Identification of the proliferations of T-cell subsets in vitro by EdU incorporation

The purpose of the experiment is to eatablish a feasible protocol for the detection of the proliferations of T cell of T-cell subsets in vitro by EdU incorporation and to optimize the conditions.
Conclusion:
We described that an optimized technology of EdU incorporation and the labeling of cell surface antigens combined with flow cytometry analysis, which could be used to detect the proliferations of T lymphocyte subsets accurately. Different factors contribute to this method include EdU concentration, EdU incubation time, fixation, permeabilization, Click reaction, wash reagents, extent of wash, which may have significant effects on final results; therefore, these factors should be scrutinized in immunofluorescence experiments. To standardize voltage settings across samples, single stained controls were included. Voltages were adjusted such that fluorescence intensity was identical for each antibody, regardless of date of acquisition.