Fcap array software version 3

IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α were quantified in cultured supernatants of spleen cells by a flow cytometer, using a Multiplex CBA kit (BD Cytometric Bead Array Th1/Th2/Th17, San Diego, USA) according to the manufacturer’s instructions. Cells were acquired with a FACSCalibur cytometer and analyzed with FCAP Array TM Software Version 3.0 (BD). IL-9 determination was assayed with CBA flex set (BD Cytometric Flex Set Th9, San Diego, USA).

IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF-α were quantified in culture supernatants of spleen cells by flow cytometry by using Multiplex CBA kit (BD Cytometric Bead Array Th1/Th2/Th17, San Diego, USA) according to manufacturer’s instructions. Cells were acquired in FACSCalibur cytometer and analyzed with FCAP Array TM Software Version 3.0 (BD).

Supernatants from overnight co-culture experiments was collected and stored at minus 80 degrees Celsius until cytokines measurement determination for human IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A using the BD^TM Cytometric Bead Array (CBA) Human Th1/Th2/Th17 Cytokines Kit (BD Biosciences), in accordance with the manufacturer’s instruction. Briefly, supernatants were incubated with beads- and PE- conjugated anti-cytokine antibodies. Each serial diluted of standard protein was also incubated with those antibodies. After 3 hours of incubation, beads were washed and acquired by FACS. Two hundred forty thousand beads per test were acquired, to capture at least 300 events per each cytokine bead. To make standard curves and measure cytokines level, data were analyzed using FCAP Array^TM Software Version 3.0 (BD Biosciences). As a control, we measured the supernatant collected from ATCs only, or MV4-11, or patient’s tumor cells only.