Normal human peripheral blood cells were unstimulated (left) or stimulated for 72 hours with CD3 and CD28 Monoclonal Antibodies, Functional Grade (Product # 16-0037-85) and (Product # 16-0289-85) (right). Cells were then stained with CD25 Monoclonal Antibody, eFluor 450 (Product # 48-0259-42) and CD278 (ISA-3) Monoclonal Antibody, NovaFluor Red 660. Total viable cells in the lymphocyte gate were used for analysis, as determined by LIVE/DEAD Blue (Product # L34962). Data was acquired on a 5-laser Cytek Aurora and unmixed with autofluorescence extraction.