Forward light scatter is a simple measure of T-cell activation and proliferation but is not universally suited for doublet discrimination.

Following activation by antigen, T cells enter the cell cycle in stages that can be defined with flow cytometric markers. We show that these markers include the increase of forward light scatter width (FSC-W) signal and the ratio of FSC area/peak. This change in light scatter precedes the first cell division and may reflect blast transformation. We show that the FSC-W parameter can be used, alone or in combination with other activation markers, to monitor the relative and absolute numbers of T cells responding to a proliferative stimulus. In contrast to dye dilution assays, the FSC-W method does not allow discrimination between consecutive cell divisions, but it has several advantages and could complement the dye dilution assay. Our findings also show that the routine elimination of doublets based on FSC signals may exclude proliferating T cells from the analysis.