Initial screening of cytokine concentrations in the supernatant of the stimulated pDCs was performed using Bio-Plex Pro Human Cytokine 17-Plex Panel (Bio-Rad), complemented by a Bio-Plex Pro Human Cytokine IP-10 (Bio-Rad) and Bio-Plex Pro Human Cytokine IFN-a2 (Bio-Rad) resulting in a total of 19 cytokine targets (IP-10, MIP-1β, TNF-α, IFN-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, G-CSF, GM-CSF, IFN-γ and MCP-1). Consecutive analyses were performed using a combination of Bio-Plex Pro Human Cytokine IP-10, Bio-Plex Pro Human Cytokine MIP-1α, Bio-Plex Pro Human Cytokine TNF-α and Bio-Plex Pro Human Cytokine IFN-a2 sets (Bio-Rad).
Multiplex assay was performed on a Luminex 200 system (Bio-Rad) in accordance with the manufacturer’s instructions. Standards were analysed in duplicates and each sample in triplicates. Washing was performed between each step using a HydroFlex microplate equipped with a magnetic plate carrier (Tecan). Analysis of the data was performed using Bio-Plex Manager (Bio-Rad).
Multiplex assay was performed on a Luminex 200 system (Bio-Rad) in accordance with the manufacturer’s instructions. Standards were analysed in duplicates and each sample in triplicates. Washing was performed between each step using a HydroFlex microplate equipped with a magnetic plate carrier (Tecan). Analysis of the data was performed using Bio-Plex Manager (Bio-Rad).