Forty-eight hours after activation, T cells were added with 1 µCi/well of [3H]-thymidine (American Radiolabeled Chemicals, St. Louis, MO, USA) for 16 hours and harvested, and the radioactivities were measured in a scintillation β-counter (MicroBeta, Trilux, PerkinElmer, Turku, Finland). All samples were quadripicated.
For apoptosis analysis, T cells were cultured in a 24-well plate in a CO2 incubator at 37℃, added with various concentrations of vitamin C up to 1 mM for 2 hours, and activated with PMA/ionomycin. After 24 hours, 1×106 cells were suspended in Annexin V binding buffer (BD Pharmingen, Franklin Lakes, NJ, USA) and incubated with Annexin V (BD Pharmingen) for 15 minutes. Propidium iodide (PI) (BD Pharmingen) was added just before flow cytometric analysis.
For the analysis of activation marker expression, human T cells in a fluorescence-activated cell sorting (FACS) tube (1×106/tube) were washed twice in cold PBS containing 0.05% bovine serum albumin (Amresco, Solon, OH, USA), incubated with anti-human CD69-FITC (BD Pharmingen) and anti-human CD25-FITC (BD Pharmingen) antibodies, 0.2 µg each on ice, and subjected to flow cytometric analysis.
Flow cytometric analysis was performed using FACS Calibur (BD Biosciences, San Diego, CA, USA).
For apoptosis analysis, T cells were cultured in a 24-well plate in a CO2 incubator at 37℃, added with various concentrations of vitamin C up to 1 mM for 2 hours, and activated with PMA/ionomycin. After 24 hours, 1×106 cells were suspended in Annexin V binding buffer (BD Pharmingen, Franklin Lakes, NJ, USA) and incubated with Annexin V (BD Pharmingen) for 15 minutes. Propidium iodide (PI) (BD Pharmingen) was added just before flow cytometric analysis.
For the analysis of activation marker expression, human T cells in a fluorescence-activated cell sorting (FACS) tube (1×106/tube) were washed twice in cold PBS containing 0.05% bovine serum albumin (Amresco, Solon, OH, USA), incubated with anti-human CD69-FITC (BD Pharmingen) and anti-human CD25-FITC (BD Pharmingen) antibodies, 0.2 µg each on ice, and subjected to flow cytometric analysis.
Flow cytometric analysis was performed using FACS Calibur (BD Biosciences, San Diego, CA, USA).