Expression of activation marker CD69 by flow cytometry

T cells and NK cells upregulate surface marker CD69 upon activation. CD69 is one of the early activation markers in T cell stimulation/ activation and thus the stimulation or activation state of the cells can be easily measured by analyzing the number/ percent of T cells expressing CD69. A comprehensive method to evaluate the expression of CD69 on the cell surface is by doing flow cytometric analysis of cells stained with a CD69 antibody. The experiment described here utilizes a Phycoerithrin (PE) conjugated CD69 antibody along with an APC CD25 antibody to determine T cell activation. The double positive cells (i.e. C69+CD25+) represent the activated cells in the population. Cells (mouse splenocytes) were stimulated by PMA (20ng/ml) and Ionomycin 1ug/ml) for 48 hours followed by staining with FITC CD8 along with CD69 PE and CD25 APC. During flow cytometric analysis, cells were initially gated on a CD8+ population and then the proportion of CD69+CD25+ were determined.