Magpix 200 instrument

Melanoma tumor cell lines were cultured in control conditions or incubated with soluble lectins (HPA, WGA, MAA) for 2h and washed. Supernatants were harvested after 20h of culture and IL1β, IL6, IL8, IL10, MCP1, MIP1α, MIP1β and TGFβ secretions were measured by LUMINEX technology using MAGPIX^®200 Instrument with xPONENT^® software (Bio-Rad, Cressier).

To study the maturation of DCs after TLR‐L stimulation, samples were cultured in complete RPMI medium at 4 × 10⁶ cells mL^–1 for 21 h with or without a single or a mixture of the TLR ligands previously described. The potential upregulation of the co‐stimulatory molecules on the 3 DC subsets was then investigated using the fluorochrome‐labelled anti‐human CD86 (BD), CD40 and CD80 (Beckman) antibodies, together with the antibodies allowing to depict the 3 DC subsets (CD11c, HLA‐DR (BD), Lin, CD45 (Biolegend), cDC1/BDCA1 (Beckman), BDCA2 and BDCA3 (Miltenyi)). Analyses were performed using LSRII Flow Cytometer and FACSDiva software v8 (BD). PBMCs and melanoma metastatic tumor supernatants were harvested after 20h of culture, and IL12p70, IFNα, IFNβ, IFNλ1 and IFNλ2 cytokine secretions were measured by LUMINEX technology using MAGPIX®200 Instrument with xPONENT® software (Bio‐Rad, Cressier).