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

Authors

  • Yanli Sun,

    1. Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China
    2. National Center for Clinical Laboratories, Beijing Hospital of the Ministry of Health, Beijing, People's Republic of China
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  • Yu Sun,

    1. Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China
    2. National Center for Clinical Laboratories, Beijing Hospital of the Ministry of Health, Beijing, People's Republic of China
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  • Guigao Lin,

    1. Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China
    2. National Center for Clinical Laboratories, Beijing Hospital of the Ministry of Health, Beijing, People's Republic of China
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  • Rui Zhang,

    1. National Center for Clinical Laboratories, Beijing Hospital of the Ministry of Health, Beijing, People's Republic of China
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  • Kuo Zhang,

    1. National Center for Clinical Laboratories, Beijing Hospital of the Ministry of Health, Beijing, People's Republic of China
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  • Jiehong Xie,

    1. National Center for Clinical Laboratories, Beijing Hospital of the Ministry of Health, Beijing, People's Republic of China
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  • Lunan Wang,

    1. National Center for Clinical Laboratories, Beijing Hospital of the Ministry of Health, Beijing, People's Republic of China
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  • Jinming Li

    Corresponding author
    1. National Center for Clinical Laboratories, Beijing Hospital of the Ministry of Health, Beijing, People's Republic of China
    • National Center for Clinical Laboratories, Beijing Hospital of the Ministry of Health, No.1 Dahua Road, Dongdan, Beijing 100730, P R China
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Abstract

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 10–50 μM, the optimal incubation time 8–12 h and the proper volume of Click volume 100 μl for labeling 1 × 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°C, −80°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. © 2012 International Society for Advancement of Cytometry

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