Th1 th2 th17 th22 13 plex

Single-cell suspensions from spleen were prepared by disrupting the tissue through a 70 μm mesh. Total splenocytes (2x10⁶) and liver leukocytes (2x10⁵) were activated using anti-CD3 Ab (4μg/ml, clone 154-2C11, BD Biosciences). Sorted (>94% purity) Vα3.2⁺/Vβ9⁺ NKT cells from liver (1x10⁵) were activated by anti-CD3/CD28 dynabeads (1:1 ratio bead:cell, Life Technologies). In all cases cells were grown in complete medium (RPMI 1640 medium supplemented with 10% FCS, 100 U/mL penicillin/streptomycin, 2.5% sodium bicarbonate (7.5% solution), 1 mM sodium pyruvate and 69 μM 1-thioglycerol). The supernatants were collected after 24h and analyzed for cytokines using the mouse Th1/Th2/Th17/Th22 13-plex (eBiosciences) according to manufacturer´s instructions.

Cytokine levels in the culture supernatants were measured by cytometric bead array (Th1Th2Th17 kit from BD Biosciences and Th1Th2Th17Th22 13-plex from eBioscience, San Diego, CA USA) or ELISA. All ELISA reagents were purchased from BD Biosciences with the exception of rIL-17 (eBioscience) and used according to manufactures instructions. NO production was measured in culture supernatants by Griess reaction as described [11 (link)].