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Clone f38 2e2

Manufactured by BioLegend
Sourced in United States

Clone F38-2E2 is a monoclonal antibody that recognizes the human CD40 antigen. CD40 is a cell surface receptor that belongs to the tumor necrosis factor receptor superfamily and is expressed on B cells, dendritic cells, macrophages, and other cell types. The antibody can be used for flow cytometry and other immunoassays to detect and analyze CD40-expressing cells.

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5 protocols using clone f38 2e2

1

Trophoblast-Decidual Cell Interactions

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Freshly isolated Tros were seeded at a density of 2 × 105 cells/ml per well in Matrigel (Coring, U.S.A)-coated 24-well plates overnight. The cells were then washed twice with phosphate-buffered saline (PBS, HyClone, U.S.A). Equal numbers of dCD14+ cells or pCD14+ cells were added to each well. In some wells, anti-HLA-C (10 μg/ml, clone W6/32; Biolegend, U.S.A), HLA-G (10 μg/ml, clone 87 G; Biolegend, U.S.A) were added. dCD14+ cells were also cultured with HTR8/Svneo cells (ATCC, www.atcc.org) or DSCs for 48 h. In some wells, dCD14+ cells (2 × 105 cells) were plated in the upper chamber (0.4 mm pore size cell culture inserts, Millipore, Germany), while Tros were plated in the lower chamber to establish indirect cell contact.
Freshly isolated dCD4+ T cells co-cultured with Tim-3+dMφs or Tim-3dMφs at a 1: 1 ratio. In some experiments, Tim-3+dMφs were pretreated with anti-Tim-3 (10 μg/ml, clone F38-2E2, BioLegend, U.S.A.), or anti-CD132 (10 μg/ml, clone TUGh4, BioLegend, U.S.A.), or anti-IL-4 (10 μg/ml, clone F38-2E2, BioLegend, U.S.A.). Phorbol 12-myrstate 13-acetate (PMA) (50 ng/ml, Biolegend, U.S.A.), ionomycin (1 μg/ml, Biolegend, U.S.A.) and brefeldin A (10 mg/ml, BioLegend, U.S.A.), were added 4 h before the end of the 48 h co-culture. The supernatants were then collected for intracellular cytokine analysis of T cells.
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2

TIGIT and TIM-3 Blockade Enhances CD8+ T-cell Function

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We selected cryopreserved PBMCs from S1 (n=10) and S2 (n=10). Samples were previously characterized by the expression of TIGIT and TIM-3 on total CD8+ T-cells. PBMCs were thawed and rested for four hours at 37 °C in a 5% CO2 incubator. Next, cells were incubated under RPMI complemented medium 10% FBS with 1 μl/ml of anti-CD28/CD49d and 1 μl/ml of Monensin A overnight at 37 °C in a 5% CO2. PBMCs are divided in the following conditions; (1) unstimulated, (2) SEB (1 μg/ml, Sigma-Aldrich) and (3) HIV-1-Gag peptide pool (2 μg/peptide/ml) in the absence or presence of αTIGIT and/or αTIM-3, and its respective isotype antibodies. For the single blockade of TIGIT (αTIGIT), we included Ultra-LEAF purified anti-human TIGIT antibody (10 μg/ml, clone A15153G, Biolegend) or its control isotype Ultra-LEAF purified mouse IgG2a antibody (10 μg/ml, MOPC-173, Biolegend). For single TIM-3 blockade (αTIM-3), we used Ultra-LEAF purified anti-human TIM-3 antibody (10 μg/ml, clone F38-2E2, Biolegend) or its respective isotype Ultra-LEAF purified mouse IgG1 antibody (10 μg/ml, MOPC-21, Biolegend). Finally, we included αTIGIT+αTIM-3 or their respective IgG2 + IgG1 isotypes for a combinational blockade. The next day, PBMCs were surface and intracellularly stained with the panel of antibodies and the methodology described in the section above.
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3

Modulating CD8+ T cell function

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dCD8+ T cells were cultured (5 × 105 per well) in the presence of anti-Tim-3 (10 μg/ml, clone F38-2E2, BioLegend, USA), anti-CTLA-4 (10 μg/ml, clone L3D10, BioLegend, USA), both of the two antibodies, or isotype control for 48 h. Brefeldin A, PMA, and ionomycin was added 4 h before the end of the culture. The cells were then collected for further analysis by flow cytometry.
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4

Decidual CD8+ T Cell Immune Modulation

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Decidual immune CD8+ T cells were cultured (5 × 105 per well) in the presence of antibodies against Tim-3 (10 μg/ml, clone F38-2E2, BioLegend), PD-1 (10 μg/ml, clone EH12.2H7, BioLegend), both Tim-3 and PD-1, or isotype control. After 48 h, the culture supernatant was collected and analyzed by flow cytometry or cytotoxicity assay.
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5

Cytokine Production in CD4+ T Cells

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dCD4+T cells were cultured (5 × 105 per well) in the presence of anti-CTLA-4 (10 μg/ml, clone L3D10, BioLegend, U.S.A.), anti-Tim-3 (10 μg/ml, clone F38-2E2, BioLegend, U.S.A.), both of the two antibodies, or isotype control for 48 h. Brefeldin A, PMA, and ionomycin was added 4 h before the end of the culture. The cells were then collected for further analysis by flow cytometry.
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