Multicolor flow cytometry analysis of the proliferations of T-lymphocyte subsets in vitro by EdU incorporation

EdU (5-ethynyl-2'-deoxyuridine) incorporation has proved advantageous in the studies of cell kinetics, DNA synthesis, and cellular proliferation in vitro and in vivo compared to [3H]thymidine incorporation and BrdU (5-bromo-2'-deoxyuridine) incorporation. Here, we describe a method that combines EdU incorporation and immunostaining with flow cytometric analysis to detect the proliferations of T lymphocyte subsets in vitro and optimized the assay's conditions. We found that the number of EdU+ cells were associated with EdU concentration, incubation time, and the volume of Click reaction solution, the best EdU concentration 1050 mu M, the optimal incubation time 812 h and the proper volume of Click volume 100 mu l for labeling 1 x 106 lymphocytes. Fixation was better to be performed before permeabilization, not together with. Furthermore, the permeabilization detergent reagent, PBS with 0.05% saponin was better than Tris buffer saline (TBS) with 0.1% Triton X-100. In addition, sufficient wash with PBS with 0.05% saponin has no influence on the staining of EdU+ cells. Also, the lymphocytes incorporating EdU could be stored at 4 degrees C, -80 degrees C, and in liquid nitrogen up to 21 days. The present study will aid in optimization of flow cytometry assay to detect the proliferations of T cell subsets by EdU incorporation and the labeling of cell surface antigens. (C) 2012 International Society for Advancement of Cytometry