Cytokine bead array th1 2 17 kit

Sorted Th cells from healthy donors were activated with anti-CD3/CD28 beads (1:5 ratio)(Life Technologies) in XVIVO-15 media for 5 days a 37°C. Then, prostaglandin E2 (10 uM), 1,25(OH)VitD (10nM) and recombinants chemokines CCL1 and CCL18 (0.5ug/mL) were added to 1x10⁵ Th in XVIVO-15 serum-free medium for 72h a 37°C. After the incubation, the supernatants were stored for further cytokine production measurement using the Cytokine Bead Array Th1/2/17 Kit (BD) and live cells were counted (CountBright Absolute Counting Beads), stained with anti-CCR8 and analyzed by flow cytometry.

Sorted Teff and Tregs from healthy donors were activated with anti-CD3/CD28 beads (1:5 ratio) (Life Technologies) and 1000 UI IL-2 for 5 days a 37°C. Then, 100 uL of OSCC and control secretomes were added to 2x10⁵ Teff or 2x10⁵ Tregs (in 100uL) in XVIVO-15 serum-free medium 48h a 37°C. After the incubation, the supernatants were stored for further cytokine production measurement using the Cytokine Bead Array Th1/2/17 Kit (BD) and the cells were counted (CountBright Absolute Counting Beads), stained with Live/Dead dye (Life Technologies), anti-CXCR3, anti-CCR4, anti-CCR6, anti-CCR8, anti-PD-1 and anti-TIGIT (all BioLegend) and analyzed by flow cytometry. For the analysis of cells after secretome co-culture, cells were washed after co-culture with secretome and cultured in new media X-VIVO15 (LONZA) serum-free medium for 48 h at 37°C with anti-CD3/CD28 beads (1:5 ratio) (Life Technologies) and 1000 UI IL-2. After the incubation, the supernatants were stored for further cytokine production measurement and the cells were stained with Live/Dead dye (Life Technologies), anti-CCR6, anti-CCR8, anti-PD-1 and anti-TIGIT (all BioLegend).

2x10⁵ sorted Teff (Treg-depleted) from healthy donors were activated with anti-CD3/CD28 beads (1:5 ratio) (Life Technologies) in XVIVO-15 media for 5 days a 37°C in the presence or absence of 1,25(OH)VitD (10nM in ethanol) or carrier (ethanol). The supernatants were stored for cytokine measurement using the Cytokine Bead Array Th1/2/17 Kit (BD) and the cells were counted (CountBright Absolute Counting Beads) and stained with Live/Dead dye (Life Technologies), anti-FOXP3, anti-GATA3, anti-Tbet and anti-RORγτ and analyzed by flow cytometry.