Soluble anti mouse cd28 clone 37

T cells were cultured in RPMI 1640 (Corning, Cat#: 10-040-CV) supplemented with 10% FBS (Gemini Bioproducts), 2 mM L-glutamine (Corning, Cat#: 25-005-CI), 10 mM HEPES (Corning, Cat#: 25-060-CI), 1% penicillin/streptomycin, 50 μg mL⁻¹ gentamycin (Quality Biological), non-essential amino acids (100X, Gibco), and 50 μM β-mercaptoethanol (Sigma). For peptide activation, splenocytes from OT-I transgenic mice were activated with OVAI peptide (100 ng mL⁻¹) at cell concentration 5 E⁶ mL⁻¹. For plate-bound anti-CD3 activation, CD8⁺ T cells were isolated (MojoSort^™ Mouse CD8 T cell Isolation Kit, Cat#: 480035, Biolegend). 2 million cells at concentration 1 E⁶ mL⁻¹ were activated in 6-well plate coated with 5 μg mL⁻¹ anti-mouse CD3 (clone 2C11, Bio X Cell) and 2 μg mL⁻¹ soluble anti-mouse CD28 (clone 37.51, Bio X Cell). To assess cell proliferation, cells were pre-labelled with Cell Proliferation Dye-eFluor450 and then activated. For co-culturing WT and GOT1^−/− CD8⁺ T cells, cells with different congenic markers were pre-labelled with Cell Proliferation Dye-eFluor450, then activated alone or mixed at 1:1 ratio. For cytokine production, cells were stimulated 4–6 h with phorbol myristate acetate (PMA, 50 ng mL⁻¹), Ionomycin (500 ng mL⁻¹) or peptide (100 ng mL⁻¹) in the presence of Golgi-stop (BD Biosciences) for intracellular staining.