Anti cd40

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-CD40 is a laboratory reagent that specifically binds to the CD40 protein, which is expressed on the surface of various cell types, including B cells, dendritic cells, and some tumor cells. This reagent can be used in research applications to study the role of CD40 signaling in immune function and disease processes.

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Close spelling variants of this product

Anti-CD40 antibody (18 citations) Anti-CD40 (1C10) (5 citations) FITC-conjugated anti-CD40 (3 citations) Anti-CD40-APC (3 citations) Functional-grade anti-CD40 (2 citations) PerCP-eFluor-labeled anti-mouse CD40 (2 citations) Anti-human CD40 PE (2 citations) Anti-CD40 (IC10, (2 citations) PE-conjugated anti-CD40 (2 citations) PE-conjugated anti-mouse CD40/MHC-II/CCR7/Foxp3/PD-L1 (2 citations)

70 protocols using anti cd40

Purification and Culture of Naive B Cells

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Naive mature B cells were purified by negative selection from RBC-lysed splenocyte cell suspensions using MACS anti-mouse CD43 microbeads (no. 130–049-801; Miltenyi Biotec) on a MidiMACS magnetic separation apparatus (Miltenyi Biotec). Purified cells were cultured in B cell media (sterile-filtered RPMI 1640 [no. 22400–089; Life Technologies] plus 15% FBS [no. 35–010-CV; Corning] plus 1% additional L-glutamine [200 mM stock] plus 1% penicillin/streptomycin mixture [no. 400–109; Gemini] plus 0.0005% 2-ME [no. BP176–100; Fisher Scientific]) and stimulated with various conditions: LPS (20 μg/ml, no. L4130; Sigma-Aldrich), LI (LPS [20 mg/ml] plus IL-4 [12.5 ng/ml, no. 404-ML-010; R&D Systems]), LII (LPS [20 μg/ml] plus IL-2 [100 U/ml, recombinant human; National Institutes of Health] plus IL-5 [5 ng/ml, no. 215–15; PeproTech]), LTD (LPS [10 μg/ml] plus TGF-β [2 ng/ml, no. 240-B-010; R&D Systems] plus anti– IgD-dextran [0.33 mg/ml, FinaBio no. 0001; Fina Biosolutions]), aCI (anti-CD40 [0.5 μg/ml, clone HM40–3, no. 16–0402-86; eBioscience] plus IL-4 [12.5 ng/ml]), and aCII (anti-CD40 [0.5 mg/ml] plus IL-4 [12.5 ng/ml] plus IL-5 [2 ng/ml]). Cell cultures were initiated at 0.5 × 10⁶ cells/ml and split 1:2 at 48 and 72 h. Flow cytometric analysis of CFSE staining was performed according to the manufacturer’s instructions (CellTrace CFSE Cell Proliferation Kit Protocol; Invitrogen).

Pucella J.N., Cols M., Yen W.F., Xu S, & Chaudhuri J. (2019). The B Cell Activation-Induced miR-183 Cluster Plays a Minimal Role in Canonical Primary Humoral Responses. Journal of immunology (Baltimore, Md. : 1950), 202(5), 1383-1396.

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Th17 Cell Differentiation Assay

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5×10 5 CD4 + CD25 -CD62L hi naïve T cells and 1×10 5 NKB cells from CIA mice were co-cultured in the presence of plate-bound anti-CD3 (5 μg/mL), anti-CD28 (1 μg/mL), TGF-β (2.5 ng/mL, Peprotech, Rocky Hill, NJ), IL-6 (20 ng/mL, Peprotech), anti-CD40 (3 μg/mL), CpG (10 μg/mL), anti-IL-4 and anti-IFN-γ (5 μg/mL, Biolegend) for 3 days. Then, the cells were harvested for Th17 cell detection, while the cell culture supernatants were collected for IL-17A analysis.

Wang P., Song J., Bai M., Zheng X., Xie Y., Tang Y., Li X., Fang X., Yuan J., Ren L., Ye H., Su Y., Wang S., Li Z., & Hu F. (2021). Natural killer-like B cells promote Th17 cell differentiation and exacerbate rheumatoid arthritis.

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Phenotypic Analysis of Stimulated PBMCs

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Human peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation from whole blood drawn from healthy volunteers. PBMCs were labelled with 5μM CellTrace Violet (CTV) and plated in 96-well U-bottom plates in the presence of F(ab’)2 fragment donkey anti-mouse IgM (anti-IgM) (5μg/mL, Jackson ImmunoResearch) with Anti-CD40 (1μg/mL, Peprotech) for 7d in X-vivo media. For some experiments PBMCs were stimulated in the presence of recombinant IFNβ, IFNγ or IFNλ (all 20μg/mL, Peprotech) as indicated. Cells were then analyzed by flow cytometry. Briefly, Human PBMCs were analyzed using following antibodies: Live/dead cell discrimination was performed using Live/Dead Fixable viability dye 455UV (Thermo Fisher). For surface staining following antibodies against CD19 (SJ25C1), CD27 (M-T271), CD38 (HB7), CD86 (IT2.2), IgD (IA6–2) and Tim-1 (1D12) were used. All data were collected on a BD Symphony A5 (BD Biosciences) and analyzed with FlowJo software (Tree Star).

McClain M.S., Cao P., Iwamoto H., Vinion-Dubiel A.D., Szabo G., Shao Z, & Cover T.L. (2001). A 12-Amino-Acid Segment, Present in Type s2 but Not Type s1 Helicobacter pylori VacA Proteins, Abolishes Cytotoxin Activity and Alters Membrane Channel Formation. Journal of Bacteriology, 183(22), 6499-6508.

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Cell Stimulation Protocols for Immunology

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We stimulated BJAB cells with 4 μg/ml anti-CD40 (Invitrogen-140409-82) and 10 μg/ml anti-IgM (Sigma-I0759) for 4 hours. We stimulated Jurkat cells with 5 μg/ml anti-CD3 (Biolegend-317315) and 100 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-P1585) for 4 hours. We stimulated THP1 cells with 1 μg/ml bacterial lipopolysaccharide (LPS) from E. coli K12 (LPS-EK Invivogen tlrl-peklps) for 4 hours. We stimulated U937 cells with 200 ng/ml LPS for 4 hours.

Nasser J., Bergman D.T., Fulco C.P., Guckelberger P., Doughty B.R., Patwardhan T.A., Jones T.R., Nguyen T.H., Ulirsch J.C., Lekschas F., Mualim K., Natri H.M., Weeks E.M., Munson G., Kane M., Kang H.Y., Cui A., Ray J.P., Eisenhaure T.M., Collins R.L., Dey K., Pfister H., Price A.L., Epstein C.B., Kundaje A., Xavier R.J., Daly M.J., Huang H., Finucane H.K., Hacohen N., Lander E.S, & Engreitz J.M. (2021). Genome-wide enhancer maps link risk variants to disease genes. Nature, 593(7858), 238-243.

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B cell activation under different stimuli

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B cells were treated with different concentrations of TI mitogens LPS (Sigma-Aldrich, Steinheim, Germany, catalog number: L2630), Polyinosinic-polycytidylic acid sodium salt (Poly I:C, Sigma, catalog number: P1530), and CpGs (mouse TLR9 agonist kit, InvivoGen, catalog number: tlrl-kit9m) or TD mitogen anti-CD40 (Invitrogen, Carlsbad, California, USA; Clone: 1C10) in combination with or without recombinant interleukin-4 (IL-4, Miltenyi Biotec, catalog number: 130-094-061) for 24 h. After testing the different CpG-ODNs in S5 Fig, CpG-ODN 1826 (1.25 μg/ml, InvivoGen, catalog code: tlrl-1826-1) was used for all further experiments in this study. For BCR activation, cells were treated with 5 μg/mL anti-IgM F(ab’)2(μ chain) fragment (Jackson ImmunoResearch, West Grove, Pennsylvania, catalog number: 115-006-075).

Honke N., Lowin T., Opgenoorth B., Shaabani N., Lautwein A., Teijaro J.R., Schneider M, & Pongratz G. (2022). Endogenously produced catecholamines improve the regulatory function of TLR9-activated B cells. PLoS Biology, 20(1), e3001513.

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Murine Naïve B Cell Activation

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WT splenic naïve B cells were isolated using CD43 Micro-Beads (Miltenyi Biotec) and stimulated with anti-CD40 (1 µg/ml, Invitrogen) or anti-IgM (1 µg/ml, Jackson ImmunoResearch) antibody alone or in combination with IL-4 (20 ng/ml, Peprotech) for up to 1 day. Total RNA was isolated from cultured B cells at indicated timepoints using the Quick-RNA Microprep Kit (Zymo Research). cDNA was synthesized with the RevertAid RT Kit (Thermo Scientific) and qRT-PCR was performed on a CFX96-C1000 Thermo Cycler (Bio-Rad) using the SensiFAST SYBR No-ROX Kit (Bioline, Meridian Bioscience). Hprt1 was used as a housekeeping gene and the standard curve method was used for quantification. The following primers were used: Bhlhe40-F CTC CTA CCC GAA CAT CTC AAA C, Bhlhe40-R CCA GAA CCA CTG CTT TTT CC, Hprt1 -F AGT GTT GGA TAC AGG CCA GAC, Hprt1-R CGT GAT TCA AAT CCC TGA AGT.

Rauschmeier R., Reinhardt A., Gustafsson C., Glaros V., Artemov A.V., Taneja R., Adameyko I., Månsson R., Busslinger M., & Kreslavsky T. (2021). Cell-intrinsic functions of the transcription factor Bhlhe40 in activated B cells and T follicular helper cells restrain the germinal center reaction and prevent lymphomagenesis.

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Stimulation and Activation of B Cells

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CH12 and primary splenic B cells (from 129Sv1 mouse strain) were cultured in RPMI media supplemented with 10% heat inactivated serum, 10 mM HEPES, 1 mM sodium pyruvate, 100 U/mL Penicillin, 100 U/mL Streptomycin, 50 μM β-mercaptoethanol, 1x non-essential amino acids.
CH12 cells were stimulated for 2 or 4 days at a density of 10⁵ cells/ml, in the presence of 50 µg/ml of LPS (Sigma) (LPS stimulation); 50 µg/ml LPS and 20 ng/ml IFN-γ (R&D) (IFN-γ stimulation); 50 µg/ml LPS and 25 ng/ml IL4 (eBiosciences) (IL4 stimulation); 50 µg/ml LPS, 10 ng/ml IL4, and 2 ng/ml TGFβ (R&D) (LIT stimulation); 1 µg/ml anti-CD40 (eBiosciences), 10 ng/ml IL4, and 2 ng/ml TGFβ (CIT stimulation). Purification and stimulation of primary splenic B cells were as described (ref. ^{40 (link)}).

Santos J.M., Oudinet C., Schöne L., Dauba A, & Khamlichi A.A. (2019). Essential role of the initial activation signal in isotype selection upon deletion of a transcriptionally committed promoter. Scientific Reports, 9, 18543.

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Isolation and Stimulation of PBMCs

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PBMCs were isolated from heparinized blood by Ficoll-Hypaque density-gradient centrifugation (Pharmacia Biotech). In some experiments, cells were stimulated with 2.5 μg/ml anti-CD40 (eBioscience) and 20 ng/ml phorbol 12-myristate 13-acetate (PMA; Calbiochem) at 37°C in an atmosphere of 5% CO₂ for 48 hours. For stimulation with cytokines, 100 ng/ml recombinant human IL-6 (rhIL-6), 10 ng/ml rhTNF, 10 ng/ml rhIL-1β, or 100 ng/ml rhIL-17A (all from R&D Systems), each in combination with 2.5 μg/ml anti-CD40, was used. To assay for soluble RANKL in culture supernatants, B cells were isolated from PBMCs using CD19 microbeads, and separation was performed with a magnetic-activated cell sorter (Miltenyi Biotec). The cells were then cultured for 48 hours in the presence of anti-CD40 and PMA. Thereafter, the medium was replaced and the cells were returned to culture at 37°C in an atmosphere of 5% CO₂ for an additional 5 days. Soluble RANKL was detected by enzyme-linked immunosorbent assay (ELISA; PeproTech), in accordance with the manufacturer’s protocol.

Meednu N., Zhang H., Owen T., Sun W., Wang V., Cistrone C., Rangel-Moreno J., Xing L, & Anolik J.H. (2016). Production of RANKL by Memory B Cells: A Link Between B Cells and Bone Erosion in Rheumatoid Arthritis. Arthritis & rheumatology (Hoboken, N.J.), 68(4), 805-816.

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Autophagy Regulation in Bone Homeostasis

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C57BL/6 mice were from The Jackson Laboratory and were maintained under specific pathogen-free conditions under protocols approved by Institutional Animal Care and Use Committees. Femurs from Atg^flox/flox and Atg^flox × CD11c-cre mice were a gift from David Lieb (Geisel School of Medicine, Dartmouth College, Hanover, NH). LPS (Escherichia coli sero-type 0111:B4) was from Sigma-Aldrich and used at 100 ng/ml. Etomoxir and 6-diazo-5-oxo-l-norleucine were purchased from Sigma-Aldrich. NOS inhibitor S-ethyl-isothiourea (SEITU, 500 μM) was purchased from Cayman Chemical. RAP (100 nM) was purchased from InvivoGen. KU 0063794 (KU, 100 nM) was purchased from Tocris Bioscience. 7-Aminoactinomycin D (7-AAD) and all Abs for FACS analysis were from BD Biosciences except for anti-CD40, which was purchased from eBioscience.

Amiel E., Everts B., Fritz D., Beauchamp S., Ge B., Pearce E.L, & Pearce E.J. (2014). Mechanistic Target of Rapamycin Inhibition Extends Cellular Lifespan in Dendritic Cells by Preserving Mitochondrial Function. Journal of immunology (Baltimore, Md. : 1950), 193(6), 2821-2830.

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CSR Efficiency Assay in CH12F3 Cells

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CH12F3 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and 50 μM of beta-mercaptoethanol. For CSR assay, healthy CH12F3 cells were seeded at 5 × 10⁴ cells/ml in the presence of 1 μg/ml anti-CD40 (eBioscience 16-0402-86), 5 ng/ml of IL-4 (R&D Systems 404-ML), and 0.5 ng/ml TGF-β1 (R&D Systems 240-B) and grown for 72 hr. Cells were stained with a FITC-conjugated anti-mouse IgA (BD Biosciences 559354) and analyzed on a LSR II flow cytometer (BD Biosciences). CSR efficiency is determined as the percentage of IgA-positive cells.

Han L., Masani S., Hsieh C.L, & Yu K. (2014). DNA Ligase I Is Not Essential for Mammalian Cell Viability. Cell reports, 7(2), 316-320.

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