Confocal microscopy was performed for detection of the uptake of exosomes, milk-exosome/cis were stained with PKH26 Dye (PKH26 Red Fluorescent Cell Linker Kits, Sigma-Aldrich, Merck KGaA, Darmstadt, GER) to evaluate the internalization by A2780CP cells. Milk-exosomes diluted in 1.5 mL Diluent C (PKH26 Red Fluorescent Cell Linker Kits, Sigma-Aldrich, Merck KGaA, Darmstadt, GER), then were mixed with the mixture containing 6 μL PKH26 dye and 1.5 mL Diluent C following the protocol of the manufacturer. After 10 min, 6 mL ultracentrifuged exosome-free-FBS was added and incubated for 5 min to bind the excess dye. Then 15 mL DMEM were added to wash the product, followed ultracentrifugation again at 1,25,000 g for 60 min at 4°C. The supernatant was removed as completely as possible and purified PKH26-labeled-milk-exosome/cis were resuspended with 1 mL DMEM. Then, 1 mL of 5,000-ng/mL PKH26-labeled-milk-exosome/cis in DMEM were incubated with A2780CP cells/3 × 104 per well in 24-well-plate containing round coverslips at 37°C under 5% CO2 condition. Anti-fluorescence-quenching agent with DAPI (6-diamidino-2-phenylindole) (DAPI Fluoromount-GTM; Yeasen Biotechnology, Shanghai, China) was used to mount the slides and label nuclei. Images were captured with a TCS SP5 confocal laser scanning microscopy (Leica Microsystems, Wetzlar, GER).
Pkh26 red fluorescent cell linker kit
Manufactured by Merck Group
Sourced in United States, Germany
The PKH26 Red Fluorescent Cell Linker Kit is a laboratory product used to fluorescently label cells. It contains reagents and protocols for the reversible incorporation of a red fluorescent dye into the cell membrane, allowing for the tracking and visualization of labeled cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Pkh67 green fluorescent cell linker kit, by Merck Group (10 mentions)
Lsm 710, by Zeiss (8 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Hoechst, by Thermo Fisher Scientific (5 mentions)
Actingreen, by Thermo Fisher Scientific (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Exoab kit 1, by System Biosciences (4 mentions)
Diluent c, by Merck Group (3 mentions)
Matrigel, by BD (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (3 mentions)
Fluorescence microscope, by Olympus (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Exoquick exosome isolation reagent, by System Biosciences (3 mentions)
Exosome spin columns mw 3000, by Thermo Fisher Scientific (2 mentions)
Xc50 camera, by Olympus (2 mentions)
Bx53 microscope, by Olympus (2 mentions)
Scid mice, by Nara Biotech (2 mentions)
218 protocols using pkh26 red fluorescent cell linker kit
1
Tracking Exosome Internalization by Confocal Microscopy
2
Lung Cancer Cell Line Characterization
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A549, H460, H661, and H1299 lung cancer cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and cultured with RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, South Logan, UT, USA). Cisplatin, 4′,6-diamidino-2-phenylindole (DAPI), and a PKH-26 Red Fluorescent Cell Linker Kit were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). FITC-conjugated CD44 antibody, APC-conjugated CD90 antibody, and corresponding isotype controls were purchased from BD Biosciences (San Jose, CA, USA). Anti-human CD44, CD90, and p53 primary antibodies were purchased from Abcam (Cambridge, MA, USA). CCR2 antibody was obtained from R&D (Minneapolis, MN, USA); CD68 antibody and Lipofectamine 2000 reagent were purchased from Invitrogen (Carlsbad, CA, USA). Secondary antibodies FITC-conjugated goat anti-rabbit IgG and Cy3-conjugated goat anti-mouse IgG were purchased from Jackson ImmunoResearch (West Grove, PA, USA). pCMV-Neo-Bam p53 R249S was a gift from Bert Vogelstein (Addgene plasmid # 16438), and pCMV-Neo-Bam p53 wt (wt p53) was a gift from Bert Vogelstein (Addgene plasmid # 16434).
3
Tracking Extracellular Vesicle Uptake in HUVEC Cells
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EVs were labelled using the PKH26 Red Fluorescent Cell Linker kit (Sigma-Aldrich) according to the manufacturer’s instructions with minor modifications. Two microgram (2 μg) of the PKH26 labelled EVs, or the same volume of the PKH26-PBS control, were resuspended in Endothelial Cell Growth Medium with 10% FDE and added to 1 × 104 HUVEC cells maintained at 37 °C in a humidified atmosphere with 5% CO2. All samples were ultracentrifuged at 110,000× g for 1 h at 4 °C before being added to the cells and unincorporated dye from exosome labelling reactions was removed by using Exosome Spin Columns (MW 3000) (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. After 24 h of incubation, uptake was stopped by washing and fixation in 3.7% PFA for 10 min. Cells were then stained with a fluorescein isothiocyanate (FITC)-conjugated phalloidin (Sigma-Aldrich) and visualized with a Nikon Eclipse E800M fluorescence microscope (Nikon, Tokyo, Japan).
4
Phagocytosis of Candida albicans by Neutrophils
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Candida albicans was grown to stationary phase in YPD medium (Sigma-Aldrich) at 30 °C with orbital shaking at 160 rpm. Labeling of C. albicans with CFSE (Invitrogen, Carlsbad, CA, USA) was performed by incubating 1 × 108 yeasts with CFSE (0.5 μM) for 1 h at 37 °C. Yeasts were then washed twice in PBS and suspended in culture medium. Neutrophils were labeled with PKH26 red fluorescent cell linker kit (Sigma-Aldrich), according to the manufacturer’s instructions. The phagocytosis assay was performed by incubating PKH26-labeled neutrophils and CFSE-labeled yeasts at a neutrophil/yeast ratio of 1:1, in medium supplemented or not with 50 mM of NaCl during 40 min at 37°C, in the absence or presence of 10% fresh autologous serum. Phagocytosis was then evaluated by flow cytometry. Phagocytosis of Zymosan particles was assessed using pH sensitive Zymosan particles labeled with Red dye, which fluorescence markedly increases as pH decreases, allowing to distinguish endocyted particles from those attached to the cell surface. It was evaluated by flow cytometry.
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Candida albicans was grown to stationary phase in YPD medium (Sigma-Aldrich) at 30 °C with orbital shaking at 160 rpm. Labeling of C. albicans with CFSE (Invitrogen, Carlsbad, CA, USA) was performed by incubating 1 × 108 yeasts with CFSE (0.5 μM) for 1 h at 37 °C. Yeasts were then washed twice in PBS and suspended in culture medium. Neutrophils were labeled with PKH26 red fluorescent cell linker kit (Sigma-Aldrich), according to the manufacturer’s instructions. The phagocytosis assay was performed by incubating PKH26-labeled neutrophils and CFSE-labeled yeasts at a neutrophil/yeast ratio of 1:1, in medium supplemented or not with 50 mM of NaCl during 40 min at 37°C, in the absence or presence of 10% fresh autologous serum. Phagocytosis was then evaluated by flow cytometry. Phagocytosis of Zymosan particles was assessed using pH sensitive Zymosan particles labeled with Red dye, which fluorescence markedly increases as pH decreases, allowing to distinguish endocyted particles from those attached to the cell surface. It was evaluated by flow cytometry.
5
Fluorescent Labeling of Exosomes
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Exosomes were labeled using the PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich). Exosomes were added to 75ul Diluent C. In parallel, 1ul PKH26 dye was added to 75ul Diluent C and incubated with the exosome solution for 5 minutes. To bind excess dye, 150ul 1% BSA was added. Exosome Spin Columns (Invitrogen) were used for fast removal of unincorporated dye from labeled exosome preparations. AML12 cells were cultured in media containing PKH26 exosomes. Cells were fixed and stained for nuclei (DAPI, blue) and images were taken under a confocal microscope (Zeiss LSM 700, Jena, Germany).
6
Tracking Cell-Scaffold Interactions for Tissue Engineering
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The cell–scaffold constructs were divided into the following groups: control group (cells on NO or O) and cells on scaffolds loaded with TGF-β3 (NO + TGF-β3 or O + TGF-β3). hCH or hMDSCs were seeded at the density of 50,000 cells per scaffold. Ten microliters of cell suspension were seeded on each scaffold and incubated for 1 h at 37 °C, 5% CO2. Further, an additional 90 μL of the medium was added, and all samples were cultured in a culture medium at 37 °C, 5% CO2.
For cell tracking experiments, hCH or hMDSC were stained with a pkh-26 red fluorescent cell linker kit (Sigma-Aldrich, USA) according to the manufacturer’s instructions. Briefly, cells were centrifuged, and 1 mL (for 2 × 107 cells) of Diluent C was added, followed by 1 mL of 2× Dye Solution, and the mixture was incubated for 1–5 min. The staining was stopped with a 1% bovine serum albumin (BSA) solution and incubated for an additional minute. Cells were washed twice and resuspended in medium for further tracking experiments. Stained hCH or hMDSCs were seeded on scaffolds and followed for 21 d. Cell–scaffold constructs were inspected via microscopy and evaluated on days 1, 3, 7, and 14.
For cell tracking experiments, hCH or hMDSC were stained with a pkh-26 red fluorescent cell linker kit (Sigma-Aldrich, USA) according to the manufacturer’s instructions. Briefly, cells were centrifuged, and 1 mL (for 2 × 107 cells) of Diluent C was added, followed by 1 mL of 2× Dye Solution, and the mixture was incubated for 1–5 min. The staining was stopped with a 1% bovine serum albumin (BSA) solution and incubated for an additional minute. Cells were washed twice and resuspended in medium for further tracking experiments. Stained hCH or hMDSCs were seeded on scaffolds and followed for 21 d. Cell–scaffold constructs were inspected via microscopy and evaluated on days 1, 3, 7, and 14.
7
Platelet Isolation and Labeling Protocol
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The complete workflow of the experiment including the time flow is given in Fig. 1 . Platelets were isolated as reported previously and labeled with the red dye PKH26 Red Fluorescent Cell Linker Kit (Sigma) as described by us25 (link). Platelets were isolated by cardiac puncture, as this method does not cause activation of platelets48 . Briefly, blood was taken from adult anesthetized (Ketamine 100 mg/kg and Xylazine 10 mg/kg (AniMedica) 12-months old C57BL/6 N mice or APP_SweDI mice. The blood was directly drawn from the heart and collected in EDTA tubes. Subsequently, the blood was centrifuged at 100 × g for 10 min at room temperature (RT) to obtain the platelet rich plasma (PRP). PGI2 (Prostaglandin, 500 nM, Sigma) was added and platelets were isolated from PRP by centrifugation at 400 × g for 10 min at RT and then resuspended in 100 µl diluent C (Sigma, PKH26 kit), then 2 µl of the diluted dye PKH26 was added, mixed and the cells were incubated for 5 min at RT. After the incubation, 1 ml of Tyrode buffer (pH 7.4) was added, the cells centrifuged at 400 × g for 10 min at RT and resuspended in 3 ml PBS/EDTA/Heparin. Labeling efficiency of the platelets was checked under the microscope and using FACS analysis (data not shown).
8
Platelets Labeling with PKH26 Dye
9
IL7 Modulates Bone Marrow B Cell
10
Exosome Labeling and Macrophage Uptake
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Exosomes were labeled using the PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich, St. Louis, USA). First, 100 μL exosome suspensions were mixed with 100 μL of Diluent C to obtain Diluent A. Diluent B was prepared by mixing the PKH26 ethanolic dye with 100 μL of Diluent C. After mixing Diluent A with Diluent B and incubating the mixture for 5 min, 200 μL of 5% bovine serum albumin (BSA, GIBCO) solution was added to stop the staining reaction. Then, PKH26-labeled exosomes were isolated by incubating the above solution with the Exo-Quick exosome precipitation solution (SBI) for 12 h at 4 °C based on the manufacturer’s protocol, and then performing centrifugation at 1500×g for 30 min. After co-culturing exosomes with macrophages for 24 h, the macrophages were fixed with 4% paraformaldehyde (Solarbio) for 20 min and their nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Solarbio). Fluorescence images showing exosome uptake by macrophages were obtained using laser scanning confocal microscopy (ZEISS, Oberkohen, Germany).
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