Autoreactive T cells for diverse human diseases undergo accelerated apoptosis when exposed to TNF unlike normal human T cells that show a small amount of proliferation. A 96 well plate based, WST-1 assay , was used to confirm cell death versus viability. This is a cell proliferation assay that indirectly measures cell death. The isolated CD8 T lymphocytes were plated into 96 well U-bottom plate at a cell concentration of 100,000 cells/well. Cells were cultured overnight at 26uC in RPMI media with 1% heat inactivated fetal calf serum. T cells were then treated with TNF for 1 hr. After 1 hour of exposure to TNF, the WST-1 reagent was added according to the manufacturers instructions. The cleavage of WST-1, by metabolically active cells, was measured by a DTX 880 Spectrophotometer at a wavelength of 405 nm. Each experiment was performed in triplicate. Test medium was used as background control. Data were presented as a percent of proliferation compared to untreated cells. Throughout this text we use the words pass and fail with respect to this assay and this will mean that if killing is observed in a diabetic sample it passes the test. CD8 T cells were isolated using magnetic bead separation from fresh human blood within 1 hour of blood donation. For isolation buffer we used Hank��s Buffered Salt Solution without calcium and magnesium containing 2% heat-inactivated Fetal GDC-0879 Bovine Serum and dasatinib. Isolated cells were stained with phycoerythrinlabeled HLA-A2 Fingolimod Tetramers containing fragments of the islet specific peptide IGRP. For negative controls we used tetramers containing fragments of the breast cancer peptide Her-2/neu. The IGRP tetramers were produced by the NIH Tetramer Core Facility whereas the Her-2/neu tetramers were purchased from Beckman Coulter. Staining was in the dark for 30 min and the cells were co-stained for FITC-CD8 during the last 10 minutes. The cells were then washed in HBSS with 2% heat-inactivated FBS and kept on ice until flow cytometry. A minimum of 25,000 CD8 T cells were analyzed on a Becton Dickinson FACSCalibur using the CellQuest acquisition program. Separate samples of cells labeled with FITC-CD8 and Propidium Iodine were analyzed to determine purity and viability for each preparation. The results were analyzed using either no gating, or gating on the main single cell CD8 population, thus excluding debris, dead or apoptotic cells, and clustered cells.