T Cell Proliferation Assay with Antioxidants

ovalbumin-specific or myelin basic protein-specific rat T cells were resuspended in medium + 5% FBS and seeded in a 96-well plate (5 × 10⁴ per well) in the presence of irradiated rat thymocytes (2 × 10⁶ per well), serving as antigen-presenting cells38 (link)54 55 (link)56 (link). Rat mononuclear splenocytes and human peripheral blood cells were resuspended in medium + 5% FBS and seeded in a 96-well plate (1 × 10⁵ per well)40 (link)57 (link). Cells were incubated with or without antioxidants: trolox (Sigma-Aldrich), vitamin C (Sigma-Aldrich), PEG-HCCs, or HCCs for 30 min at 37 °C and 5% CO₂. ovalbumin-specific and myelin basic protein-specific rat T cells were stimulated with 10 μg/mL ovalbumin or guinea pig myelin basic protein (Sigma-Aldrich), respectively36 (link)38 (link)58 (link). Rat mononuclear splenocytes and human peripheral blood cells were stimulated with 1 μg/mL concanavalin A or phytohemagglutinin, respectively36 (link)59 (link). Cells were cultured for 72 h at 37 °C and 5% CO₂, and 1 μCi [³H] thymidine (MP Biomedicals) was added to each well during the final 16–18 h of incubation. Cells were then lysed by freezing and DNA was harvested on fiberglass filters using a cell harvester (Inotech Biosystems International). [³H] thymidine incorporation was measured on a β-scintillation counter (Beckman Coulter).