Magnisort mouse cd4 naive t cell enrichment kit

Total and naive CD4⁺ T cells were isolated using the MagniSort Mouse CD4 T cell Enrichment Kit and MagniSort Mouse CD4 Naive T cell Enrichment Kit (Invitrogen), respectively. 5 × 10⁵ cells in 200 µl RPMI-1640 medium (PAN Biotech) supplemented with 10% FCS (Biochrom), 2% sodium pyruvate (Sigma), 1× non-essential amino acids (Gibco), 0,01% β-mercaptoethanol (Roth), 1% penicillin/streptomycin (Lonza) and 2 mM L-glutamine (Lonza) were cultivated in 96-well U-bottom cell culture plates (Greiner) coated with 4 µg/ml anti-CD3 (BD Pharmingen) and supplemented with 1 µg/ml anti-CD28 (BD Pharmingen). For differentiation of the naive CD4⁺ T cells into Th1 lymphocytes, the culture was additionally supplemented with 10 ng/ml mIL-12 (Invitrogen) and 5 μg/ml anti-IL-4 (Invitrogen). Th2 cells were differentiated with 10 ng/ml IL-4, 5 µg/ml anti-IL-12, and 5 µg/ml anti-IFNγ. For Th17 differentiation 5 ng/ml TGFβ, 40 ng/ml IL-6, 10 ng/ml IL-23, 2 µg/ml anti-IFNγ, and 2 µg/ml anti-IL-4 were added to the medium, for regulatory T cell differentiation 5 ng/ml TGFβ, 20 ng/ml IL-2, 5 µg/ml anti-IL-12, 5 µg/ml anti-IFNγ, and 5 µg/ml anti-IL-4 were used. Iron sources ferric cloride FeCl₃, ferric sulfate Fe₂(SO₄)₃, and ferric citrate FeC₆H₅O₇ were added at a concentration of 5 µM for 48 h.

Naive CD4⁺ and CD8⁺ T cells were sorted using the MagniSort Mouse CD4 Naive T cell Enrichment Kit (8804-6824-74, Thermo Fisher Scientific) and the Naive CD8a⁺ T Cell Isolation Kit (30-096-543, Miltenyi Biotec), respectively. The sorted cells were stimulated with anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) antibodies in 96-well plates (1 × 10⁵ cells per well) for flow cytometric analysis, or stimulated with anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) antibodies in 6-well plates (5 × 10⁶ cells per well) for immunoblot analysis or metabolic analysis.