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Anti cd40

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Anti-CD40 is a laboratory reagent used in immunological research. It is an antibody that binds to the CD40 receptor, which is expressed on the surface of various immune cells, including B cells, dendritic cells, and macrophages. The primary function of Anti-CD40 is to serve as a tool for studying the role of CD40 signaling in immune responses and related biological processes.

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Lab products found in correlation

Anti cd86, by BD (12 mentions) Anti cd80, by BD (10 mentions) Anti cd11c, by BD (8 mentions) Lipopolysaccharides (lps), by Merck Group (7 mentions) Interleukin 4 (il 4), by Thermo Fisher Scientific (5 mentions) Facscalibur flow cytometer, by BD (5 mentions) Anti hla dr, by BD (4 mentions) Interleukin 4 (il 4), by R&D Systems (3 mentions) Anti cd83, by BD (3 mentions) Easysep mouse b cell isolation kit, by STEMCELL (2 mentions) Mouse recombinant il 4, by Bio-Techne (2 mentions) Mouse recombinant il 21, by Bio-Techne (2 mentions) Tgf β, by R&D Systems (2 mentions) Igm pe, by Southern Biotech (2 mentions) Celltrace violet, by Thermo Fisher Scientific (2 mentions) Anti igm f ab 2, by Southern Biotech (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Anti cd3, by BD (2 mentions) Facscan flow cytometer, by BD (2 mentions) Facsverse instrument, by BD (2 mentions)

Close spelling variants of this product

Anti-CD40-PE (18 citations) Anti-CD40-FITC (13 citations) Anti-mouse CD40 (9 citations) Anti-CD40-APC (9 citations) Anti-CD40 (hm40-3 clone; (6 citations) APC mouse anti-human CD40 (6 citations) Anti-CD40 antibody (6 citations) Anti-CD40 (HM40–3; (5 citations) Anti-CD40 (clone 3/23) (4 citations) PE-conjugated anti-CD40 (4 citations)

48 protocols using anti cd40

1

Induction of IgG1 and IgG3 Class Switching in Mouse Splenic B Cells

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Mouse splenic B cells were isolated using EasySep™ Mouse B Cell Isolation Kit (Stem Cell) and stimulated with 20 μg/ml LPS (Sigma-Aldrich) for CSR to IgG3. For CSR to IgG1, B cells were stimulated with 20 μg/ml LPS (Sigma-Aldrich) and 10 ng/ml of mouse recombinant IL-4 (Bio-Techne, # 404-ML), or 1 μg/ml anti-CD40 (BD Biosciences) and 10 ng/ml of mouse recombinant IL-4 (Bio-Techne, # 404-ML), or 1 μg/ml anti-CD40 (BD Biosciences), 10 ng/ml of mouse recombinant IL-4 (Bio-Techne, # 404-ML) and 10 ng/ml of mouse recombinant IL-21 (Bio-Techne, # 594-ML).
For the proliferation assay, cells were stained with CellTrace™ Far Red Cell Proliferation Kit (Thermofisher) for 5 min at 37 °C and cultured with indicated stimulation. Cells were analyzed at day 0 and day 3 using flow cytometry.
Huang H., Li Y., Zhang G., Ruan G.X., Zhu Z., Chen W., Zou J., Zhang R., Wang J., Ouyang Y., Xu S, & Ou X. (2023). The RNA-binding protein hnRNP F is required for the germinal center B cell response. Nature Communications, 14, 1731.
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2

Splenic B cells activation assay

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Splenic B cells were cultured (106 cells/ml) in RPMI (Euroclone) + 10% FCS (Euroclone) and 2-mercaptoethanol (50 mM; Life Technologies) for 72h with 20 μg/ml LPS (Sigma) ± 10 ng/ml IL-4 (Sigma) or for 48h with 10 μg/ml anti-CD40 (BD Pharmingen) ± 10 ng/ml IL-4 (Sigma). To perform fast stimulation 5x106 B cells were resuspended in 400ul PBS without FCS and treated for 5 and 10 minutes with 10μg/ml anti-CD40 (BD Pharmingen) ± 10 ng/ml IL-4 or for 1, 5, 10 minutes with 10 μg/ml anti-Mouse IgM (Jackson Immuno Research); the incubation was performed in water bath at 37°C followed by the addiction of ice-cold PBS to stop the stimulation; cells were immediately pelleted and lysed. DLBCL cell lines OCI-Ly1, OCI-Ly18 and Pfeiffer were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Germany). OCI-Ly1 were maintained in IMDM (Invitrogen) + 20% FBS + 2-mercaptoethanol (50 mM); OCI-Ly18 in RPMI + 10% FBS + 2-mercaptoethanol (50 mM); Pfeiffer in RPMI + 10% FBS + Sodium Pyruvate 1 mM. The cells were maintained in an incubator at 37°C in a modified atmosphere with 5% CO2. Testing for Mycoplasma infection was carried out on a monthly basis. All procedures of handling were carried out under a sterile hood.
Quotti Tubi L., Mandato E., Canovas Nunes S., Arjomand A., Zaffino F., Manni S., Casellato A., Macaccaro P., Vitulo N., Zumerle S., Filhol O., Boldyreff B., Siebel C.W., Viola A., Valle G., Mainoldi F., Casola S., Cancila V., Gulino A., Tripodo C., Pizzi M., Dei Tos A.P., Trentin L., Semenzato G, & Piazza F. (2023). CK2β-regulated signaling controls B cell differentiation and function. Frontiers in Immunology, 13, 959138.
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3

Cell Cycle and Chromatin Regulation Protocols

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For the cell cycle experiments, cells were stalled in S phase and G2/M phase, using 0.1 mM Hydroxyurea (Sigma-Aldrich, H8627-1G) for 20 h and 10 ng/ml Nocodazole (Sigma-Aldrich, M1404-2MG) for 8 h, respectively.
For the chromatin conformation experiments, cells were incubated with 10 nM Trichostatin A (Sigma-Aldrich, T8552-1MG) for 24 h to inhibit HDAC activity and thus induce hyperacetylation and decondensation of the chromatin.
For immune activation, CH12F3 cells were incubated with a cytokine cocktail (CIT) consisting of 2.5 μg/ml anti-CD40 (BD Biosciences, 553788), 10 ng/ml IL-4 (R&D Systems, 404-ML-050), and 50 ng/ml TGFβ (R&D Systems, 240-B-010). Primary B cells were incubated with 5 μg/ml Lipopolysaccharide solution (InvitrogenTM, 15526286) and 10 ng/ml IL-4. Class switch recombination was assessed by IgM to IgA switching for CH12F3 cells, and IgM to IgG1 switching for primary B cells.
Morrish R.B., Hermes M., Metz J., Stone N., Pagliara S., Chahwan R, & Palombo F. (2019). Single Cell Imaging of Nuclear Architecture Changes. Frontiers in Cell and Developmental Biology, 7, 141.
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4

Multiparametric Flow Cytometry Analysis

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FITC-labelled anti-IA/IE, PE-labelled anti-Vβ11, anti-TNFα, anti-CD45.2, anti-CD40, APC-conjugated anti-CD80, anti-IFNγ, anti-CD69, PeCy7-labelled anti-CD11c, PercypCy5.5-labeled anti-CD44, anti-IL-2 and APC-Cy7-conjugated anti-CD62L were obtained from BD Pharmingen (San Diego, CA, USA). PE- labelled anti-CD86, PE-efluor 610-labelled anti-CD25 and Pacific blue conjugated-Annexin V were from Biolegend (San Diego, CA, USA). Alexa 405-conjugated anti-CD4 was obtained from CALTAG lab (Buckingham, UK). APC-conjugated anti-IA/IE and PE-labelled anti-CCR7 were obtained from eBioscience (San Diego, CA, USA). All stainings were performed in presence of mouse Fc Block (BD Biosciences). Dead cells were stained using the Live/Death detection kit with a near-infrared dye (Invitrogen). The samples were analysed using CyAn ADP Analyser (Beckman Coulter, Brea, CA, USA) and the data were analysed using FlowJo version 9.2 (Tree Star inc, Ashland, Oregon, USA).
Nasi A., Bollampalli V.P., Sun M., Chen Y., Amu S., Nylén S., Eidsmo L., Rothfuchs A.G, & Réthi B. (2017). Immunogenicity is preferentially induced in sparse dendritic cell cultures. Scientific Reports, 7, 43989.
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5

CpG-Induced Cytokine Profiling in DLN

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To quantify cytokine production in response to CpG injection, DLN were dissected 24 hours post CpG injection and cultured for an additional 24 h ex vivo in complete RPMI (Gibco). Cytokine levels of IL-12p70, were determined by Luminex (BioRad) according to the manufacturer’s protocol.
To address cytokine production by sorted Ly6Chi Monocytes, MHCIIhi DCs and MHCIIint DCs, these cells were sorted from the pooled DLN of 40 mice injected 24 hours earlier with 20 µl of CpG (100 µg/ml). 2 × 105 cells were cultured in a volume of 200 µl either without further stimulation or in the presence of 10 ng/ml LPS (Invivogen) and 1 µg/ml anti-CD40 (BD Biosciences). Cytokine levels, were determined by Luminex (BioRad) according to the manufacturer’s protocol.
De Koker S., Van Hoecke L., De Beuckelaer A., Roose K., Deswarte K., Willart M.A., Bogaert P., Naessens T., De Geest B.G., Saelens X., Lambrecht B.N, & Grooten J. (2017). Inflammatory monocytes regulate Th1 oriented immunity to CpG adjuvanted protein vaccines through production of IL-12. Scientific Reports, 7, 5986.
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6

Lymphocyte Development and CSR Analysis

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Lymphocyte development and in vitro CSR were performed as described before (10 (link),31 (link),32 ). Hematopoietic cells from 5–8 week mice were stained and analyzed on a FACS Calibur flow cytometer (BD Biosciences). The antibodies are: PE-CD4 (clone GK1.5, BD Pharmingen, 553730), FITC-CD8α (clone 53–6.7, BioLegend, 100705), APC-TCRβ (clone H57-597, BD Pharmingen, 553174), PE/Cy7 TER-119 (clone TER-119, BioLegend, 116222), FITC-CD43 (clone S7, BD Pharmingen, 553270), PE-Cy5-B220 (clone RA3-6B2, BD Pharmingen, 553091) and PE-IgM (Southern Biotech, 1020-09). For in vitro CSR, CD43− splenic cells (anti-CD43 magnetic beads, Miltenyi, 130-049-801) were cultured (~1 × 106 cells ml−1) in RPMI (Gibco, 11875-093), serum supplements (10 (link),31 (link),32 ), IL-4 (20 ng/mL; R&D, 404-ML-050), and anti-CD40 (1 μg/mL; BD Bioscience, 553721) and analyses with FITC-conjugated IgG1 (clone A85-1, BD Pharmingen, 553443) and PECy5-conjugated B220 (clone RA3-6B2, BD Pharmingen, 553091). FlowJo software package was used for data analyses
Milanovic M., Houghton L.M., Menolfi D., Lee J.H., Yamamoto K., Li Y., Lee B.J., Xu J., Estes V.M., Wang D., Mckinnon P.J., Paull T.T, & Zha S. (2020). The cancer-associated ATM R3008H mutation reveals the link between ATM activation and its exchange. Cancer research, 81(2), 426-437.
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7

Isolation and Culture of Naïve B Cells

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After PBMC isolation, B cells were enriched by positive selection using magnetic columns according to the manufacturer’s instructions (EasySepTM, StemCell Technology, Vancouver, BC, Canada). The cells were stained with anti-CD19-PerCP-Cy5.5, anti-CD27-PE, anti-IgM-APC, and anti-IgD-FITC (all antibodies from BD Biosciences, San Diego, CA, USA). The B cells, defined as CD19+CD27−IgD+IgM−, were sorted with FACSAria II cell sorter (Beckton-Dickinson, San Jose, CA, USA). After sorting, the cells were labeled with 5 µM of CFSE dye (Invitrogen, Eugene, OR, USA) before the culture, according to manufacturer’s instructions. The cells were cultured in AIM-V serum-free medium in the presence of CpG-ODN2006 (InvivoGen, San Diego, CA, USA) (2.5 µg/mL) and anti-CD40 (BD Bioscience, San Diego, CA, USA) (2 µg/mL) in a 96-well U bottom plate at 37 °C in a humidified 5% CO2 incubator. On day 5, the cells were harvested and acquired using FACSCelesta (Beckton-Dickinson, San Jose, CA, USA).
Kasahara T.D, & Gupta S. (2024). IgD+IgM− B Cells in Common Variable Immunodeficiency. Pathogens, 13(2), 136.
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8

Hypoxia Modulates Ig Isotype Switch in B Cells

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Splenic B cells were purified (90–95%) by depleting T cells using biotinylated anti-Thy1.2 mAb followed by streptavidin-conjugated microbeads. For IgG1, B cells (0.5×106 cells/ml) were activated with LPS or F(ab’)2 anti-IgM (Southern Biotechnology) and anti-CD40 (BD Pharmingen), cultured with BAFF and IL-4. For IgG2c, B cells (0.5×106 cells/ml) were activated with LPS or anti-IgM and anti-CD40, cultured with BAFF and IFN-γ. For IgA, B cells (0.5×106 cells/ml) were activated with LPS (1 µg/ml) or anti-IgM and anti-CD40 and cultured with BAFF (10 ng/ml), TGF-β (5 ng/ml), IL-4 (10 ng/ml), IL-5 (10 ng/ml), and all-trans retinoic acid (RA) (10 nM) in IMDM medium supplemented with 10% FBS, Pen/Strep, L-glutamine, and β-mercaptoethanol. To analyze the partitioning of cell division, purified B cells were stained with CellTrace™ Violet (Thermo Fisher Scientific) according to manufacturer’s instruction or CFDA-SE as described previously33 . Cells were cultured (4 d) at pO2 of 21%, 5% or 1% after which surface Ig was analyzed by flow cytometry. In comparisons of all three [O2] (oxygen tensions), experiments were performed by dividing one common pool of B cells and using two separate hypoxia chambers maintained at constant pO2 using nitrogen.
Cho S.H., Raybuck A.L., Stengel K., Wei M., Beck T.C., Volanakis E., Thomas J.W., Hiebert S., Haase V.H, & Boothby M.R. (2016). Germinal Center hypoxia and regulation of antibody qualities by a hypoxia response system. Nature, 537(7619), 234-238.
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9

Phenotypic Characterization of Monocyte-Derived Dendritic Cells

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Before and after differentiation, the cells were collected, washed and resuspended in Dulbecco’s phosphate-buffered saline (D-PBS, HyClone). To analyze surface antigens, anti-CD1a (BD Biosciences, San Jose, CA), anti-CD11c (BD Biosciences), anti-CD14 (Miltenyi Biotec Gmbh), anti-CD40 (BD Biosciences), anti-CD80 (Miltenyi Biotec Gmbh), anti-CD83 (Miltenyi Biotec Gmbh), anti-CD86 (Miltenyi Biotec Gmbh) and anti-CD209 (BD Biosciences) fluorescence monoclonal antibodies were used. Matched labeled isotypes were used as controls. The labeled cells were analyzed using flow cytometry (Accuri C6, BD Biosciences). The screening criterion of monocyte-derived DCs was defined as CD40+CD209+ cells in this study [55 (link), 56 (link)].
Yao C.L, & Tseng T.Y. (2023). The synergistic and enhancive effects of IL-6 and M-CSF to expand and differentiate functional dendritic cells from human monocytes under serum-free condition. Journal of Biological Engineering, 17, 6.
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10

Immune Cell Immunophenotyping Protocol

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Cells were harvested and counterstained immunophenotypically using anti‐CD83, anti‐CD80, anti‐CD86, anti‐CD40 and anti‐CD11c (BD Pharmingen).
Gao W., Liu H., Yuan J., Wu C., Huang D., Ma Y., Zhu J., Ma L., Guo J., Shi H., Zou Y, & Ge J. (2016). Exosomes derived from mature dendritic cells increase endothelial inflammation and atherosclerosis via membrane TNF‐α mediated NF‐κB pathway. Journal of Cellular and Molecular Medicine, 20(12), 2318-2327.
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