Cytometric bead array flex kit

The level of cytokines was quantified in the culture supernatants collected from either DC generation cultures on day 7 or DC and T cell co-cultures on day 5 using cytometric bead array flex kit, according to the manufacturer's instructions (BD Biosciences). DC supernatants were assessed for IL-6, IL-10, IL-12p70, and TGF-ß1 whilst DC-T cell co-culture supernatants for IL-6, IFNγ, TNFα and IL-2. Data was acquired on a FACS Canto II and analyzed with FCAP software (BD Biosciences).

Mice were euthanized as described above and blood was collected via cardiac puncture. Serum was separated from whole blood using Microtainer serum separator tubes (BD Biosciences) by spinning clotted whole blood at 10,000 × g for 2 min. Serum concentration of IFNγ, TNF, and KC was determined using the Cytometric Bead Array Flex kit (BD Biosciences). Capture beads and undiluted serum samples were mixed and applied to a 96-well 1.2 μm Multiscreen Filter plate (EMD Millipore), then incubated at room temperature for 1 h. Detection beads were added to the filter plate and incubated for an additional 1 h. After washing with wash buffer, the beads were transferred to a 96-well round bottom plate and analyzed on a FACSCanto II flow cytometer (BD Biosciences). Quantification of data was performed using the FCAP Array software (BD Biosciences).

Cytokines were measured in plasma samples, including IFN-β, IL-18 (ELISA, R&D Systems, Minneapolis, MN), IL-1β (Cytometric bead array flex kit, BD Biosciences, San Jose, CA), TNF, IL-6, and IFN-γ (LEGENDplex bead-based immunoassay kit, BioLegend, San Diego, CA).