Fcap arraytm software v3

Cytokine quantification of Th1/Th2/Th17 cytokines; IL-2, IL-4, IL-6, IL-10, TNF, IFNγ, and IL-17A was performed with the aid of the CBA Human Th1/Th2/Th17 cytokine kit (BD Biosciences, San Diego, CA United States) and using the supernatants of cells that had been stimulated with either OMVs or PHA for 24 h, following the manufacturer’s instructions. Samples were read by using the LSRFortessa^TM cytometer (BD Biosciences) with CBA template, a total of 2000 events were recorded, and the resulting data was analyzed with the FCAP Array^TM software v3.0 (BD Biosciences).

Plasma levels of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17A, IL-17F, TNF-α, TNF-β, IFN-γ, IL-22, and IL-12 p70 were quantified by a Human 14-plex kit (AimPlex, Cat# C191114) and a bead-based array protocol. All experimental processes were conducted according to the manufacturer’s instructions. Antibody-beads were added into the 96 wells, standards and samples were dispensed into the wells, and all plasma samples and standards were incubated on the arrays at room temperature with gentle shaking. Biotinylated antibodies were then added after several washes and the arrays were incubated at room temperature. The streptavidin-PE cocktail was added. After another series of washes and incubations, the reading buffer was added, and results were collected via a flow cytometer. Finally, the cytokine concentration was calculated using the FCAP arrayTM software V3.0 (BD Company).