For each neat sample two CBA flex set reactions were set up, each measuring ten different cytokines/chemokines. Reactions were carried out as per the manufacturer's instructions.
To determine the levels of cytokines in the circulation, 12.5 µl of serum was analyzed using the BD™ CBA Flex sets according to the manufacturer's instructions.
Recombinant IL-6 and IL-8 used to generate standard curves were provided with the BD CBA flex set kit. Receptor expression statistics in total PBMC, CD4+ cells, and FOXP3 overexpressing cells were done using a paired, two tailed t-test.
A CBA flex set assay of seven different beads detecting cytokines and chemokines (FGF2, MIP-1β, IL-1β, VEGF, TNF, angiogenin and GM-CSF) was used (Becton Dickinson, Sweden).
Concentrations of MIP-1α, MIP-1β, MIG and RANTES were determined from serum samples collected at day 3 post CB3/LPS treatment with rIL-6 or DMEM treatment at days 0, 1 and 2 PI using a BD CBA flex set kit according to manufacturers' instructions (BD Biosciences).
Serum cytokine and chemokine levels were measured at day 4 PI using a mouse inflammation CBA kit (BD Bioscience) allowing for simultaneous detection IL-6, IL-10, MCP-1, IFN-γ, TNF-α and IL-12p70 from a single sample or a chemokine flex set (BD Bioscience) customized to allow for simultaneous detection of CXCL9 (MIG), CCL3 (MIP1α), CCL4 (MIP1β) and CCL5 (RANTES) from a single sample.
Bronchoalveolar lavage (BAL) was performed 4 weeks post-infection, and cytokine levels in BAL fluid were determined using Cytometric Bead Array and Flex sets according to the manufacturer's instructions (BD Biosciences).
The amount of IL-1β present in the culture supernatants of NK cells was measured using the cytometric bead array kit (BD Biosciences) in combination with human IL-1β Flex set according to the manufacturer's protocol.
The quantification of interleukin IL-6 and IL-8 in the supernatants of cultured epithelial cells was determined by using the cytometric bead array for Human IL-6 and IL-8 flex sets (Cat. 558 276 and 558 277 respectively, BD Bioscience, San Jose, CA, USA).
Quantitation of mouse cytokine content incorporating Th1, Th2 cytokines and chemokines (IFNγ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, GM-CSF, KC, MCP-1, MIG, and TNFα) were simultaneously determined using a multiplexed bead assay (Cytometric Bead Array Flex sets [CBA]) according to the manufacturer's recommended protocol (Becton Dickinson).
The levels of human interleukin 6 (IL 6), IL8, tumour necrosis factor α (TNF-α) and monocyte chemo-attractant protein 1 (MCP-1) from HK-2 supernatants was measured using the BDTM Cytometric Bead Array cytokine Flex sets (bead based immunoassay) according to the manufacturer's instructions.
