Human OX40 Ligand/TNFSF4 Antibody

Detection of OX40 Ligand/ TNFSF4 in Human Mature Dendritic Cells by Flow Cytometry. Human mature dentritic cells differentated from human peripheral blood mononuclear cell derived CD14⁺cells treated with 20 ng/mL Recombinant Human IL-4 (Catalog # 204-IL) and 50 ng/mL Recombinant Human GM-CSF (Catalog # 215-GM) for 7 days and 1 µg/mL LPS, 20 ng/mL Recombinant Human TNF-a (Catalog # 210-TA), and 20 ng/mL Recombinant Human IL-1 beta /IL-1F2 (Catalog # 201-LB) for last 24 hours were stained with Mouse Anti-Human OX40 Ligand/TNFSF4 Monoclonal Antibody(Catalog # MAB10541, filled histogram) or isotype control (Catalog # MAB002, open histogram), followed by Goat F(ab)2 Anti-Mouse IgG (H+L) Allophycocyanin (Catalog # F0101B).

OX40 Ligand/TNFSF4 in Human PBMCs. OX40 Ligand/TNFSF4 was detected in immersion fixed human peripheral blood mononuclear cells (PBMCs) stimulated with LPS using Mouse Anti-Human OX40 Ligand/ TNFSF4 Monoclonal Antibody (Catalog # MAB10541) at 25 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (orange; Catalog # NL007) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

IL‑8 Secretion Induced by OX40 Ligand/TNFSF4 and Neutralization by Human OX40 Ligand/TNFSF4 Antibody. Recombinant Human OX40 Ligand/TNFSF4 (Catalog # 1054-OX) induces IL-8 secretion in the HT1080 human fibrosarcoma cell line transfected with human OX40 in a dose-dependent manner (orange line) as measured by Human IL-8 Duoset (Catalog # DY208). IL-8 Secretion elicited by Recombinant Human OX40 Ligand/TNFSF4 (10 ng/mL) is neutralized (green line) by increasing concentrations of Mouse Anti-Human OX40 Ligand/TNFSF4 Monoclonal Antibody (Catalog # MAB10541). The ND₅₀ is typically 0.1-0.6 µg/mL.

Detection of Mouse OX40 Ligand/TNFSF4 by Flow Cytometry Th2 polarization induced by SEA is mediated via Dectin-2 and Syk signaling in vivo.(A–D) WT or CD11c delta Syk mice were injected with SEA (panel A, B), SEA delta alpha -1/ omega -1 (panel C), or omega -1 (panel D) in the hind footpad, and draining pLNs were analyzed 7 d later. (A, C, D) pLN cells were restimulated with PMA/Ionomycin in the presence of brefeldin A, and CD4+ T cells were stained for indicated intracellular cytokines and percentage cytokine-positive CD4+ T cells enumerated. Based on these data, the ratio between the percentage IL-4+ over IFN-gamma + CD4+ T cells was determined as a measure for overall skewing towards Th2. (B) pLN cells were restimulated with SEA for 72 h, and cytokine levels in culture supernatants were determined. (A–D) Bar graphs represent mean ± SEM of 5 to 6 mice per group and are representative of 2 (panel A–C) or 1 (panel D) experiment. (E) BMDCs cultured from BM from WT, Dectin-1−/−, or Dectin-2−/− mice were pulsed overnight with SEA delta alpha -1/ omega -1 and injected into hind footpads after which CD4+ T-cell responses were analyzed as in panel A. Representative flow cytometry plots of intracellular staining of CD4+ T cells are depicted, of which the data are enumerated in bar graphs representing mean ± SEM of 2 independent experiments with 3 to 6 mice per group. (F) SEA binding and uptake was determined as in Fig 4F. (G) ROS production by indicated BMDCs was determined as described in Fig 5E, 1 h after stimulation with SEA delta alpha -1/ omega -1. Based on MFI, bar graphs represent fold change relative to control condition, which is set to 1. (H) BMDCs were stimulated as indicated for 18 h after which expression of OX40L was analyzed by flow cytometry. Representative plots are depicted, of which the data are enumerated in bar graphs and shown as fold change relative to control condition, which is set to 1, based on percentage positive cells. (F–H) Bar graphs represent mean of duplicates or triplicates ± SEM of 2 independent experiments. “*”: P < 0.05; “**” and “##”: P < 0.01; “***” and “###”: P < 0.001 for significant differences with the control conditions (*) or between-test condition (#) based on unpaired analysis (unpaired Student t test). Underlying data can be found in S1 Data. omega -1, omega-1; BMDC, bone marrow–derived DC; CD4, cluster of differentiation 4; H2-DCFDA, 2',7'-dichlorodihydrofluorescein diacetate; IFN-gamma, interferon gamma ; IL-4, interleukin 4; MFI, mean fluorescence intensity; nd/pLN, nondraining/popliteal lymph node; OX40L, OX40 ligand; PBS, phosphate buffered saline; PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species; SEA, soluble egg antigen; Syk, spleen tyrosine kinase; Th2, T helper 2; WT, wild-type. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pbio.2005504), licensed under a CC-BY license. Not internally tested by R&D Systems.