rabbit anti ox40l antibody  (Cell Signaling Technology Inc)

Journal: International Journal of Nanomedicine

Article Title: Enhanced Antitumor Immunity Through T Cell Activation with Optimized Tandem Double-OX40L mRNAs

doi: 10.2147/IJN.S479434

Figure Lengend Snippet: In vitro transfection of the diOX40L mRNA. (A) Reverse transcription (RT) quantitative (q)PCR assay of OX40L expression on patient-derived HCC. Data were processed using GraphPad Prism 8 and are presented as the mean ± SD. The P values were determined using a t -test. ***, P < 0.001. ( B) Western blot analysis of OX40L expression in HEK293T cells after the indicated treatments for 24 hours. ( C) Western blot analysis of OX40L expression in HEK293T cells after the diOX40L mRNA transfected for 24, 48, and 72 hours. ( D) Immunofluorescence imaging of OX40L expression in HEK293T cells treated with noncoding and diOX40L mRNA for 24 hours, 48 hours, and 72 hours. OX40L protein was stained in green, and DAPI in blue. ( E) Flow cytometry analysis of cell uptake efficiency by H22 subcutaneous tumors after intratumoral injection with different mRNA agents. ( F) The percentages were used to present the flow cytometry data. ( G) The mean fluorescence intensity (MFI) was used to present the flow cytometry data. Data of ( E, F ) were processed using GraphPad Prism 8 and are presented as the mean ± SD. The P values were analyzed by One-way ANOVA with multiple comparisons. *, P < 0.1; **, P < 0.01; ***, P < 0.001.

Article Snippet: Blots were incubated for 1 hour at room temperature in blocking buffer containing 5% bovine serum albumin (BSA; Solarbio, SW3015); incubated overnight at 4°C with rabbit anti-OX40L antibody (1:1500 dilution, CST, 14991) and mouse anti-β-actin antibody (1:1500 dilution, CST, 3700); and subsequently incubated with HRP-conjugated goat anti-rabbit (1:5000 dilution) and HRP-conjugated goat anti-mouse (1:5000 dilution) antibodies for 1.5 h at room temperature.

Techniques: In Vitro, Transfection, Reverse Transcription, Expressing, Derivative Assay, Western Blot, Immunofluorescence, Imaging, Staining, Flow Cytometry, Injection, Fluorescence

Journal: Frontiers in Immunology

Article Title: IL-33-primed human mast cells drive IL-9 production by CD4 + effector T cells in an OX40L-dependent manner

doi: 10.3389/fimmu.2024.1470546

Figure Lengend Snippet: OX40L induced by IL-33 in MCs promotes IL-9 production in Th cells. (A) Ligand–receptor interaction inference and visualization. The top 30 strongest predicted interactions visualized by the circle plot depicting links between predicted ligands upregulated in MCs upon IL-33 treatment with their associated receptors expressed in memory CD4 + T cells activated with anti-CD3/CD28 beads. The arrow thickness is proportional to the computed L–R score. Multiple ICAM-1/integrin interactions have the highest score but are not represented to avoid overloading the diagram. Some gene symbols have been replaced by more commonly used protein names. (B) Expression of indicated costimulation molecules and TNFSF molecules by MC treated or not with IL-33 according to the RNAseq dataset. (C, D) OX40L expression on the MC surface following IL-33 treatment analyzed by flow cytometry; representative histogram (C) and pooled data from n = 8 donors are shown; bars represent mean ± SEM; each point corresponds to an experiment, Wilcoxon matched-pairs signed rank test. (E, F) Analysis of cytokine production by memory CD4 + T cell stimulated with anti-CD3/CD28-coated beads in the presence of MCs or MC IL-33 or MC IL-33 plus anti-OX40L blocking mAb or its isotype matched control. Representative dot plots of indicated intracellular cytokine stainings and pooled data from six independent experiments are shown. Bars represent mean ± SEM, and each point corresponds to an independent experiment, Friedman test followed up by pairwise Wilcoxon signed-rank tests. * p < 0.05, ** p < 0.01.

Article Snippet: They were incubated with mouse anti-tryptase (IgG1 kappa, clone AA1, Dako) and anti-OX40L (polyclonal rabbit IgG, Invitrogen) at RT for 1 h in blocking buffer.

Techniques: Expressing, Flow Cytometry, Blocking Assay, Control

Journal: Frontiers in Immunology

Article Title: IL-33-primed human mast cells drive IL-9 production by CD4 + effector T cells in an OX40L-dependent manner

doi: 10.3389/fimmu.2024.1470546

Figure Lengend Snippet: Antigen-presenting MCs primed with IL-33 promote IL-9 production in CD4 + T cells in an OX40L-dependent manner. (A) Experimental setting of coculture experiments. MCs were stimulated with IFN-γ for 48 h, treated with IL-33 for 4 h, and then loaded with a cocktail of SAg. MCs were cocultured for 6 days with freshly isolated human effector/memory CD4 T cells. (B) CD4 + T-cell number at the end of the coculture (C–E) Immunological synapse (IS) formation between SAg-pulsed MC IL-33 and memory CD4+ T cells, typical example of OX40L enrichment at the IS (C) , fluorescence intensity profile of channels corresponding to CD4, OX40, and OX40L staining averaged along the rectangle drawn in (C, D) , quantification of MC/CD4 + T-cell conjugates showing OX40L enrichment or not at the IS ( n ~ 50) (E) . (F, G) Intracellular flow cytometry analysis of CD4 + T-cell cytokine production (F) , in the presence of anti-OX40L blocking mAb or rabbit IgG control (G) . (H) Amount of IL-9 and IL-13 produced by FACS-sorted CD4 + T cells after 6 days of coculture and restimulated by PMA/ionomycin. Box-and-whiskers plots in the style of Tukey or mean ± SEM; each point corresponds to an independent experiment. * p < 0.05, ** p < 0.01, *** p < 0.001, ****p<0.0001; ns, not significant.

Article Snippet: They were incubated with mouse anti-tryptase (IgG1 kappa, clone AA1, Dako) and anti-OX40L (polyclonal rabbit IgG, Invitrogen) at RT for 1 h in blocking buffer.

Techniques: Isolation, Fluorescence, Staining, Flow Cytometry, Blocking Assay, Control, Produced

Journal: Frontiers in Immunology

Article Title: IL-33-primed human mast cells drive IL-9 production by CD4 + effector T cells in an OX40L-dependent manner

doi: 10.3389/fimmu.2024.1470546

Figure Lengend Snippet: OX40L induced by IL-33 in MCs promotes IL-9 production in Th cells. (A) Ligand–receptor interaction inference and visualization. The top 30 strongest predicted interactions visualized by the circle plot depicting links between predicted ligands upregulated in MCs upon IL-33 treatment with their associated receptors expressed in memory CD4 + T cells activated with anti-CD3/CD28 beads. The arrow thickness is proportional to the computed L–R score. Multiple ICAM-1/integrin interactions have the highest score but are not represented to avoid overloading the diagram. Some gene symbols have been replaced by more commonly used protein names. (B) Expression of indicated costimulation molecules and TNFSF molecules by MC treated or not with IL-33 according to the RNAseq dataset. (C, D) OX40L expression on the MC surface following IL-33 treatment analyzed by flow cytometry; representative histogram (C) and pooled data from n = 8 donors are shown; bars represent mean ± SEM; each point corresponds to an experiment, Wilcoxon matched-pairs signed rank test. (E, F) Analysis of cytokine production by memory CD4 + T cell stimulated with anti-CD3/CD28-coated beads in the presence of MCs or MC IL-33 or MC IL-33 plus anti-OX40L blocking mAb or its isotype matched control. Representative dot plots of indicated intracellular cytokine stainings and pooled data from six independent experiments are shown. Bars represent mean ± SEM, and each point corresponds to an independent experiment, Friedman test followed up by pairwise Wilcoxon signed-rank tests. * p < 0.05, ** p < 0.01.

Article Snippet: Cells were blocked/permeabilized in blocking buffer for 30 min at RT and subsequently stained with mouse anti-OX40 mAb (IgG1 kappa, clone ACT35, BD Pharmingen), rabbit anti-OX40L (polyclonal rabbit, PA5-116057, Invitrogen), and anti-CD4 (polyclonal goat, AF-379-NA, R&D Systems).

Techniques: Expressing, Flow Cytometry, Blocking Assay, Control

Journal: Frontiers in Immunology

Article Title: IL-33-primed human mast cells drive IL-9 production by CD4 + effector T cells in an OX40L-dependent manner

doi: 10.3389/fimmu.2024.1470546

Figure Lengend Snippet: Antigen-presenting MCs primed with IL-33 promote IL-9 production in CD4 + T cells in an OX40L-dependent manner. (A) Experimental setting of coculture experiments. MCs were stimulated with IFN-γ for 48 h, treated with IL-33 for 4 h, and then loaded with a cocktail of SAg. MCs were cocultured for 6 days with freshly isolated human effector/memory CD4 T cells. (B) CD4 + T-cell number at the end of the coculture (C–E) Immunological synapse (IS) formation between SAg-pulsed MC IL-33 and memory CD4+ T cells, typical example of OX40L enrichment at the IS (C) , fluorescence intensity profile of channels corresponding to CD4, OX40, and OX40L staining averaged along the rectangle drawn in (C, D) , quantification of MC/CD4 + T-cell conjugates showing OX40L enrichment or not at the IS ( n ~ 50) (E) . (F, G) Intracellular flow cytometry analysis of CD4 + T-cell cytokine production (F) , in the presence of anti-OX40L blocking mAb or rabbit IgG control (G) . (H) Amount of IL-9 and IL-13 produced by FACS-sorted CD4 + T cells after 6 days of coculture and restimulated by PMA/ionomycin. Box-and-whiskers plots in the style of Tukey or mean ± SEM; each point corresponds to an independent experiment. * p < 0.05, ** p < 0.01, *** p < 0.001, ****p<0.0001; ns, not significant.

Article Snippet: Cells were blocked/permeabilized in blocking buffer for 30 min at RT and subsequently stained with mouse anti-OX40 mAb (IgG1 kappa, clone ACT35, BD Pharmingen), rabbit anti-OX40L (polyclonal rabbit, PA5-116057, Invitrogen), and anti-CD4 (polyclonal goat, AF-379-NA, R&D Systems).

Techniques: Isolation, Fluorescence, Staining, Flow Cytometry, Blocking Assay, Control, Produced