monomeric soluble cd40l  (R&D Systems)

Journal: Cells

Article Title: Melanoma-Derived Extracellular Vesicles Bear the Potential for the Induction of Antigen-Specific Tolerance

doi: 10.3390/cells8070665

Figure Lengend Snippet: Melanoma cell-derived sEVs modulate the immune receptor expression on APCs. ( a ) Upper panel: The cell surface expression of MHC class I, II, CD40, and CD86 of CD14+ primary human monocytes (HuMo) was measured by flow cytometry (green lines). Overlaid are the histograms after exposure to A375 melanoma-derived sEVs for 48 hours (filled violet peaks; representative of n = 6). Lower panel: HuMo were differentiated into DCs (HuDC) and exposed to sEVs for 48 hours and the cell surface expression was analyzed by flow cytometry, as above (representative of n = 3). ( b ) The mean fluorescence intensities (MFI) of the surface expression of MHC class I, II, CD40, and CD86 after exposure to melanoma-derived sEVs was related to the MFI of unexposed cells. Both monocytes and DCs derived from either the THP-1 cell line (left diagram) or primary human CD14+ cells (right diagram) were used in the experiments. At least three independent experiments were conducted for each cell type and significant differences were marked by asterisks (Student’s t-test). ( c ) The mRNA expression levels in DC cells derived from the peripheral blood of healthy donors treated with A375-derived sEVs relative to untreated control cells. Mean values and standard deviations are shown from three independent experiments. The differences between samples after sEV exposure and untreated controls did not reach statistical significance (Student’s t -test).

Article Snippet: For the stimulation of cytokine production, cells were incubated at a density of 5 × 10 5 cells per mL in serum-free medium (AIM V, Gibco–Thermo Fisher Scientific, Life Technologies Polska, Warsaw, Poland) for 24 h with LPS (Lipopolysaccharide (E. coli 0111:B4), 1 µg/mL; Sigma-Aldrich, St Louis, MO, USA) and CD40 ligand (100 ng/mL, soluble, human, recombinant Mega CD40L, Enzo Life Sciences, Lausen, Switzerland).

Techniques: Derivative Assay, Expressing, Flow Cytometry, Fluorescence

Journal: Cells

Article Title: Melanoma-Derived Extracellular Vesicles Bear the Potential for the Induction of Antigen-Specific Tolerance

doi: 10.3390/cells8070665

Figure Lengend Snippet: sEVs affect the cytokine secretion of APCs. THP-1 DC cells were stimulated to secrete cytokines by incubation with LPS and CD40L. The addition of melanoma cell-derived sEVs (A375, DMBC12 or DMBC21) increased the expression levels of IL-12 and IL-6, while secretion of VEGF was less affected. Mean values and standard deviations are shown from three independent experiments. Significant differences in the presence versus absence of sEVs are marked with asterisks.

Article Snippet: For the stimulation of cytokine production, cells were incubated at a density of 5 × 10 5 cells per mL in serum-free medium (AIM V, Gibco–Thermo Fisher Scientific, Life Technologies Polska, Warsaw, Poland) for 24 h with LPS (Lipopolysaccharide (E. coli 0111:B4), 1 µg/mL; Sigma-Aldrich, St Louis, MO, USA) and CD40 ligand (100 ng/mL, soluble, human, recombinant Mega CD40L, Enzo Life Sciences, Lausen, Switzerland).

Techniques: Incubation, Derivative Assay, Expressing

Journal: Cells

Article Title: Melanoma-Derived Extracellular Vesicles Bear the Potential for the Induction of Antigen-Specific Tolerance

doi: 10.3390/cells8070665

Figure Lengend Snippet: The down-regulation of receptor molecules on APCs could be partially caused by TGF-β. ( a ) Incubation of PBMCs differentiated into DCs with 20 pg/mL recombinant TGF-β reduced the expression of CD40 and MHC class I and II molecules, measured by flow cytometry. Reduced expression of all four antigens after incubation with sEVs derived from A375 melanoma cells was reversed in the presence of anti-TGFβ antibodies (1 µg/mL). The expression of MHC molecules, CD40 and CD80, was evaluated by flow cytometry. The mean fluorescence intensities (MFI) relative to untreated control cells are shown. Significant differences between samples treated with sEVs or with sEVs and anti-TGF-β antibodies are marked by asterisks. ( b ) TGF-β was measured by a cytometric bead assay in A375 cell-derived sEVs. A sample without sEVs served as a negative control.

Article Snippet: For the stimulation of cytokine production, cells were incubated at a density of 5 × 10 5 cells per mL in serum-free medium (AIM V, Gibco–Thermo Fisher Scientific, Life Technologies Polska, Warsaw, Poland) for 24 h with LPS (Lipopolysaccharide (E. coli 0111:B4), 1 µg/mL; Sigma-Aldrich, St Louis, MO, USA) and CD40 ligand (100 ng/mL, soluble, human, recombinant Mega CD40L, Enzo Life Sciences, Lausen, Switzerland).

Techniques: Incubation, Recombinant, Expressing, Flow Cytometry, Derivative Assay, Fluorescence, Negative Control