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AIM-positive CD4 T cells and CD8 T cells were detected at least 3 h after stimulation. Furthermore, examination of the multiple combination of each marker revealed that minimum contribution of CD134 on the total pool of AIM-positive cells at this setting, suggesting the essential and non-essential AIMs to maximize the detected number of AIM-positive cells. Taken together, this optimized method will be a useful tool for the simultaneous monitoring the T-cell receptor stimulation-dependent activation of CD4 and CD8 T cells