Rpmi 1640 with l glutamine

Naive CD8 T (CD44^lowCD62L^high) cells were prepared using a Naive CD8⁺ T-cell Isolation kit (cat#130-096-543; Miltenyi Biotec, San Diego, CA, USA). Naive CD8 T cells (1.5 × 10⁶) were stimulated with immobilized anti-TCR-β mAb (3 μg/ml, H57-597; BioLegend) and anti-CD28 mAb (1 μg/ml, 37.5; BioLegend) for 2 days in the presence of IL-2 (10 ng/ml, Pepro Tech). The cells were then transferred to a new plate and further cultured in the presence of IL-2 (10 ng/ml). The cells were cultured in RPMI 1640 with l-glutamine (cat#189-02025; Wako Chemicals) supplemented with 10% fetal bovine serum, 2 mM l-glutamine (16948-04; Nacalai Tesque, Kyoto, Japan), 1 mM sodium pyruvate (cat#06977-34; Nacalai Tesque), 1% MEM nonessential amino acids (cat#06344-56; Nacalai Tesque), 10 mM HEPES (cat#15630-080; Thermo Fisher Scientific, Waktham, MA, USA), 55 μM 2-Mercaptoethanol (cat#21985-023; Thermo Fisher Scientific), and 1% penicillin-streptomycin (cat#26253-84; Nacalai Tesque). For glutamine-deprived conditions, the cells were cultured in RPMI 1640 without l-glutamine (cat#183-02165; Wako Chemicals) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 1% MEM nonessential amino acids, 10 mM HEPES, 55 μM 2-Mercaptoethanol, and 1% penicillin-streptomycin.