Pe conjugated anti mouse cd86

Manufactured by BioLegend

Sourced in United States

PE-conjugated anti-mouse CD86 is a monoclonal antibody that binds to the CD86 antigen expressed on the surface of activated B cells, macrophages, and dendritic cells in mice. The antibody is conjugated with the fluorescent dye Phycoerythrin (PE) for detection and analysis purposes.

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6 protocols using pe conjugated anti mouse cd86

Analysis of Murine Dendritic Cell Activation

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On the 33rd day, the harvested splenocytes were fixed in 100 μL of fixation buffer (BioLegend) for 30 min at 4 °C and washed twice with 1 mL of wash buffer (PBS containing 0.1% BSA). After centrifugation at 400× g for 5 min at 4 °C, the cells were stained with specific antibodies, including FITC-conjugated anti-mouse CD11c, PE-conjugated anti-mouse CD86, and anti-mouse CD80 antibodies (Biolegend), for 30 min at 4 °C. The PE rat IgG2a,κ isotype control antibody (Biolegend) was used as a negative control [21 (link)]. The samples were acquired with a forward side scatter threshold of 80,000 and a live gate on CD11c-positive events by a BD Accuri™ 5 flow cytometer (BD Biosciences). The MFI of cells was calculated using BD Accuri™ C6 system software (version 1.0.264.15).

Lin C.C., Chuang K.C., Chen S.W., Chao Y.H., Yen C.C., Yang S.H., Chen W., Chang K.H., Chang Y.K, & Chen C.M. (2022). Lactoferrin Ameliorates Ovalbumin-Induced Asthma in Mice through Reducing Dendritic-Cell-Derived Th2 Cell Responses. International Journal of Molecular Sciences, 23(22), 14185.

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Intracellular Staining and Flow Cytometry Analysis

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For intracellular staining studies, cells were incubated with Brefeldin A for 5 h at 37°C. Antibody reagents were obtained from Biolegend: AF647-conjugated anti-mouse CD45 (30-F11), PE-conjugated and AF647-conjugated anti-mouse CD11b (M1/70), Brilliant Violet 421-conjugated anti-mouse CD11c (N418), PE-Cy7-conjugated anti-mouse CD4 and PE-conjugated anti-mouse CD4 (GK1.5), AF647-conjugated anti-mouse CD19 (6D5), PE-Cy7-conjugated anti-mouse F4/80 (BM8), Brilliant Violet 421-conjugated anti-mouse CD3 (17A2), PE-Cy7-conjugated anti-mouse Gr-1 (RB6-8C5), PE-conjugated and AF647-conjugated anti-mouse CD8b (YTS156.7.7), AF488-conjugated anti-mouse/human CD44 (IM7), PE-Cy7-conjugated anti-mouse CD62L (MEL-14), AF647-conjugated anti-mouse CD69 (H1.2F3), PE-conjugated anti-mouse IFNγ (XMG1.2), PE-conjugated anti-mouse IL12/IL23p40 (C15.6), PE-conjugated anti-mouse CD86 (GL-1), PE-conjugated anti-mouse CD80 (16-10A1), AF647-conjugated anti-mouse FoxP3 (MF-14), PE-conjugated anti-mouse CD25 (PC61) and CD16/32 blocking antibody (93). IL12p35 (4D10p35) eflour660 was obtained from eBioscience. FACS analysis was performed using a Miltenyi 8-color MACSQuant and data were analyzed using FlowJo (TreeStar).

Sanders K.L., Fox B.A, & Bzik D.J. (2015). Attenuated Toxoplasma gondii stimulates immunity to pancreatic cancer by manipulation of myeloid cell populations. Cancer immunology research, 3(8), 891-901.

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Synthesis and Characterization of PEG-Peptide Conjugate

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20 kDa 4-arm PEG-azide was purchased from JenKem Technology USA (Beijing, China). Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and sodium ascorbate (NaAsc) were purchased from Sigma-Aldrich (St. Louis, MO). Copper (II) sulfate pentahydrate (CuSO₄ • 5 H₂O) was purchased from Acros Organics (Geel, Belgium). Alkyne functionalized peptide bearing an N-terminal 4-pentynoic acid (homopropargyl, hp) modification, hpPLP_139–151 (hp-HSLGKWLGHPDKF-OH), was obtained from Biomatik, USA, LLC (Wilmington, DE). All reagents were used as received without further purification. For in vitro cell assays and in vivo studies, female 4–6-week-old SJL/J (H-2) mice were purchased from Envigo Laboratories (Indianapolis, IN). For EAE induction, incomplete Freund’s adjuvant (IFA) and heat-killed mycobacterium tuberculosis H37RA were purchased from Difco (Sparks, MD). Additionally, pertussis toxin was purchased from List Biological Laboratories (Campbell, CA). For use in flow cytometry, TruStain fcX (anti-mouse CD16/32), R-phycoerythrin (PE)/Cy7-conjugated anti-mouse CD3, PE-conjugated anti-mouse CD86, FITC-conjugated anti-mouse CD80, AlexaFluor647-conjugated anti-mouse CD19, and BV421-conjugated anti-mouse CD11c were purchased from BioLegend (San Diego, CA).

Christopher M.A., Johnson S.N., Griffin J.D, & Berkland C. (2020). Autoantigen Tetramer Silences Autoreactive B Cell Populations. Molecular pharmaceutics, 17(11), 4201-4211.

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Macrophage Polarization Dynamics

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Metformin, LPS, AICAR, and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma (St. Louis, MO). Recombinant murine IL-13 was purchased from PeproTech (Rocky Hill, NJ). Mouse recombinant M-CSF and antibodies against AMPKα1 and p-AMPKα1 were from Cell Signaling Technology (Beverly, MA, USA). Antibody against Actin was purchased from Santa Cruz Biotechnology (CA, USA). Antibodies for flow cytometry including PE-conjugated anti-mouse CD206, PE-conjugated anti-mouse CD86 and FITC-conjugated anti-mouse F4/80 were purchased from Biolegend (San Diego, CA, USA). For immunofluorescence, first antibodies including anti-mouse F4/80, anti-CD31, anti-αSMA, and FITC-conjugated anti-mouse CD206 were from eBioscience, Abcam, Sigma and Biolegend respectively, while secondary antibodies including anti-Rat and anti-mouse were from life technology. FITC Annexin V Apoptosis Detection Kit I and matrigel were purchased from BD (San Jose, CA, USA). JetPrime transfection agent was obtained from Polyplus. Clodronate liposomes and PBS liposomes were purchased from ClodronateLiposomes.com (Amsterdam, Netherlands).

Ding L., Liang G., Yao Z., Zhang J., Liu R., Chen H., Zhou Y., Wu H., Yang B, & He Q. (2015). Metformin prevents cancer metastasis by inhibiting M2-like polarization of tumor associated macrophages. Oncotarget, 6(34), 36441-36455.

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Evaluating Macrophage Activation Markers

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RAW 264.7 cells and mouse peritoneal macrophages (5 × 10⁵cells/well) were incubated in 24-well plates with 7.5 µg/ml BC01 for
12, 24, or 48 h. The cells were harvested and washed with sterile PBS
and were incubated with FITC-conjugated anti-mouse CD40 (Clone:
HM40-3, Biolegend, USA), APC-conjugated anti-mouse CD80 (Clone:
16-10A1, Biolegend, USA), PE-conjugated anti-mouse CD86 (Clone: GL-1,
Biolegend, USA), and Percp/Cy5.5-conjugated anti-mouse I-A/I-E (Clone:
M5/114.15.2, BioLegend, USA) for 30 min in the dark. Finally, the
cells were fixed with 1% paraformaldehyde and were analyzed using a
FACScan flow cytometer. In the experiments designed to block TLR9, the
RAW 264.7 cells were pre-treated with a specific 2 µM ODN 2088
inhibitor for 12 h at 37°C.

Li J., Fu L., Wang G., Subbian S., Qin C, & Zhao A. (2019). Unmethylated CpG motif-containing genomic DNA fragment of Bacillus calmette-guerin promotes macrophage functions through TLR9-mediated activation of NF-κB and MAPKs signaling pathways. Innate Immunity, 26(3), 183-203.

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Comprehensive Molecular and Cellular Analysis Protocol

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Flow cytometry antibodies and reagents: FITC-conjugated anti-mouse CD11b, APC-conjugated anti-mouse CD45, PE-conjugated anti-mouse LAMP1 and PE-conjugated anti-mouse CD86 were purchased from Biolegend (USA), anti-CD16/CD32 Fc Block and myelin removal reads were purchased from Miltenyi Biotec (Germany). Western blot and immunofluorescence (IF) antibodies: IBA-1 (R&D systems, USA); V GLUT1 (Santa Cruz Biotechnology, USA); LAMP1 and CD68 were purchased from Abcam (United Kingdom); albumin, PSD95 and PKM2 were purchased from Proteintech (China). β-catenin, Cyclin-D1, c-Myc, ENO1, PFKFB3, HK1, LDHA, PDK1, GLUT1 were purchased from Cell Signaling Technology (USA), horseradish peroxidase (HRP)-linked goat anti-rabbit IgG (Fcmacs, China), NeuN (Santa Cruz Biotechnology), donkey anti-rat IgG H&L (Alexa Fluor® 647) (Abcam), goat anti-mouse IgG-TRITC (Abcam), rabbit anti-goat IgG-FITC (Fcmacs). ELISA kit: Maltose and Glucose assay kit (RayBiotech, USA), l-lactate assay kit (Cayman, USA), interleukin (IL)-6 (Fcmacs, China), IL-1β (Fcmacs, China). TRIzol reagent and SYBR green dye were bought from Invitrogen (Carlsbad, USA). PKM2 overexpression plasmid was purchased from Nanjing Jereh Company (China), and RFectPM Eukaryotic DNA Transfection Kit was purchased from Changzhou EMI Company (China). β-catenin protein inhibitor (KYA1797K) was purchased from MCE Company (USA).

Lu L., Wang H., Liu X., Tan L., Qiao X., Ni J., Sun Y., Liang J., Hou Y, & Dou H. (2021). Pyruvate kinase isoform M2 impairs cognition in systemic lupus erythematosus by promoting microglial synaptic pruning via the β-catenin signaling pathway. Journal of Neuroinflammation, 18, 229.

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