Pkh26 red fluorescent cell linker kit for general cell membrane labeling

Manufactured by Merck Group

Sourced in Germany, United States

The PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling is a laboratory product that allows for the fluorescent labeling of cell membranes. The kit contains a cell membrane linker dye that binds to lipid regions, enabling the visualization of cells under fluorescent microscopy.

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Lab products found in correlation

Exoquick precipitation kit, by System Biosciences (2 mentions) Human hela cells, by American Type Culture Collection (1 mentions) Biorevo bz 9000 microscope, by Keyence (1 mentions) 96 well suspension culture plate, by Greiner (1 mentions) Dexamethasone d1756, by Merck Group (1 mentions) Cd81 antibody, by Santa Cruz Biotechnology (1 mentions) Ab108337, by Abcam (1 mentions) Calnexin, by Cell Signaling Technology (1 mentions) Pkh67 green fluorescent cell linker kit for general cell membrane labeling, by Merck Group (1 mentions) Fitc conjugated goat anti rabbit igg h l secondary antibody, by Thermo Fisher Scientific (1 mentions) Grp94, by Cell Signaling Technology (1 mentions) Cd63 antibody, by Santa Cruz Biotechnology (1 mentions) Lamin a c, by Cell Signaling Technology (1 mentions) B27 supplement, by Thermo Fisher Scientific (1 mentions) Ultra low attachment plate, by Corning (1 mentions) Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions) Epidermal growth factor (egf), by Merck Group (1 mentions) Insulin, by Merck Group (1 mentions) Heparin, by Merck Group (1 mentions) Accumax, by Merck Group (1 mentions)

Close spelling variants of this product

PKH26 (838 citations) PKH26 Red Fluorescent Cell Linker (38 citations) PKH26 kit (31 citations) PKH26 Red (28 citations) PKH26 Fluorescent Cell Linker (9 citations) PKH26 labeling kit (8 citations) PKH26 Red Fluorescent Cell Linker Kits for General Cell Membrane Labeling (5 citations) Red Fluorescent Cell linker for General Cell Membrane (2 citations)

16 protocols using pkh26 red fluorescent cell linker kit for general cell membrane labeling

HeLa Cell Membrane Labeling with PKH26

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Human HeLa cells (ATCC, Rockville, MD, USA) were grown in Dulbecco’s minimal essential medium supplemented with 10% fetal bovine serum, 1% glutamine, 100U of penicillin and streptomycin (Celbio S.r.l., Milan, Italy), in a humidified air atmosphere containing 5% CO2. Cells were seeded onto glass coverslips in six-multiwell plates (1×10^{5 (link)} per well) 48 h before being incubated with PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma-Aldrich, Catalog Nos. MINI26 and PKH26GL) to stain the plasma membrane. After staining according to manufacturer’s recommendations, the medium containing PKH26 dye was removed and replaced with fresh medium. The cells were observed as fresh preparations by fluorescence microscopy or processed for transmission electron microscopy (see below) either immediately after staining or after 30 min, 1 h and 3 h.
Some cells were stained in suspension with PKH26 and immediately observed as fresh preparations by fluorescence microscopy.

Grecchi S, & Malatesta M. (2014). Visualizing Endocytotic Pathways at Transmission Electron Microscopy via Diaminobenzidine Photo-Oxidation by a Fluorescent Cell-Membrane Dye. European Journal of Histochemistry : EJH, 58(4), 2449.

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Tumor spheroid co-culture with infected MSCs

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PaCaDD or MIA-PaCa2 cells were seeded in DMEM cell culture medium containing 2% heat-inactivated FCS and 0.25% methylcellulose (PureCol, Biomaterials, Fremont, USA) in a 96-well suspension culture plate (Greiner Bio-One, Frickenhausen, Germany) with a density of 10,000 cells/well for spheroid formation. The cells were used for co-incubation experiments with infected MSCs 72 h later. MSCs were stained with the PKH26 red fluorescent cell linker kit for general cell membrane labeling (Sigma-Aldrich, Munich, Germany), and 5 × 10⁴ MSCs/well were seeded in a 24-well plate in DMEM cell culture medium supplemented with 2% FCS. The MSCs were infected 24 h later with a virus titer of 2000 TCID₅₀. Two hours after infection, the MSCs were overlaid with a gel layer of methylcellulose, Matrigel and collagen (PureCol, Biomaterials, Fremont, USA) at a ratio of 1:1:1 in DMEM supplemented with 2% FCS. PaCaDD183 or MIA-PaCa2 spheroids were seeded on top of the gel layer and co-incubated with MSCs for 16 h. Next, the spheroids were transferred with a cut tip of a 10 μl pipette into new 96-well suspension culture plates. Forty-two hours after infection, images of non-fixed spheroids were acquired under a BIOREVO BZ-9000 microscope (Keyence, Neu-Isenburg, Germany).

Kaczorowski A., Hammer K., Liu L., Villhauer S., Nwaeburu C., Fan P., Zhao Z., Gladkich J., Groß W., Nettelbeck D.M, & Herr I. (2016). Delivery of improved oncolytic adenoviruses by mesenchymal stromal cells for elimination of tumorigenic pancreatic cancer cells. Oncotarget, 7(8), 9046-9059.

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Exosome Isolation and Membrane Labeling

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Exosome isolation reagent (from cell culture media) was purchased from Thermo Fisher scientific (4478359, China). PKH67 Green Fluorescent Cell Linker Kit for General Cell Membrane Labeling (MINI67-1KT, China) and PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (PKH26GL-1KT, China) were purchased from Sigma. Lipopolysaccharide (LPS, Escherichia coli 0111: B4, L4391), β‐glycerophosphate (G9422), L‐ascorbic acid 2‐phosphate (49752), and dexamethasone (D1756) were from Sigma, China. Antibodies used in this study were rabbit polyclonal antibody against alkaline phosphatase (ALP) (ab108337, Abcam), CD63 Antibody (sc-5275, SANTA CRUZ Biotechnology), CD81 Antibody (sc-166029, SANTA CRUZ Biotechnology), calnexin (2679 T, cell Signaling Technology), Grp94 (20292 T, cell Signaling Technology), and Lamin A/C (4777 T, cell Signaling Technology). Secondary antibody used for immunofluorescence staining was fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (H + L) Secondary Antibody (31635, Thermo Fisher Scientific, China).

Zhao Q., Zhang Y., Xiao L., Lu H., Ma Y., Liu Q, & Wang X. (2021). Surface engineering of titania nanotubes incorporated with double-layered extracellular vesicles to modulate inflammation and osteogenesis. Regenerative Biomaterials, 8(3), rbab010.

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Thymosphere Culture and Enumeration

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Spheres were grown in ultralow attachment plates (Corning) at variable densities in MEBM medium (Lonza) supplemented with B27 supplement (GIBCO), 0.5 μg/ml hydrocortisone (Sigma), 5 μg/ml insulin (Sigma), 4 μg/ml heparin (Sigma), 20 ng/ml bFGF (Invitrogen), and 20 ng/ml EGF (Sigma). For precise enumeration of thymospheres under various experimental conditions, we determined the optimal plating dilution to be 10⁵ nonhematopoietic cells (NHC)/ml. Unless indicated otherwise, spheres were collected on day 7–8 of culture. PKH-26 labeling was performed on fresh thymic digests using PKH-26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma) at a concentration of 1 × 10⁻⁹ M PKH-26 dye per 1 × 10⁵ cells according to the manufacturer’s protocol. Imaging of spheres was performed with a motorized inverted Cell Observer Z1 (Zeiss).
For the sphere re-formation assay, thymospheres were collected by size selection on 35 μm filters, dissociated into single cells both enzymatically (10 min in Accumax, Sigma) and mechanically by gentle pipetting, filtered through 35 μm filters, and plated in the thymosphere-culturing medium as described above at a density of 10⁴ and 10³ cells per ml.

Ucar A., Ucar O., Klug P., Matt S., Brunk F., Hofmann T.G, & Kyewski B. (2014). Adult Thymus Contains FoxN1− Epithelial Stem Cells that Are Bipotent for Medullary and Cortical Thymic Epithelial Lineages. Immunity, 41(2), 257-269.

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Labeling and Harvesting Cell Sheets

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Prior to cell sheet formation MSC (passage 2–4) were labeled by PKH26 Red dye (PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling, Sigma-Aldrich, Milwaukee, WI, USA). The cells were detached with 0.05% trypsin, labeled and seeded at 3 × 106/well density in a 6-well culture plate (Corning, Corning, NY, USA). Forming CSs were maintained in DMEM, 10% FBS, penicillin/streptomycin for at least 48 h prior to experiments. The transgene expression was verified by enzyme-linked immunosorbent assay to assess SCF concentration in conditioned media. The Mouse SCF ELISA Kit (ab100740, Abcam, Cambridge, MA, USA) was used. An optical density was measured using VictorTM X3 Multi Label Plate Reader (Perkin Elmer Inc, Waltham, MA, USA). The cell sheet was harvested by incubation in Versene solution (Paneco, Moscow, Russia) until it self-detached within 5–7 min.

Dergilev K.V., Shevchenko E.K., Tsokolaeva Z.I., Beloglazova I.B., Zubkova E.S., Boldyreva M.A., Menshikov M.Y., Ratner E.I., Penkov D, & Parfyonova Y.V. (2020). Cell Sheet Comprised of Mesenchymal Stromal Cells Overexpressing Stem Cell Factor Promotes Epicardium Activation and Heart Function Improvement in a Rat Model of Myocardium Infarction. International Journal of Molecular Sciences, 21(24), 9603.

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Labeling MSCs with PKH26 Dye

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To generate red-labeled MSCs we used the PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma-Aldrich) as per manufacturer procedures. In brief, in RT a 2 × cell suspension was prepared by re-suspending pre-washed 2 × 10⁷ MSCs in serum free medium in 1 ml of Diluent C. A 2 × dye solution prepared by adding 4 μl of PKH26 ethanolic dye solution to 1 ml of Diluent C and mixed well. Rapidly add the 1ml of 2 × cell suspension to 1 ml of 2 × dye solution and immediately mix the sample by pipetting. Leave for < 5 min and stop the reaction by adding 2 ml of serum and incubate 1 min to allow binding of excess dye. Centrifuge cells at 400 × g for 10 min followed by washing 3 times with 10 ml complete medium to remove any unbound dye. Red-labeled cells were injected in mice circulation by injection through the left cardiac ventricle.

Ananthula S., Sinha A., Gassim M.E., Batth S., Marshall GD J.r., Gardner L.H., Shimizu Y, & ElShamy W.M. (2016). Geminin overexpression-dependent recruitment and crosstalk with mesenchymal stem cells enhance aggressiveness in triple negative breast cancers. Oncotarget, 7(15), 20869-20889.

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EPDENs Uptake by ADMSCs Visualization

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Fragaria-derived EPDENs were stained with PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma-Aldrich), according to the manufacturer’s instructions, with minor modifications, as previously described [22 (link)]. Unincorporated dye from EPDEN labeling reactions was removed using Exosome Spin Columns (MW 3000) (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. PKH26-labeled EPDENs (2 µg/L × 10⁴ cells), or the same volume of the PKH26-PBS control, were added to semiconfluent ADMSCs. After 4 h and 24 h of incubation, uptake was stopped by washing and fixation in 3.7% PFA for 10 min. Cells were then stained with a FITC-conjugated phalloidin (Sigma-Aldrich) and visualized with a Nikon Eclipse E800M fluorescence microscope (Nikon, Tokyo, Japan).

Perut F., Roncuzzi L., Avnet S., Massa A., Zini N., Sabbadini S., Giampieri F., Mezzetti B, & Baldini N. (2021). Strawberry-Derived Exosome-Like Nanoparticles Prevent Oxidative Stress in Human Mesenchymal Stromal Cells. Biomolecules, 11(1), 87.

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Phagocytosis of Heat-Induced Cell Corpses

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Cell death was induced by treatment of thymocytes at 47°C for 20 min, and then, cells were incubated at 37°C for 3 h before subjecting the cell corpses to the phagocytosis assay. Before the phagocytosis assay in vivo, cell corpses were stained with the PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma-Aldrich) according to the manufacturer’s instructions. Then, 5 × 10⁷ PKH26-stained cell corpses were intravenously administered to mice, and mouse spleens were collected 2 h after cell corpse administration. Immune cells engulfing PKH26-positive cell corpses were detected using flow cytometry.

Shibata T., Sato R., Taoka M., Saitoh S.I., Komine M., Yamaguchi K., Goyama S., Motoi Y., Kitaura J., Izawa K., Yamauchi Y., Tsukamoto Y., Ichinohe T., Fujita E., Hiranuma R., Fukui R., Furukawa Y., Kitamura T., Takai T., Tojo A., Ohtsuki M., Ohto U., Shimizu T., Ozawa M., Yoshida N., Isobe T., Latz E., Mukai K., Taguchi T., Hemmi H., Akira S, & Miyake K. (2023). TLR7/8 stress response drives histiocytosis in SLC29A3 disorders. The Journal of Experimental Medicine, 220(9), e20230054.

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Tracking Implanted DFAT Cells in Organs

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DFAT cells from the ICR mice were labeled with PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma Chemical, St. Louis, MO, USA) as described previously [13 (link)]. In total, 10⁵ labeled DFAT cells were infused through the posterior orbital venous plexus of the ICR mice. At 1 h, 24 h and 1 week after the injection, kidney, aorta, liver and lungs were removed and fixed in 3% formalin in phosphate-buffered saline (PBS) (Kanto Chemical, Tokyo, Japan) and embedded in paraffin. Sections were observed under a fluorescence microscope (IX73; Olympus, Tokyo, Japan), and images were obtained with a digital imaging system (Fig. 1).
Fig. 1

Distribution of implanted DFAT cells. In total, 10⁵ PKH26-labeled DFAT cells were infused through the posterior orbital venous plexus in ICR mice. One hour, 24 h, and one week after the infusion, kidney, aorta, liver and lungs were removed and fixed in 3% formalin in PBS and embedded in paraffin. Arrowheads indicate trapped DFAT cells. Bar = 50 μm. DFAT dedifferentiated fat

Utsunomiya K., Maruyama T., Shimizu S., Matsumoto T., Endo M., Kobayashi H., Kano K., Abe M, & Fukuda N. (2022). Implantation of dedifferentiated fat cells ameliorated antineutrophil cytoplasmic antibody glomerulonephritis by immunosuppression and increases in tumor necrosis factor-stimulated gene-6. Stem Cell Research & Therapy, 13, 319.

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Fluorescent Labeling of MSCs

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For live cell imaging, MSCs were labeled with PKH26® Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma Aldrich) prior encapsulation in PRP gels. After detaching and counting of cells, the desired amount of cells were pipetted in a new falcon tube, washed with serum-free αMEM and centrifuged at 400g for 5 min. Cells were resuspended in Diluent C (Sigma) and filtered using a 40 μm cell strainer, and dye working solution was added (2 μl PKH26 Red/1 × 10⁶ MSCs) and incubated for 5 min at room temperature. The labeling reaction was stopped by addition of FBS (SeraPlus), and cells were washed three times with medium and the cell count was determined.

Zahn J., Loibl M., Sprecher C., Nerlich M., Alini M., Verrier S, & Herrmann M. (2017). Platelet-Rich Plasma as an Autologous and Proangiogenic Cell Delivery System. Mediators of Inflammation, 2017, 1075975.

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