1 4 dithiothreitol dtt

All procedures involving mice were performed according to protocols approved by the Laboratory Animal Care and Use Committees of Wright State University, Kumamoto University and University of the Ryukyus according to the NIH guidelines. Mouse spleen dissection and T cell isolation was performed as previously described in detail⁵³ . Each spleen was weighed on a scale shortly after dissection. For splenocyte culture, the spleen was crushed between two glass slides to release splenocytes which were passed through 40 µm nylon cell strainer (Fisher Scientific, Fair Lawn, NJ) and cultured in RPMI-1640 (Lonza, Walkersville, MD) containing 2 mM L-glutamine, supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA), 1 mM 1,4-dithiothreitol (DTT; Research Products International, Mount Prospect, IL), 50 IU/ml penicillin and 50 µg/ml streptomycin (MP Biomedicals, Irvine, CA). T cells were purified from splenocytes using nylon wool fiber columns (Polysciences, Inc., Warrington, PA) and cultured in supplemented RPMI-1640. The purified T-cell preparation had approximately 42% CD4 and 35% CD8 cells as determined by flow cytometry (data not shown).