Pkh26 membrane dye

Manufactured by Merck Group

Sourced in United States

PKH26 membrane dye is a fluorescent dye used for labeling cell membranes. It is a lipophilic dye that binds to the lipid regions of the cell membrane, allowing for the visualization and tracking of labeled cells.

Automatically generated - may contain errors

Lab products found in correlation

Spelling variants of this product

PKH26 (838 citations) PKH26 dye (97 citations) PKH26 red fluorescent membrane linker dye (10 citations) PKH26 (red) membrane dye (6 citations) PKH26 fluorescent cell membrane dye (3 citations)

12 protocols using pkh26 membrane dye

Exosomal miRNA Intercellular Transfer

Check if the same lab product or an alternative is used in the 5 most similar protocols

Exosomes were fluorescently labeled using PKH26 membrane dye (Sigma-Aldrich, USA) according to the manufacturer’s instructions. Then the labeled exosomes were washed with PBS and then incubated with the recipient cells MRC-5 for 48 h to determine the uptake of exosomes.
For the tracing of exosomal miRNAs, the HCT116 cells were transfected with FAM-labeled miR-146a-5p or miR-155-5p and co-cultured with MRC-5 cells for 48 h. The transfer of the FAM-labeled miRNAs was observed by confocal microscopy TCS SP5 (Leica, Germany). The nucleus was stained with DAPI.

Wang D., Wang X., Song Y., Si M., Sun Y., Liu X., Cui S., Qu X, & Yu X. (2022). Exosomal miR-146a-5p and miR-155-5p promote CXCL12/CXCR7-induced metastasis of colorectal cancer by crosstalk with cancer-associated fibroblasts. Cell Death & Disease, 13(4), 380.

+ Open protocol

+ Expand

Measuring Dendritic Cell Phagocytosis

Check if the same lab product or an alternative is used in the 5 most similar protocols

NIH/3T3 were stained with the PKH-26 membrane dye (Sigma Aldrich, Cat#PKH26-GL) following the manufacturer’s instructions. 1x10⁵ MutuDC were plated in 96 round bottom-well plates together with PKH-26⁺ 3T3s at different 3T3:MutuDC ratios (1:2, 1:4, 1:8, 1:16, 1:32). The co-cultures were incubated in the presence of prazosin (10 μM) or DMSO for 2 h or 5 h at 37°C, 5% CO2, or left on ice for 5 h. Cells were then stained with anti-CD11c-APC (clone HL3, BD PharMingen Cat#550261) and violet live/dead Dye (ThermoFisher Cat#L34955) and fixed to prevent further uptake.
Percentage of PKH-26⁺ MutuDCs (CD11c⁺ cells) was determined. Phagocytic index was calculated by subtracting the percentage of PKH-26⁺ cells in CD11c⁺ gate obtained at 4°C from the percentage of this subset measured at 37°C after 2 h or 5 h of incubation.

Kozik P., Gros M., Itzhak D.N., Joannas L., Heurtebise-Chrétien S., Krawczyk P.A., Rodríguez-Silvestre P., Alloatti A., Magalhaes J.G., Del Nery E., Borner G.H, & Amigorena S. (2020). Small Molecule Enhancers of Endosome-to-Cytosol Import Augment Anti-tumor Immunity. Cell Reports, 32(2), 107905.

+ Open protocol

+ Expand

Exosome Isolation and Characterization Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

PKH26 membrane dye and exososme inhibitor GW4869 were purchased from Sigma-Aldrich. Trans-well chambers were purchased from Merck Millipore. PrestoBlue Cell Viability Reagent were purchased from Invitrogen. jetPEI^® transfection reagent were purchased from Polyplus-transfection^®. EIF3C polyclonal antibodies were purchased from Sigma. S100A11,CD63,TSG101,CD31,CD34,CD81,GM130,HA, Actin polyclonal antibodies were purchased from GeneTex; CD9 and ALIX polyclonal antibodies were purchased from Abcam, and horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse secondary antibody were ordered from System Biosciences. pcDNA5/FRT EIF3C plasmid was kindly given by Dr. Hershey JW.

Lee H.Y., Chen C.K., Ho C.M., Lee S.S., Chang C.Y., Chen K.J, & Jou Y.S. (2018). EIF3C-enhanced exosome secretion promotes angiogenesis and tumorigenesis of human hepatocellular carcinoma. Oncotarget, 9(17), 13193-13205.

+ Open protocol

+ Expand

T Cell Phenotypic and Functional Analyses

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following reagents and fluorochrome-conjugated monoclonal antibodies were used in this study for T cell phenotypic and functional analyses: PKH26 membrane dye (Sigma), APC- anti-CD4, APC-anti-CD8a, PE-anti-CD4, PE-anti-CD25, FITC or APC-anti-TNFα, PE-anti-ICOS-L FITC-Annexin-V (all from eBioscience), FITC-anti-CXCR4, APC-anti-CXCR7, PE-anti-hCCR4 (all from R&D Systems), and FITC-anti-CD279/PD-1 (Biolegend, San Diego, CA). Intracellular expression of TNFα was detected following fixation and permeabilization (fixation/permeabilization buffer concentrate, Cat. No. 00-5123-43, diluent, Cat. No. 00-5223-56, and permeabilization buffer, Cat. No. 00-8333-56, all from eBioscience). All samples were analyzed with a FACSCalibur flow cytometer, using CellQuest software (BD Biosciences). For analysis of tumor ascites CD14⁺ cell phenotype, the following monoclonal antibodies were used: FITC-anti-CD14, PE-anti-CD11b (both from R & D Systems), PE-anti-CD11c, PE-anti-B7-H1, PerCP-anti-CD14 (all from BD Biosciences), FITC-anti-CD279/PD-1, FITC-anti-CD83, FITC-anti-CD80, PE-anti-CD86, APC-anti-CD14, and PE-IL-1β (all from eBioscience). Intracellular expression of IL-1β was assayed following overnight incubation of CD14⁺ cells in the presence of Brefeldin A, followed by fixation and permeabilization as described above.

Goyne H.E., Stone P.J., Burnett A.F, & Cannon M.J. (2014). Ovarian tumor ascites CD14+ cells suppress dendritic cell-activated CD4+ T cell responses through IL-10 secretion and indoleamine 2,3-dioxygenase. Journal of immunotherapy (Hagerstown, Md. : 1997), 37(3), 163-169.

+ Open protocol

+ Expand

CD8 T Cell Cytotoxicity Assay with Tbc1d10c

Check if the same lab product or an alternative is used in the 5 most similar protocols

CFSE-loaded OT-I;Tbc1d10c^+/+ and OT-I;Tbc1d10c^−/− CD8 T cells were activated for 24 h by OVA peptide stimulation as described above. MC38 cells were pre-incubated with 10 ng/ml murine recombinant Ifn-γ (R&D Systems) and OVA_257-264 peptide for 24 hrs and subsequently stained with PKH26 membrane dye as per manufacturer’s protocol (Sigma Aldrich). FACS-sorted CD8 CFSE+ T cells were then plated with OVA-coated MC38 cells for 12 hrs at the following effector-to-target cell ratios: 1:2, 1:1, 2:1, and 4:1. In some cases, siRNA-transduced OT-I CD8 T cells were incubated at a 2:1 effector-to-target ratio. For each ratio, each T cell effector genotype was analyzed in triplicate. Cultures were harvested and stained with Fixable Viability Dye as per manufacturer’s Protocol (Invitrogen), and MC38-specific lysis was quantified by flow cytometry. For control values, MC38 cells were either boiled for 5 minutes (Maximal lysis) or incubated without CD8 T cells (nonspecific lysis). Specific lysis was calculated as follows: Specific Lysis = [(Sample Lysis) – (nonspecific Lysis)] ÷ [(Maximal Lysis) – (nonspecific Lysis)].

Cohen A.O., Woo S.H., Zhang J., Cho J., Ruiz M.E., Gong J., Du R., Yarygina O., Jafri D.Z., Bachelor M.A., Finlayson M.O., Soni R.K., Hayden M.S, & Owens D.M. (2022). Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response. Oncoimmunology, 11(1), 2141011.

+ Open protocol

+ Expand

Exosome Uptake by HUVECs Visualization

Check if the same lab product or an alternative is used in the 5 most similar protocols

Exosomes were fluorescently labeled using PKH26 membrane dye (Sigma). After that, labeled exosomes were co-cultured with HUVECs for 24 h at 37°C. Later on, exosomes that absorbed by HUVECs were observed by a fluorescence microscope. DAPI (blue) was used to label nuclei.

Dai G., Yang Y., Liu S, & Liu H. (2022). Hypoxic Breast Cancer Cell-Derived Exosomal SNHG1 Promotes Breast Cancer Growth and Angiogenesis via Regulating miR-216b-5p/JAK2 Axis. Cancer Management and Research, 14, 123-133.

+ Open protocol

+ Expand

T Cell Proliferation Assay with Myeloid Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Splenocytes were stained with the PKH26 membrane dye (Sigma-Aldrich) following the manufacturer’s instructions. For the T cell proliferation assay, the PKH26-stained splenocytes were plated at 2.5 × 10⁴/well on a 96-well plate in RPMI supplemented with 10% FBS, 1% (v/v) penicillin/streptomycin solution (Gibco), 1 mM sodium pyruvate, and 50 µM β-mercaptoethanol. Splenocytes were either unstimulated and cultured as a monoculture or splenocytes were stimulated with murine CD3/CD28 Dynabeads (Thermofisher Scientific, Darmstadt, Germany; 1:1 ratio) and 30 U/mL murine rIL-2 (Peprotech) and cultured as a monoculture or as a co-culture with purified CD11b⁺ Gr⁺ bone marrow cells in a 1:1 or 1:0.5 cell ratio. The cells were incubated for 3 d at 37 °C with 5% CO₂, then subsequently the T cells were assessed for their proliferation rate and cell number by flow cytometry. Cells were stained for CD3-FITC, CD4-Alexa Fluor 700, CD8α-APC antibodies (all Biolegend), and DAPI and collected in TruCount tubes (BD Bioscience). Proliferation was normalized to T cells stimulated with CD3/CD28 Dynabeads and rIL-2, which were set to 100%.

Hofstee M.I., Heider A., Häckel S., Constant C., Riool M., Richards R.G., Moriarty T.F, & Zaat S.A. (2021). In Vitro 3D Staphylococcus aureus Abscess Communities Induce Bone Marrow Cells to Expand into Myeloid-Derived Suppressor Cells. Pathogens, 10(11), 1446.

+ Open protocol

+ Expand

Visualizing Exosome Cellular Uptake

Check if the same lab product or an alternative is used in the 5 most similar protocols

Exosomes were stained with PKH26 membrane dye (Sigma, CAT. MIDI26–1KT). After culturing with the labeled exosomes for 3 h, the cells were fixed and stained with Hoechst. The cellular uptake of exosomes was observed on a Leica TCS-SP5 LSM electron microscope (JEM-1220, JEOL, Ltd, Japan). For the in vitro experiments, 1 × 10⁵ receptor cells were co-cultured with 10 mg of exosomes.

Han M., Hu J., Lu P., Cao H., Yu C., Li X., Qian X., Yang X., Yang Y., Han N., Dou D., Zhang F., Ye M., Yang C., Gu Y, & Dong H. (2020). Exosome-transmitted miR-567 reverses trastuzumab resistance by inhibiting ATG5 in breast cancer. Cell Death & Disease, 11(1), 43.

+ Open protocol

+ Expand

Exosome Internalization Assay Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

PKH26 membrane dye (PKH26GL, Sigma-Aldrich, Germany) was utilized to fluorescently label exosomes for labeling purposes. The labeled exosomes were collected by ultracentrifugation after being rinsed with PBS. For the exosome internalization assay, labeled exosomes were mixed for 24 h with recipient cells. The cell skeleton of HUVECs was stained with PhalloidiniFluor 488 Reagent (Abcam, UK) and the nucleus was stained with DAPI (Solarbio, USA). Confocal microscopy pictures were taken using a Zeiss (Germany) laser scanning microscope. Cells (2 × 10⁵) were subject to subsequent experiments after stimulation with 1 × 10⁹ exosomes for 48 h.

Mao Y., Wang J., Wang Y., Fu Z., Dong L, & Liu J. (2024). Hypoxia induced exosomal Circ-ZNF609 promotes pre-metastatic niche formation and cancer progression via miR-150-5p/VEGFA and HuR/ZO-1 axes in esophageal squamous cell carcinoma. Cell Death Discovery, 10, 133.

+ Open protocol

+ Expand

Exosome Uptake Visualization in MSCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

For exosome-uptaking experiment, purified exosomes derived from OPM2 (OPM2-exo) were stained using PKH26 membrane dye (Sigma, USA). Stained exosomes were washed in 2 ml of PBS, collected by ultracentrifugation as demonstrated above, and resuspended in filtered PBS. 10 μg of the PKH26-stained exosomes or the same volume of the PKH26-PBS control was added and incubated for 24 h. The binding of OPM2-exo to the MSCs was observed with a fluorescence microscope (Germany). OPM2 cells were washed twice with PBS, stained with Hoechst 33342 for 5 min, and washed twice with PBS before being photographed.

Cheng Q., Li X., Liu J., Ye Q., Chen Y., Tan S, & Liu J. (2017). Multiple Myeloma-Derived Exosomes Regulate the Functions of Mesenchymal Stem Cells Partially via Modulating miR-21 and miR-146a. Stem Cells International, 2017, 9012152.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!