BMSCs were labeled with PKH26 red fluorescent linker dye (Sigma-Aldrich). The Bio 3D conduit was created from PKH26-labeled BMSCs according to the method described above and transplanted into the nerve gap through an identical surgical procedure. Eight weeks after surgery, the regenerated nerve was removed and immunohistochemical staining was performed as described above with S-100, p75-NTR, and GFAP antibody.
Pkh26 red fluorescent linker dye
Manufactured by Merck Group
Sourced in United States
PKH26 is a red fluorescent lipophilic dye that can be used to label the cell membrane of living cells. It is a useful tool for tracking and monitoring cell populations during in vitro and in vivo experiments.
Automatically generated - may contain errors
5 protocols using pkh26 red fluorescent linker dye
1
Nerve Regeneration using Labeled BMSCs
2
Tracking Extracellular Vesicle Uptake in Fibroblasts
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The EVs were concentrated by ultrafiltration and PBS was removed. Then, 125 lL of diluent C and 125 lL of PKH26 red fluorescent linker dye (Sigma-Aldrich) were added and the samples were incubated for 5 min at room temperature. The labeling process was completed by the addition of 1% BSA in PBS. The excess dye was removed by three rounds of ultrafiltration.
For the uptake assay, 2.5 9 10 4 /well of NIH3T3 fibroblasts were seeded in a 24-well plate with a coverslip (Matsunami Glass, Osaka, Japan) and cultured for 24 h. The cells were labeled with CellTracker Green CMFDA Dye (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and then treated with the PKH26-labeled EVs (5 lgÁmL À1 ) or an equal volume of PBS. After 24 h, the cells were washed with PBS and fixed with 4% paraformaldehyde. Images of the cells were captured using a laser scanning confocal microscope and Pascal LSM software (LSM5 Pascal; Zeiss, Jena, Germany).
For the uptake assay, 2.5 9 10 4 /well of NIH3T3 fibroblasts were seeded in a 24-well plate with a coverslip (Matsunami Glass, Osaka, Japan) and cultured for 24 h. The cells were labeled with CellTracker Green CMFDA Dye (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and then treated with the PKH26-labeled EVs (5 lgÁmL À1 ) or an equal volume of PBS. After 24 h, the cells were washed with PBS and fixed with 4% paraformaldehyde. Images of the cells were captured using a laser scanning confocal microscope and Pascal LSM software (LSM5 Pascal; Zeiss, Jena, Germany).
3
Labeling ADMSCs with PKH26 Dye
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The characterized CD34-ve, CD29+ve, CD105+ve ADMSCs were labelled with a PKH26 red fluorescent linker dye (Sigma-Aldrich, German, catalogue no. MINI26). Final concentrations of 2 × 10−6 M PKH26 dye and 1 × 107 cells/ml in a 2 ml total volume were stained following the manufacturer's instruction.
After harvesting, the cells in the above count were resuspended in 1 ml of diluent C (supplied with the kit), with pipetting to ensure complete dispersion. 1 ml of cell suspension was rapidly added to 1 ml of PKH26 dye, with immediately mixing by pipetting. The sample was incubated at 25°C for 2-5 minutes with gentle inversion of the tube to assure mixing. Then, the reaction was stopped by adding serum for 1 min. The cells were centrifuged at 2000 rpm for 10 minutes at 25°C, and 10 ml of complete culture medium was added to the cells.
After harvesting, the cells in the above count were resuspended in 1 ml of diluent C (supplied with the kit), with pipetting to ensure complete dispersion. 1 ml of cell suspension was rapidly added to 1 ml of PKH26 dye, with immediately mixing by pipetting. The sample was incubated at 25°C for 2-5 minutes with gentle inversion of the tube to assure mixing. Then, the reaction was stopped by adding serum for 1 min. The cells were centrifuged at 2000 rpm for 10 minutes at 25°C, and 10 ml of complete culture medium was added to the cells.
4
Fluorescent Labeling of Small EVs
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sEVs were incubated with PKH67 green or PKH26 red fluorescent linker dye (Sigma-Aldrich) (1:500 dilution) in a final volume of 100 μL at RT for 10 min. Reactions were stopped by adding 100 μL of 5% BSA in PBS. sEVs were precipitated using Exoquick-TC solution (System Biosciences) and resuspended in PBS. As described further below, sEVs were also labeled with exo-FITC dye (Systems Biosciences).
5
Mesenchymal Stem Cells Proliferation with EPOa
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Mesenchymal stem cells growth was divided into two equal fractions, each fraction represented a group: -group 1: a control group of MSCs which were not treated with EPOa; -group 2: MSCs that were treated with EPOa (Eprex ® injectable solution 10.000 IU/mL, Janssen-Cilag Pty Ltd.). MSCs were seeded at 4 × 10 3 cells/cm 2 in tissue culture plates and incubated with the culture medium supplemented with EPOa (40 IU/mL) for 24 h [15] (link). The cells were examined for cell proliferation assay using the MTT (3-[4,5-dimethylthiazol-2-yl]-2.5-diphenyl tetrazolium bromide) cell proliferation kit (Trevigen Inc., Gaithersburg, MD, USA) as per manufacturer's protocol. Then MSCs were labelled with PKH26 red fluorescent linker dye supplied by Sigma (Saint Louis, Missouri, USA) for homing detection, and used for injection in the treated groups. MSCs 1 × 10 6 in 1 mL of phosphate buffer saline, pH 7.4, were administered to the groups treated with cells for each 1 cm 2 of the burn area.
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