpmi 1640 culture medium, by Thermo Fisher Scientific (2 mentions)
Celltracker orange cmtmr, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Cambrex (2 mentions)
Streptomycin, by Cambrex (2 mentions)
Pkh67, by BD (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Celltracker red cmtpx, by Thermo Fisher Scientific (2 mentions)
Lsm 780 confocal microscope, by Zeiss (2 mentions)
Nunc lab tek 2 chamber slide, by Thermo Fisher Scientific (2 mentions)
34 protocols using pkh67
1
Exosome Uptake by Vascular Smooth Muscle Cells
2
Trogocytosis Measurement in γδ T Cells
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Daudi cells were stained with the lipophilic green-emitting dye PKH67 (Sigma-Aldrich). Pure resting γδ T cells were stained with PKH67 or with cytoplasmic Cell Tracker™ Orange-CMTMR [5-(and-6)-(((4-chloromethyl) benzoyl) amino) tetramethylrhodamine), Molecular Probes, Oregon, USA] according to the manufacturer’s instructions. CMTMR+ γδ T cells were cocultured with PKH67+ cells (Daudi cells or autologous γδ T cells) at 37 °C in RPMI 1640 culture medium (Invitrogen, Cergy Pontoise, France) supplemented with 10% fetal calf serum (Hy1, Thermo Scientific, USA), 100 µg/ml streptomycin, and 100 IU/ml penicillin (Cambrex Biosciences, Rockland, ME, USA). The cocultures were performed in 96-well U-bottom culture plates at a cell ratio of 1:1 with a total of 200,000 cells per well. CD107a or IgG1 was added to the coculture, and brefeldin A (10 µg/ml) was added after 2 h of coculture. After 4 h of coculture, the cells were washed with 0.5 mM PBS/EDTA and then stained with 7-AAD and DAPI to identify dead cells. Trogocytosis was measured as the acquisition of PKH67 fluorescence by CMTMR+ γδ T cells, which was characterized by an increase in the mean fluorescence intensity of PKH67 by flow cytometry.
3
Tracking Extracellular Vesicle Uptake
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The fluorescent dye PKH67 (10 mM; Sigma-Aldrich) was added to the M2-EVs suspension, which was incubated for 4 min at room temperature. The stained EVs were washed with PBS via ultracentrifugation at 100,000 Â g for 70 min, after which the pellet was resuspended in PBS. BMMs (2 Â 10 4 cells/well) were stained with PKH26 and cocultured with the same number of BMSCs. PKH26-labeled BMMs and unlabeled BMSCs were incubated with PKH67-labeled M2-EVs (10 mg/mL; green labeled with PKH67) for 24 h. Images were acquired every hour using a high-content screening system (Thermo Scientific CellInsight CX7 LZR).
4
Exosome Labeling and Hepatocyte Uptake
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Following membrane labeling with PKH67 as the manufacturer’s instruction (Invitrogen), the labeled exosomes were washed in PBS and centrifuged 100,000 g for 20 min at 4°C. The purified exosomes were re-suspended in serum-free medium. Hepatocytes were then seeded into a single layer in glass bottom dishes (Cellvis, Mountain View, CA, USA), co-cultured with PKH67-labeled exosomes (30, 60, or 120 min), washed with PBS three times, and fixed in 4% paraformaldehyde. The fixed hepatocytes were stained with DiI (Invitrogen), washed with PBS, and imaged by confocal microscopy (TCS SP8; Leica, Wetzlar, Germany).
5
Visualizing VSMC-EV Internalization
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VSMCs and internalized EVs were labelled using the membrane dye PKH67 (Invitrogen) and were rinsed and re‐suspended in serum‐free medium. Next, VSMCs were seeded on a single‐layer glass chassis (Cellvis) and co‐cultured with the PKH67‐labelled vesicles for periods of 0, 1, 6 and 24 h, followed by three rinses with PBS, fixation in 4% paraformaldehyde, staining with DiI and ultimately observation under confocal microscopy (Leica TCS Sp8).
6
Exosome Uptake by Cancer Cells
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8 × 104 of SGC7901S cells were seeded in 12-well plates and incubated at 37°C with red fluorescent dye CM-Dil (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 1 h, washed with PBS, and centrifuged at 110,000 × g at 4°C for 70 min to remove excess dye. Exosomes from SGC7901R cells were pre-labeled with the green fluorescent dye PKH-67 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 1 h, washed with PBS, and centrifuged at 110,000 × g at 4°C for 70 min to remove excess dye. Unlabeled exosomes were used as a negative control. The CM-Dil-labeled SGC7901S cells were incubated with PKH-67-labeled exosomes or unlabeled control exosomes for 4 h. Then, SGC7901S cells were fixed with 4% paraformaldehyde at room temperature for 1 h. Nuclear staining was performed with DAPI (40, 6-diamidino-2-phenylindole) at room temperature for 10 min. Incorporation of exosomes into targeted SGC7901S cells was visualized by fluorescence microscopy (Zeiss AG, Germany).
7
Exosome Labeling and Nuclear Staining
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PKH67 (Sigma-Aldrich, USA) (1 μM) was used to label exosomes according to manufacturer’s instructions. 24 h after PKH67-labeled exosomes were incubated with HCT116OxR, 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, USA) was used for cell nuclear staining. The slides were fluorescently visualized with a laser scanning microscope Axio-Imager-LSM800 (ZEISS, Germany). Rhodamine-conjugated secondary antibody (Cell Signaling Technology, USA) for γ-H2AX protein and DAPI for nuclear staining. The slides were visualized for immunofluorescence with a laser scanning microscope (Zeiss, Germany).
8
Exosome Labeling and Uptake Visualization
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PKH67 (Sigma-Aldrich, USA; 1 μM) was used to label exosomes according to the manufacturer’s instructions. PKH67-labeled exosomes were cocultured with HLECs for 24 h, followed by cell nuclear staining with DAPI (Invitrogen, USA). The HLECs cells were fluorescently visualized with a laser scanning microscope Axio-Imager-LSM800 (Zeiss, Germany).
9
DOPA-PtdSer Nanoparticles for Arthritis
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Dioleoylphosphatydic acid (DOPA) and dioleoyl phosphatidylserine (PtdSer) were purchased from A.V.T. (Shanghai) Pharmaceutical Co., Ltd. (Shanghai, China). Metformin, cyclohexane, Igepal CO-520 and zinc nitrate hexahydrate (Zn(NO3)2·6(H2O)) were obtained from SigmaAldrich (St. Louis, MO, USA). Low-molecular-weight-heparin (LMWH, MW 3800~5000, Mw/Mn (PDI) = 1.34) was purchased from Melonepharma (Dalian, China). Recombinant murine macrophage colony-stimulating factor (M-CSF) (315-02) and recombinant human TNF (300-01 A) were purchased from PeproTech (Rocky Hill, USA). RPMI 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), modified Eagle’s medium, trypsin EDTA, type I collagenase, fetal bovine serum (FBS), and PBS were obtained from Gibco (USA). DiR (D12731) and PKH67 were purchased from Invitrogen (USA). The anti-CD206 antibody, anti-iNOS antibody, anti-Ly-6G antibody, and anti-myeloperoxidase (MPO) antibody were purchased from BioLegend. All siRNAs used in the CIA model were synthesized by Genepharma (Shanghai, China), and the sequences used were as follows: (i) IRF5: 5-dTdT-CUG CAG AGA AUA ACC CUG A-dTdT-3 (sense) and 5-dTdT UCA GGG UUA UUC UCU GCA G dTdT-3 (antisense). A dye was introduced at the 5′-end of the antisense strand of siIRF5. (ii) Negative control scrambled siRNA (siN.C).
10
Visualizing EXO Trafficking in Cells
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To monitor EXO trafficking, purified EXOs were labeled with the membrane-labeling dye PKH67 (Invitrogen, USA) according to the manufacturer’s instructions and were then washed and resuspended in serum-free medium. Then, the PKH67-labeled EXOs were cocultured with CM-Dil-labeled cells for 12 h. Afterward, the cells were fixed with 4% PFA, and the nuclei were labeled with DAPI (Sigma, USA) at 37 °C for 5 min. Then, the stained cells were washed and observed under a microscope (Zeiss, Imager Z2, Oberkochen, Germany).
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