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α cd40

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α-CD40 is a monoclonal antibody that binds to the CD40 antigen, a member of the tumor necrosis factor receptor superfamily. CD40 is expressed on various cell types, including B cells, dendritic cells, and some cancer cells. The α-CD40 antibody can be used in research applications to study the role of CD40 in immune responses and cell signaling.

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Lab products found in correlation

Lipopolysaccharides (lps), by Merck Group (4 mentions) α cd44, by BD (3 mentions) Tgf β, by R&D Systems (3 mentions) Facs lysis buffer, by BD (2 mentions) α cd4, by BioLegend (2 mentions) Collagenase type 4, by Thermo Fisher Scientific (2 mentions) α cd45, by BioLegend (2 mentions) α cd69, by BioLegend (2 mentions) α cd103, by BioLegend (2 mentions) α ifnar1, by BioLegend (2 mentions) Gentlemacs, by Merck Group (2 mentions) α cd8α, by BioLegend (2 mentions) Dnase 1, by Merck Group (2 mentions) αcd11c, by Thermo Fisher Scientific (2 mentions) α igm, by BioLegend (2 mentions) Mouse recombinant il 4, by Merck Group (2 mentions) Lipopolysaccharides (lps), by Thermo Fisher Scientific (2 mentions) αi a 1 e, by BioLegend (1 mentions) αcd80, by BioLegend (1 mentions) Facscanto 2, by BD (1 mentions)

11 protocols using α cd40

1

Immune Activation of DC2.4 Cells

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DC2.4 cells were seeded in a six-well plate (0.5 × 106 cells per well) and cultured for 24 hours. Then, CpG, R848, cGAMP, CGR, and NVs were added and further cultured for 24 hours. The cells were collected and stained with αI-A/I-E, αCD40, αCD80, and αCD86 antibodies (BioLegend). The cells were washed, and the expression levels of costimulatory factors were detected by flow cytometry (BD FACSCanto II).
Cheng F., Su T., Zhou S., Liu X., Yang S., Lin S., Guo W, & Zhu G. (2023). Single-dose injectable nanovaccine-in-hydrogel for robust immunotherapy of large tumors with abscopal effect. Science Advances, 9(28), eade6257.
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2

Comprehensive Immune Cell Profiling of Mouse Tissues

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At the designated times after infection, mice were euthanized, and tissues were perfused with PBS by cardiac injection. Lungs and/or dLN were harvested and single cell suspensions were created by enzymatic digestion with type IV collagenase (Fisher/Worthington) and DNase I (Sigma) and GentleMacs (Miltemyi) processing.
For flow cytometry staining, single cell homogenates were blocked in 2% rat serum for 10 min at room temperature, incubated with specified antibodies for 30 min on ice, fixed with BD FACS Lysis buffer for 10 min at room temperature, resuspended in PBS, and run on the LSRII (BD). Samples were analyzed using FlowJo software (BD).
The following anti-mouse antibodies conjugated to fluorochromes were utilized for flow cytometry: (company, clone) α-CD3ε (eBioscience/Invitrogen, 145–2C11), α-CD4 (Biolegend, GK1.5), α-CD8α (Biolegend, 53–6.7), α-CD11b (eBioscience/Invitrogen, M1/70), α-CD11c (eBioscience/Invitrogen, N418), α-CD19 (eBioscience/Invitrogen, 1D3), α-CD40 (Biolegend, HM40–3), α-CD44 (BD, IM7), α-CD45.1 (eBioscience/Invitrogen, A20), α-CD45.2 (Biolegend, 104), α-CD69 (Biolegend, H1.2F3), α-CD103 (Biolegend, 2E7), α-F4/80 (eBioscience/Invitrogen, BM8), α-IFNAR1 (Biolegend, MAR1–5A3), α-IFNγ (eBioscience/Invitrogen, XMG1.2), α-MHCII (eBioscience/Invitrogen, M5/114.15.2), α-TNFα (eBioscience/Invitrogen, MP6-XT22).
Hemann E.A., Green R., Turnbull J.B., Langlois R.A., Savan R, & Gale M J.r. (2019). Interferon-λ modulates dendritic cells to facilitate T cell immunity during infection with influenza A virus. Nature immunology, 20(8), 1035-1045.
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3

B Cell Proliferation Assay with Macrophages

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Cell-sorted PMs from diseased-BWF1 and aged-matched control mice were cocultured with sorted aged-matched control peritoneal B cells (CD19 +) in the presence of diverse stimuli. Sorted total B cells were stained with 5 μM CFSE (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions and resuspended at 2 × 106 cells/mL. The coculture was performed with 20,000 macrophages and 100,000 B cells per well in a P96 plate. The stimuli used were 2 ng/mL LPS from E. coli O111:B4 (Sigma-Aldrich), 1 μg/mL CpG ODN 1826 (Invivogen) or 2 μg/mL α-CD40 + 2 ng/mL IL-4 (Biolegend). The cells were cultured for four days and then stained for flow cytometry with CD19, CD138, and viability dye to exclude dead cells. Proliferation was analyzed by CFSE dilution.
Tejon G., Valdivieso N., Flores-Santibañez F., Barra-Valdebenito V., Martínez V., Rosemblatt M., Sauma D, & Bono M.R. (2022). Phenotypic and functional alterations of peritoneal macrophages in lupus-prone mice. Molecular Biology Reports, 49(6), 4193-4204.
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4

B Cell Enrichment and Activation Assay

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For B cell enrichment, splenocytes were incubated with antibody cocktail containing biotin-labeled αCD90, αCD11c, and αCD11b (eBioscience). Anti-biotin microbeads (Miltenyi Biotec) were used to deplete CD90+ T-cells, CD11c+ DCs, and CD11b+ macrophages using MACS separation. Purified B-cells were left unstimulated or were stimulated with α-IgM (Jackson ImmunoResearch) at 10μg/ml plus IL-4 (Peprotech) at 20ng/ml, α-CD40 (BD Biosciences) at 10μg/ml plus IL-4 at 20ng/ml, LPS (Sigma Aldrich; 055:B5) at 5μg/ml and CpG (Invioogen; ODN 1826) at 5μg/ml. Kyn/Trp ratio was determined with High-Performance Liquid Chromatography (HPLC) analysis of culture medium as previously described (28 (link)). Cytokine protein concentrations in the culture medium were measured using Ready-Set-Go! ELISA kits (eBioscience). Cell proliferation was determined by FACS analysis after labeling cells with 0.5μM 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen). Cell survival was quantified on flow cytometer by examining annexin V staining. To test the surface expression profiles of activation markers, B cells were stained with α-CD86 (BD Pharmingen), α-MHC class II (eBioscience), α-IgM (Biolegend), α-CD44 (BD Pharmingen), α-IgM (Biolegend), α-CD40 (Biolegend). Events were collected on LSR II flow cytometer and all results were analyzed with FlowJo software (TreeStar).
Shinde R., Shimoda M., Chaudhary K., Liu H., Mohamed E., Bradley J., Kandala S., Li X., Liu K, & McGaha T.L. (2015). B cell-intrinsic Indoleamine 2,3-dioxygenase1 regulates humoral immunity to T cell independent antigens. Journal of immunology (Baltimore, Md. : 1950), 195(5), 2374-2382.
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5

B Cell Class Switching to IgA

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CH12 cells were stimulated to undergo CSR to IgA by treatment with 1–5 μg/ml αCD40 (BioLegend), 5 ng/ml TGFβ (R&D Systems), and 5 ng/ml of mouse recombinant IL-4 for 48 hr. For class switching analysis, cell suspensions were stained with fluorochrome-conjugated anti-IgA (Southern Biotech) and samples were acquired on an LSRFortessa cell analyzer (BD Biosciences).
Balasubramanian S., Andreani M., Andrade J.G., Saha T., Sundaravinayagam D., Garzón J., Zhang W., Popp O., Hiraga S.I., Rahjouei A., Rosen D.B., Mertins P., Chait B.T., Donaldson A.D, & Di Virgilio M. (2022). Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region. eLife, 11, e75047.
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6

Class Switch Recombination Protocols

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B lymphocytes were isolated from mouse spleens using anti-CD43 MicroBeads (Miltenyi Biotec) and stimulated to undergo class switching with 25 μg/ml LPS (Sigma-Aldrich) and 5 ng/ml of mouse recombinant IL-4 (Sigma-Aldrich) for CSR to IgG1; 25 μg/ml LPS, 10 ng/ml BAFF (PeproTech) and 2 ng/ml TGFβ (R&D Systems) for CSR to IgG2b; or 25 μg/ml LPS only for CSR to IgG3. Mice 7 to 20 weeks old were used in age-matched groups for splenocytes isolation and CSR assays. CH12 cells (Nakamura et al., 1996 (link)) were stimulated to undergo CSR to IgA by treatment with 5-15 μg/mL αCD40 (BioLegend), 5 ng/ml TGFβ and 5 ng/ml of mouse recombinant IL-4 for 48 h. pMX-ZMYND8-3XFlag retroviral vector was generated by cloning the cDNA for murine ZMYND8 into pMX-IRES-GFP with a C-terminal 3XFlag tag. For CH12 infections with pMX-based retroviral vectors, cells were subjected to 2 rounds of infection with amphotrophic retroviral supernatant, with or without selection with 1 μg/ml of Puromycin (Sigma-Aldrich), followed by activation for 48 h before analysis for CSR efficiency.
Delgado-Benito V., Rosen D.B., Wang Q., Gazumyan A., Pai J.A., Oliveira T.Y., Sundaravinayagam D., Zhang W., Andreani M., Keller L., Kieffer-Kwon K.R., Pękowska A., Jung S., Driesner M., Subbotin R.I., Casellas R., Chait B.T., Nussenzweig M.C, & Di Virgilio M. (2018). The Chromatin Reader ZMYND8 Regulates Igh Enhancers to Promote Immunoglobulin Class Switch Recombination. Molecular Cell, 72(4), 636-649.e8.
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7

Isolation and Analysis of cDC Subsets

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cDC subsets were sorted from the MLN of WT mice in steady state or following i.p. injection of αCD40 (25 μg; Biolegend) and LPS (20 μg; Sigma-Aldrich) or Poly(I:C) (100 μg; InvivoGen) using a FACSAria III Cell Sorter (BD Bioscience). Briefly, MLN CD11c+ cells were enriched using anti-CD11c magnetic cell separation beads (Miltenyi Biotec) and then stained for surface markers prior to sorting. Total RNA was isolated with an RNA Nano-prep kit (Agilent), cDNA was prepared and amplified using Ovation Pico WTA systemV2 (TECAN), and quantitative PCR to quantify p28 expression was performed with Kapa SYBR FAST (Sigma-Aldrich). The following primers were used: b-actin sense, 5′-CCG​GGA​CCT​GAC​AGA​CTA-3′; b-actin antisense, 5′-GTT​TCA​TGG​ATG​CCA​CAG​GAT-3′; p28 sense, 5′-ATC​TCG​ATT​GCC​AGG​AGT​GA-3′; and p28 antisense, 5′-GTG​GTA​GCG​AGG​AAG​CAG​AGT-3′ (Hooper et al., 2017 (link)).
Ahmadi F., Junghus F., Ashworth C., Lappalainen A., Mörbe U., Kotarsky K, & Agace W.W. (2022). cDC1-derived IL-27 regulates small intestinal CD4+ T cell homeostasis in mice. The Journal of Experimental Medicine, 220(3), e20221090.
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8

Immune Cell Analysis by Flow Cytometry

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To determine percentages of immune cell subsets single cell suspensions from spleen, bone marrow and peritoneal lavage were incubated with α-B220, α-CD5, α-Thy1.2, α-CD4, α-F4/80, α-CD11c, α-CD93 (AA4.1) (all from eBioscience), α-CD21, α-CD24, α-CD8, α-CD11b (all from BD Biosciences), α-CD23, α-IgM, α-CD40, α-Ly6G (all from Biolegend). For flow cytometric study >105 events were collected on LSRII flow cytometer and all results were analyzed with FlowJo software (TreeStar).
Shinde R., Shimoda M., Chaudhary K., Liu H., Mohamed E., Bradley J., Kandala S., Li X., Liu K, & McGaha T.L. (2015). B cell-intrinsic Indoleamine 2,3-dioxygenase1 regulates humoral immunity to T cell independent antigens. Journal of immunology (Baltimore, Md. : 1950), 195(5), 2374-2382.
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9

Induction of Class-Switched Ig Isotypes

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CH12 cells were stimulated to undergo CSR to IgA by treatment with 1–5 µg/ml αCD40 (BioLegend), 5 ng/ml TGFβ (R&D Systems), and 5 ng/ml mouse recombinant IL-4 for 48 h. B lymphocytes were stimulated to undergo class switching with 5 µg/ml LPS (Sigma-Aldrich) and 5 ng/ml mouse recombinant IL-4 (Sigma-Aldrich) for CSR to IgG1; 5 µg/ml LPS only for CSR to IgG3; 5 µg/ml LPS, 10 ng/ml BAFF (PeproTech), and 2 ng/ml TGFβ for CSR to IgG2b; or 5 µg/ml LPS, 10 ng/ml BAFF, 2 ng/ml TGFβ, and 1.5 ng/ml recombinant murine IL-5 (PeproTech) for CSR to IgA. For class switching analysis, cell suspensions were stained with fluorochrome-conjugated anti-IgG1, anti-IgG3 (BD Biosciences), anti-IgG2b (BioLegend), or anti-IgA (Southern Biotech).
Delgado-Benito V., Berruezo-Llacuna M., Altwasser R., Winkler W., Sundaravinayagam D., Balasubramanian S., Caganova M., Graf R., Rahjouei A., Henke M.T., Driesner M., Keller L., Prigione A., Janz M., Akalin A, & Di Virgilio M. (2020). PDGFA-associated protein 1 protects mature B lymphocytes from stress-induced cell death and promotes antibody gene diversification. The Journal of Experimental Medicine, 217(10), e20200137.
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10

Comprehensive Immune Cell Profiling of Mouse Tissues

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At the designated times after infection, mice were euthanized, and tissues were perfused with PBS by cardiac injection. Lungs and/or dLN were harvested and single cell suspensions were created by enzymatic digestion with type IV collagenase (Fisher/Worthington) and DNase I (Sigma) and GentleMacs (Miltemyi) processing.
For flow cytometry staining, single cell homogenates were blocked in 2% rat serum for 10 min at room temperature, incubated with specified antibodies for 30 min on ice, fixed with BD FACS Lysis buffer for 10 min at room temperature, resuspended in PBS, and run on the LSRII (BD). Samples were analyzed using FlowJo software (BD).
The following anti-mouse antibodies conjugated to fluorochromes were utilized for flow cytometry: (company, clone) α-CD3ε (eBioscience/Invitrogen, 145–2C11), α-CD4 (Biolegend, GK1.5), α-CD8α (Biolegend, 53–6.7), α-CD11b (eBioscience/Invitrogen, M1/70), α-CD11c (eBioscience/Invitrogen, N418), α-CD19 (eBioscience/Invitrogen, 1D3), α-CD40 (Biolegend, HM40–3), α-CD44 (BD, IM7), α-CD45.1 (eBioscience/Invitrogen, A20), α-CD45.2 (Biolegend, 104), α-CD69 (Biolegend, H1.2F3), α-CD103 (Biolegend, 2E7), α-F4/80 (eBioscience/Invitrogen, BM8), α-IFNAR1 (Biolegend, MAR1–5A3), α-IFNγ (eBioscience/Invitrogen, XMG1.2), α-MHCII (eBioscience/Invitrogen, M5/114.15.2), α-TNFα (eBioscience/Invitrogen, MP6-XT22).
Hemann E.A., Green R., Turnbull J.B., Langlois R.A., Savan R, & Gale M J.r. (2019). Interferon-λ modulates dendritic cells to facilitate T cell immunity during infection with influenza A virus. Nature immunology, 20(8), 1035-1045.
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