Iscove s modified dulbecco medium

Naïve T cells (CD4⁺ CD25⁻ CD44^low) were flow cytometry purified from spleen and lymph nodes of fate reporter mice and activated with plate-bound anti-CD3 (clone: 2C11 eBioscience) and anti-CD28 (clone: 37.51, Biolegend) (coated with 0.5 μg/ml and 5 μg/ml, respectively) in the presence of lineage polarizing cytokines as follows: IL-12 (5 ng/ml) for T_H1, IL-4 (10 ng/ml) for T_H2, IL-4 (10 ng/ml) and TGFβ (5 ng/ml) for T_H9, TGFβ (10 ng/ml) for iTreg, IL-6 (20 ng/ml) and TGFβ (1 ng/ml) and IL1β (10 ng/ml), IL-23 (20 ng/ml) (all from R&D Systems) or FICZ (250 nM; Enzo) for T_H17 cells. Alternatively, CD4+ cells were purified using EasySep Mouse CD4+ T Cell Enrichment Kit (STEMCELL Technologies) from spleen of fate reporter mice and activated as described above. Cells were cultured with anti-IFNγ (10 μg/ml, clone: XMG1.2, eBioscience), anti-IL-4 (10 μg/ml, clone: 11B11), IL-6, IL1β and FICZ, and with TGFβ (for T_H17 cells) or with anti-TGFβ (10 μg/ml, clone: 1D11) for “T_H22” cells. All cytokines were of mouse origin. Cells were cultured in Iscove’s modified Dulbecco medium (Sigma) supplemented with 5% FCS, 0.002M L-glutamine, 100U/ml penicillin, 100μg/ml streptomycin and 5×10⁻⁵M β-mercaptoethanol. Cells were analysed for intracellular cytokines on day 4.

Primary samples were cultured at 37°C and 5% CO2 in serum-free
media (SFM) consisting of Iscove's modified Dulbecco medium
(Sigma-Aldrich) supplemented with bovine serum albumin (BSA)/insulin/transferrin
(BIT9500; StemCell Technologies), 1 mM streptomycin/penicillin, 0.1 mM
2-mercaptoethanol, and a physiological growth factor (PGF) cocktail comprising
of 0.2 ng/mL SCF/GM-CSF/MIP-α, 1.0 ng/mL G-CSF/IL6 (PeproTech EC Ltd) and
0.05 ng/mL LIF (StemCell Technologies).