These cell separation methods were further standardized and automated, using the Biomek FX robotic platform for achieving improved uniformity of the cell preparations as outlined below. For the direct isolation of CD8 T cells from fresh blood, antibody labeled with 3.5 mm diameter super-paramagnetic beads were used. Before each experiment, the beads were washed once with PBS buffer with 1% FBS to Pazopanib remove the sodium azide stabilizing agent from the bead buffer. The beads were then suspended in an equivalent volume of PBS buffer with 1% FBS and placed into a 2 ml sample vial. The volume of each subjects�� whole blood sample drawn was recorded and used later for CD8 cell concentration determination. Following a wash step, the blood samples were re-suspended to 15 ml volume with HBSS buffer in 50 ml conical tubes and placed in a custom tube holder on the instrument for processing and transfer to the 2 ml sample vials. Following attachment and CD8 cell isolation, the beads were detached from the cells using the detach-a-bead reagent supplied in the CD8 isolation kit. This reagent consists of a polyclonal antibody directed against the antigen recognition site of the CD8 antibody coated on the magnetic beads. This reagent detaches the antibody/bead complex from the cells by means of competition for the CD8 antibody binding site, essentially leaving a virgin cell. The entire isolation process was performed on ice except for the steps of removing the attached beads. All lymphocyte cell isolations were analyzed by flow cytometry using a FACSCalibur. Cell quality was determined by quantifying cell viability, purity and yield. For most flow cytometry studies, flow gates were set ����open���� for inclusion of all cells. The open gate included cells of all sizes but excluded cell debris, red blood cells, fragmented cells, and apoptotic bodies. The open gate was chosen because cells undergoing cell death, especially by apoptosis, can be detected based on changes in light scattering properties and cell size. For measurement of T-cell death, Propidium Iodide staining was used in LEE011 combination with AnnexinV. Death was labeled as early or late apoptosis or necrosis. T-cell subsets were identified using anti-CD3 antibody. This subset-specific antibody was linked to phycoerythrin. For the detection of the rare autoreactive T cells of type 1 diabetes, we used two methods.