Rpmi medium

Assays with human primary T cells were conducted by Pharmaron. Human peripheral blood mononuclear cells for these assays were sourced commercially from Sailybio (Cat. No. SLB-HP200A), with ethical approvals and informed consent for the collection. Human primary T cells were isolated from fresh peripheral blood mononuclear cells (Sailybio) using the human Pan T Cell Isolation Kit (Miltenyi Biotec) and resuspended in RPMI medium 1640 containing 10% FBS, 1% penicillin/streptomycin, and 10 ng/mL human IL-2 (R&D Systems). Mouse primary T cells were isolated from fresh spleen cells using the mouse Pan T Cell Isolation Kit (Miltenyi Biotec) and resuspended in RPMI medium 1640 containing 10% FBS, 1% penicillin/streptomycin, and 10 ng/mL mouse IL-2 (R&D Systems). Compounds R80 and T35 were added as appropriate to various concentrations. Cells were seeded into 96-well plates and incubated at 37 °C and 5% CO₂ for 1 h. Human or mouse anti-CD3/anti-CD28 Dynabeads (Thermo Fisher Scientific) (78 (link)) and 100 μM cytidine (where appropriate) were then added, and cells were incubated at 37 °C and 5% CO₂ for 5 d. CTG reagent (Promega) was added to cells and incubated at room temperature for 30 min, after which luminescence was recorded using a Perkin-Elmer Envision microplate reader.