Pe anti mouse cd86 antibody

Manufactured by BioLegend

Sourced in United States

The PE anti-mouse CD86 Antibody is a fluorescently-labeled antibody that binds to the CD86 surface protein expressed on mouse cells. CD86 is a co-stimulatory molecule involved in antigen presentation and T cell activation. This antibody can be used to detect and quantify CD86-positive cells in mouse samples using flow cytometry.

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9 protocols using pe anti mouse cd86 antibody

Ultrasound-Mediated Dendritic Cell Maturation

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BMDCs were extracted from the bone marrow of 5-week-old female Balb/c mice to study DC maturation in vitro. 4T1 cells were pretreated for 12 h with PBS, US only, MH, MHS, MH + US and MHS + US (MH/MHS = 100 μg/mL, US = 1.0 MHz, 1.0 W/cm², 50% duty cycle, 5 min). Afterwards, 1 × 10⁶ immature DC cells were co-cultured with 1 × 10⁵ pretreated 4T1 cells in a transwell system for 24 h. FCM was used to examine the maturation of DC cells after staining with APC anti-mouse CD80 Antibody (Biolegend, Cat# 104714, Clone No.16-10A1), PE anti-mouse CD86 Antibody (Biolegend, Cat# 105007, Clone No.GL-1), and FITC anti-mouse CD11c Antibody (Biolegend, Cat# 117306, Clone No. N418) antibodies (The antibody concentration was 1:100 diluted with PBS). Cell culture supernatants were also collected. And ELISA kits were used to measure the pro-inflammatory cytokines IL-2 and IL-12p70 secreted by DCs.

Yu J., Zhou B., Zhang S., Yin H., Sun L., Pu Y., Zhou B., Sun Y., Li X., Fang Y., Wang L., Zhao C., Du D., Zhang Y, & Xu H. (2022). Design of a self-driven probiotic-CRISPR/Cas9 nanosystem for sono-immunometabolic cancer therapy. Nature Communications, 13, 7903.

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Evaluating DC Maturation In Vitro

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To evaluate DC maturation in vitro, bone marrow cells were obtained from BALB/c mice and cultured in complete 1640 medium, supplemented with mouse recombinant macrophage colony stimulating factor (GMCSF, 20 ng/ml, Cat# 576304, Biolegend) and IL-4 (10 ng/ml, Cat# 574304, Biolegend) for 6 days to generate bone marrow-derived DCs. Then, IR-780 treated CT26 cancer cells were co-cultured with the DCs for 24 h. Next, the cells were collected and stained with APC anti-mouse CD11c Antibody (Cat# 117310, Biolegend), Brilliant Violet 421™ FITC anti-mouse CD80 Antibody (Cat# 104706, Biolegend), PE anti-mouse CD86 Antibody (Cat# 105008, Biolegend), or anti-mouse I-A/I-E Antibody (Cat# 107632, Biolegend), and analyzed by flow cytometry.

Jiang Q., Zhang C., Wang H., Peng T., Zhang L., Wang Y., Han W, & Shi C. (2019). Mitochondria-Targeting Immunogenic Cell Death Inducer Improves the Adoptive T-Cell Therapy Against Solid Tumor. Frontiers in Oncology, 9, 1196.

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Profiling Tumor Immune Landscape by Flow Cytometry

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CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells and CD3⁺CD4⁺FOXP3⁺ T cells, CD11c ⁺CD86⁺CD80⁺ dendritic cells as well as CD45⁺CD11b⁺F4/80⁺CD206⁺ M2 like macrophage and CD45⁺CD11b⁺F4/80⁺CD86⁺ M1 like macrophage in the tumor tissues or spleen were isolated and analyzed using flow cytometry. The antibodies involved in the experiment include FITC anti-mouse CD11c Antibody (Biolegend, Cat# 117306, Clone No. N418), PE anti-mouse CD86 Antibody (Biolegend, Cat# 105007, Clone No.GL-1), APC anti-mouse CD80 Antibody (Biolegend, Cat# 104714, Clone No.16-10A1), FITC anti-mouse CD3 (Biolegend, Cat# 100203, Clone No.17A2), PE anti-mouse CD4 (Biolegend, Cat# 100407, Clone No.GK1.5), APCanti-mouseCD8a (Biolegend, Cat# 100712, Clone No.53-6.7), AlexaFluor488 anti-mouse FOXP3 (Biolegend, Cat# 320012, Clone No.150D), FITC anti-mouse/human CD11b Antibody (Biolegend, Cat# 101205, Clone No. M1/70), APC anti-mouse CD45 (Biolegend, Cat# 103112, Clone No.30-F11), PerCP anti-mouse F4/80 Antibody (Biolegend, Cat# 123126, Clone No.BM8) and PE/Cyanine7 anti-mouse CD206 (Bioegend, Cat# 141720, Clone No.C068C2). The antibody concentration was 1:100 diluted with PBS.
IL-2, IL-12p70, TNF-α, and IFN-γ in primary tumors were also examined with ELISA kits. And tumors were stained for immunofluorescence of CD3⁺CD4⁺ and CD3⁺CD8⁺ proliferated CTLs in 4T1 tumor tissue slices of the primary tumor.

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Macrophage Subpopulation Analysis

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100 μL of suspension fluid was added to 0.25 μg APC anti-mouse F4/80 antibody (BioLegend), 1.0 μg PE anti-mouse CD86 antibody (BioLegend), and 0.125 μg FITC anti-mouse CD206 antibody (BioLegend), respectively. Homogenized solutions were placed in the dark at 4°C for 30 minutes, followed by addition of 200 μL FACS buffer, and centrifuged at 4°C, 300 ×g for 5 minutes. After removal of supernatant, the cellular solutions were washed with FACS buffer, followed by addition of 1 mL FACS buffer to resuspend cellular samples. Finally, cellular aggregates were broken up and analyzed with BD FACSCanto II, APC anti-mouse F4/80 antibody-specific M1 macrophage (F4/80⁺), APC anti-mouse F4/80 antibody, and FITC anti-mouse CD206 antibody-specific M2 macrophage (F4/80⁺, CD206⁺). FlowJo software was used to analyze the percentage of macrophages in the lung tissues and the proportions of M1 and M2 macrophages in the lung and tumor tissue samples.
Percentage of macrophages: macrophages (F4/80⁺)/cells.
Percentage of M1 macrophages: M1 (F4/80⁺, CD86⁺)/macrophages (F4/80⁺).
Percentage of M2 macrophages: M2 (F4/80⁺, CD206⁺)/macrophages (F4/80⁺).

Wang W.J., Wu Y.S., Chen S., Liu C.F, & Chen S.N. (2015). Mushroom β-Glucan May Immunomodulate the Tumor-Associated Macrophages in the Lewis Lung Carcinoma. BioMed Research International, 2015, 604385.

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Immunization Protocol for SARS-CoV-2 Spike RBD

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Fmoc-amino acids were obtained from GL Biochem (Shanghai, China). 2-Cl-trityl chloride resin was obtained from Nankai Resin Co. Ltd. (Tianjin, China). Commercially available reagents were used without further purification unless noted otherwise. Deionized water was used for all experiments. All other chemicals were reagent grade or better. Horseradish peroxidase-conjugated goat anti-mouse IgG, IgG1, IgG2b, and IgG2c (1030-05, 1071-05, 1091-05, and 1078-05, 1:5000 dilution) were obtained from Southern Biotech (USA). Mouse IFN-γ, IL-5, TNF-α and IL-6 ELISA kits (430807, 431204, 430907, and 431307) were obtained from Biolegend (USA). Recombinant murine GM-CSF and IL-4 (315-03 and 214-14) were purchased from Peprotech (USA). FITC anti-mouse CD83 Antibody, FITC anti-mouse CD80 Antibody, and PE anti-mouse CD86 Antibody (121505, 104705, and 159204, 0.5 µg per million cells in 100 µL volume for usage) were purchased from Biolegend (USA). Imject Alum Adjuvant was obtained from Thermofisher (USA). RBD-Fc (Z03513, SARS-CoV-2 Spike protein, RBD, mFc Tag, CHO-expressed) and RBD-H (Z03479, SARS-CoV-2 Spike protein, RBD, His Tag,) were purchased from Genscript Biotech (Nanjing, China). This research followed institutional guidelines, and all animal experiments were approved by the Institutional Animal Care and Use Committee of Westlake University.

Xu T., Wang J., Zhao S., Chen D., Zhang H., Fang Y., Kong N., Zhou Z., Li W, & Wang H. (2023). Accelerating the prediction and discovery of peptide hydrogels with human-in-the-loop. Nature Communications, 14, 3880.

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Synthesis of Multifunctional Biomaterials

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Ovalbumin was purchased from Sigma-Aldrich (USA). Pam₃CSK₄ and Poly (I:C) were purchased from Invitrogen (USA). Fetal bovine serum, the cell culture media of RPMI 1640 and DMEM, and penicillin-streptomycin (100 × ) were purchased from Gibco-Thermo Fisher (USA). PE/Cyanine7 anti-mouse CD11c antibody, PE anti mouse CD86 antibody and other flow cytometry antibodies are purchased from Biolegend (USA). Glucomannan (GM) from Konjac was purchased from Shimizu Chemical Corporation (Japan). The 8-arm PEG amine and PEG-8DBCO were purchased from Ponsure (Shanghai, China). diSulfo-Cy5 DBCO (Methyl) were purchased from Confluore Biological Technology (China). Hyaluronic acid with a molecular weight of 800 kDa to 1 MDa, 1-Tetradecylamine, n-hexylamine and other chemicals were purchased from Aladdin (Shanghai, China), unless otherwise indicated.

Xie D., Han C., Chen C., Liao Z., Campos de Souza S., Niu Y., Mano J.F., Dong L, & Wang C. (2024). A scaffold vaccine to promote tumor antigen cross-presentation via sustained toll-like receptor-2 (TLR2) activation. Bioactive Materials, 37, 315-330.

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Nanomedicine-Enabled Immunotherapy Evaluation

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Bovine serum albumin (BSA), manganese chloride tetrahydrate (MnCl₂∙4H₂O), and sodium hydroxide (NaOH) were purchased from Aladdin, Shanghai, China. PFOB, glutathione (GSH), coumarin-6 (Cou-6), and Hoechst 33342 were obtained from Sigma–Aldrich, St. Louis, USA. GSH assay Kit, cell counting Kit-8 (CCK-8), 4ʹ,6-diamidino-2-phenylindole (DAPI), DiD, and lysosomal staining lysotracker red were procured from KeyGen Biotech, Nanjing, China. Dulbecco's modified Eagle's medium (high glucose) (DMEM), trypsin, penicillin/streptomycin, and fetal bovine serum (FBS) were purchased from Gibco, CA, USA. Calreticulin rabbit monoclonal antibody, 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (DCFH-DA), and ATP Assay Kit were purchased from Beyotime, Nantong, China. HMGB1 ELISA Kit was bought from Arigo Biolaboratories, Taiwan, China. Anti-SPARC Antibody was purchased from St. John's Laboratory, UK. TUNEL Assay Kit, anti-Calreticulin antibody, and anti-HMGB1 antibody were purchased from Abcam, Cambridge, UK. FITC anti-mouse CD11c antibody, PE anti-mouse CD86 antibody, APC anti-mouse CD80 antibody, FITC anti-mouse CD3 antibody, APC anti-mouse CD4 antibody, and PE anti-mouse CD8 antibody were purchased from BioLegend, CA, USA. All other reagents were of analytical grade and used without further purification.

Kuai X., Zhu Y., Yuan Z., Wang S., Lin L., Ye X., Lu Y., Luo Y., Pang Z., Geng D, & Yin B. (2021). Perfluorooctyl bromide nanoemulsions holding MnO2 nanoparticles with dual-modality imaging and glutathione depletion enhanced HIFU-eliciting tumor immunogenic cell death. Acta Pharmaceutica Sinica. B, 12(2), 967-981.

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Osteoimmunology Evaluation after BMP-2 and NIR

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To evaluate the local osteoimmunology after treatment, mice with cranial defection were treated with BMP-2@BC + NIR irradiation or PBS. The heads were photographed on the third day, seventh day and the fourteenth day after treatment. The blood clots and adjacent local tissues in the defect of the mouse skull were removed for flow cytometric analysis. Flow cytometry antibodies included: FITC anti-mouse F4/80 Antibody (BioLegend, Cat# 123108), PE anti-mouse CD80 Antibody (BioLegend, Cat# 104708), APC anti-mouse CD206 Antibody (BioLegend, Cat# 141708), PE anti-mouse CD86 Antibody (BioLegend, Cat# 105008) and PE anti-mouse I-A/I-E (BioLegend, Cat# 107608). Local cytokines were measured through enzyme-linked immunosorbent kits (ELISA) including Mouse IL-6 ELISA MAX™ Deluxe (BioLegend, Cat# 431304), Mouse TNF-α ELISA MAX™ Deluxe (BioLegend, Cat# 430904), Mouse IL-10 ELISA MAX™ Deluxe (BioLegend, Cat# 431414), and Mouse IL-4 ELISA MAX™ Deluxe (BioLegend, Cat# 431104).

Fan Q., Bai J., Shan H., Fei Z., Chen H., Xu J., Ma Q., Zhou X, & Wang C. (2021). Implantable blood clot loaded with BMP-2 for regulation of osteoimmunology and enhancement of bone repair. Bioactive Materials, 6(11), 4014-4026.

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Murine Bone Marrow Dendritic Cell Differentiation

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Murine bone marrow cells derived from femurs and tibiae were collected and suspended with Tris-NH 4 Cl solution for 2 ~ 4 minutes at room temperature to lyse erythrocytes. After washed with PBS solution for twice, the bone marrow cells were cultured for 7 days at a density of 1×10 6 cells/ml in RPMI medium (Thermo, USA) supplemented with 10% FBS (Gibco, Carlsbad, California, USA), 10 units/ml penicillin, 10 μg/ml streptomycin, 2 mM L-glutamine (Gibco, USA) (hereafter termed complete medium) and 25 ng/ml murine GM-CSF (Miltenyi Biotech, Germany) at 37°C in a humidi ed atmosphere with 5% CO 2 . On day 3, oating cells were discarded and fresh medium containing 25 ng/ml GM-CSF was added. Cells were further differentiated for 4 days with GM-CSF containing complete RPMI 1640 medium. Floating Lymphocytes were collected and stained using FITC anti-mouse CD11c antibody (Biolegend), PE-Cy5 antimouse CD80 antibody (Biolegend), and PE anti-mouse CD86 antibody (Biolegend). Then the cells were washed twice with PBS and subjected to FCM analysis.

Yang X., Li Y., Wang W., Li C., Zhao D., Hu G., Rao H., Hao J., & Han X. (2020). Antigens from H22 cell mutation induced by lentinan are correlated with HCC immunoprophylaxis.

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