Anti cd40 antibody

Manufactured by R&D Systems

The Anti-CD40 antibody is a lab equipment product. It is a monoclonal antibody that specifically binds to the CD40 receptor on the surface of cells. The CD40 receptor is involved in various immune responses and cellular processes. The primary function of the Anti-CD40 antibody is to facilitate the study and analysis of CD40 and its role in biological systems.

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Spelling variants of this product

Anti-CD40 (21 citations) CD40 antibody (2 citations) Anti-human CD40/TNFRSF5 antibody (2 citations)

7 protocols using anti cd40 antibody

Activation of NK and B cells for cytokine analysis

Cited 3 times

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NK cells were cultured in TheraPeak X-VIVO-10 medium (Lonza) supplemented with 10% human serum AB (Sigma Aldrich) and 1ng/mL IL-15 (Biolegend). B cells were cultured in RPMI containing 5% fetal bovine serum and 1% penicillin-streptomycin. Following isolation, each cell population was divided into two groups: a control population cultured in normal medium, and an activated population cultured in control medium supplemented with additional components. NK cell activating medium was supplemented with 10 ng/mL IL-12 (Invivogen), 50 ng/mL IL-15, and 50 ng/mL IL-18 (Biolegend) (51 (link), 52 (link)). B cell activating medium was supplemented with 5 ug/mL anti-CD40 antibody (R&D systems) and 20 ng/mL IL-4 (R&D Systems) (6 , 53 (link)).
The cells were cultured separately in activating or control medium for a number of hours depending on the lymphocyte subtype; B cells were activated for 72 hours, and NK cells for 24 hours (34 (link), 45 (link), 53 (link)). Cells were seeded at a density of 1 million cells/mL medium. At the end of the activation time, a sample of growth medium from each group was taken for cytokine analysis. A summary of the isolation and activation conditions used is provided in Table 1.

Schmitz R.L., Tweed K.E., Rehani P., Samimi K., Riendeau J., Jones I., Maly E.M., Guzman E.C., Forsberg M.H., Shahi A., Capitini C.M., Walsh A.J, & Skala M.C. (2023). Autofluorescence lifetime imaging classifies human lymphocyte activation and subtype. bioRxiv.

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Proliferation of Human B Cells

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Fifty thousand purified human B cells were seeded in a 96-well plate and treated with the indicated reagents for 3 days. Then, 1 μCi [³H]-thymidine was added to each well and incubated for an additional 24 h. The cells were harvested, and ³H-thymidine incorporation was assessed with a liquid scintillation counter (Perkin Elmer). Anti-CD40 antibody (5 μg/ml, R&D Systems, Minneapolis, MN), anti-IgM antibody (1 μg/ml, Jackson ImmunoResearch Inc., West Grove, PA), SAC (formalin-fixed Staphylococcus aureus, Cowan I strain, 1:10000 dilution, MERCK), CTLA-4-Ig, and L6-Ig control protein were also used in the study.

Liu P.C., Ssu C.T., Tsao Y.P., Liou T.L., Tsai C.Y., Chou C.T., Chen M.H, & Leu C.M. (2020). Cytotoxic T lymphocyte-associated antigen-4-Ig (CTLA-4-Ig) suppresses Staphylococcus aureus-induced CD80, CD86, and pro-inflammatory cytokine expression in human B cells. Arthritis Research & Therapy, 22, 64.

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B Cell Activation and Differentiation

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Spleens were harvested at day 8 post-immunization. Splenocytes were collected, red blood cells were lysed and cells were CD43-depleted using CD43 microbeads (Miltenyi Biotec). Single-cell suspensions of CD43^- B splenocytes from wt, and LSRµKI mice were cultured 3 days at 1x10⁶ cells/ml in RPMI 1640 with 10% fetal calf serum, 5μg/ml LPS with or without, 20ng/ml IL-4, 2ng/ml TGFβ and 2 ng/ml INFγ (PeproTech, Rocky Hill, NJ), anti-CD40 antibody (2.5μg/ml) (R&D systems) and anti-κ light chain antibody (2.5µg/ml, Southern Biotechnology) for BCR cross-linking. Culture samples were harvested at day 4 for RNA extraction. Culture supernatants were then recovered and stored at -20°C until used.

Denis-Lagache N., Oblet C., Marchiol T., Baylet A., Têteau O., Dalloul I., Dalloul Z., Zawil L., Dézé O., Cook-Moreau J., Saintamand A., Boutouil H., Khamlichi A.A., Carrion C., Péron S., Le Noir S., Laffleur B, & Cogné M. (2023). Attempts to evaluate locus suicide recombination and its potential role in B cell negative selection in the mouse. Frontiers in Immunology, 14, 1155906.

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B cell activation and gene expression

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B cells were placed on PLB containing anti-human Igκ or anti- rat Igκ antibodies and incubated at 37°C with 5% CO₂ and 75% humidity for 1 h. RPMI-10 culture media containing anti-CD40 antibody (2ug/mL, R&D systems), IL-4 (40ng/mL, R&D systems), and IL-21 (2ug/mL Biolegend), or RPMI-10 culture media alone was added to the cells, cells were incubated at 37°C for an additional 2 h and harvested. Cells were lysed, RNA was reverse transcribed, and cDNA was used to run TaqMan Gene Expression Assays using TaqMan Gene Expression Cells-to-C_T kit (Thermo Fisher) on CFX connect Real-Time PCR Detection System (Bio Rad, check model name again). TaqMan Gene Expression Assays detecting IRF-4 (Hs00180031_m1, Thermo Fisher) and ACTB (Hs1060665_g1, Thermo Fisher) were used to measure levels of mRNA and ACTB was used for normalization. Data were analyzed by Prism software (GraphPad).

Kwak K., Quizon N., Sohn H., Saniee A., Manzella-Lapeira J., Holla P., Brzostowski J., Lu J., Xie H., Xu C., Spillane K.M., Tolar P, & Pierce S.K. (2018). Intrinsic properties of human germinal-center B cells set antigen-affinity thresholds. Science immunology, 3(29), eaau6598.

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Multiparametric Evaluation of Angiogenesis

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Matrigel sections were washed in 1x PBS, blocked for 1 h at RT in blocking buffer (1% BSA, 10% goat serum, 1xPBS) and incubated with anti-CD31 (BD Pharmigen, 553370), anti-smooth muscle actin conjugated to Cy3 (C6198, Sigma), and anti-CD40 antibody (R&D Systems, AF440) overnight at 4 °C. The next day the sections were incubated with Alexa Fluor secondary goat antibodies for 1 h at RT and counterstained with DAPI (Thermo Scientific) to visualize the nuclei. Imaging was performed using a Zeiss confocal microscope with a 10x objective. Vascularization was evaluated in 5 random fields per plug, 2 plugs per mouse, using ImageJ. H&E staining was performed using standard methods and visualized with a light microscope (Nikon Eclipse 80i).

Lee A., Papangeli I., Park Y., Jeong H.N., Choi J., Kang H., Jo H.N., Kim J, & Chun H.J. (2017). A PPARγ-dependent miR-424/503-CD40 axis regulates inflammation mediated angiogenesis. Scientific Reports, 7, 2528.

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B Cell Activation and Cytokine Quantification

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1 × 10⁶ splenocytes or 0,5 × 10⁶ B cells were stimulated with 5 μg/mL of lipopolysaccharide (LPS) (InVivoGen) with or without 20 ng/mL of IL-4 (Miltenyi) or with 5 μg/mL anti-CD40 antibody (R&Dsystems) and 20 ng/mL of IL-4 for 3 days in RPMI supplemented with 10% fetal calf serum, 0.05 mM 2-mercaptoethanol, 100 U/mL penicillin-streptomycin, 1 mM sodium pyruvate, and non-essential amino acids as recommended (Gibco). Supernatants were used for ELISA quantification assay and cells were analyzed by flow cytometry at day 3.

Bonaud A., Clare S., Bisio V., Sowerby J.M., Yao S., Ostergaard H., Balabanian K., Smith K.G, & Espéli M. (2020). Leupaxin Expression Is Dispensable for B Cell Immune Responses. Frontiers in Immunology, 11, 466.

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Coculture of RA T cells and Healthy B cells

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CD4⁺ T cells were purified from PBMCs of RA patients using Human CD4⁺ T Cell Isolation Kit (Miltenyi) and then cultured with plate-coated anti-CD3 antibody (5 μg/ml) and anti-CD28 antibody (2 μg/ml) plus IGU or DMSO in RPMI-1640 containing 10% FBS for 3 days. CD19⁺ B cells were purified from PBMCs of healthy donors using Human CD19⁺ B cell Isolation Kit (Miltenyi). In a 96-well round-bottom plate, CD19⁺ B cells were co-cultured with RA-CD4⁺ T cells which were treated with or without IGU at a ratio of 5:1. A total of 2 μg/ml anti-CD3/CD28, 0.5 μg/ml anti-CD40 antibody (R&D Systems), 0.1 μM CpG (Miltenyi) were added to RPMI 1640 complete medium, which was then added to the cells at 200 μl per well. After 7 days, the supernatant was separated from the cells via centrifugation. Both cells and cell culture supernatant were collected for further experiments.

Bai Z., Lu Z., Liu R., Tang Y., Ye X., Jin M., Wang G, & Li X. (2022). Iguratimod Restrains Circulating Follicular Helper T Cell Function by Inhibiting Glucose Metabolism via Hif1α-HK2 Axis in Rheumatoid Arthritis. Frontiers in Immunology, 13, 757616.

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