Multicolor flow cytometry analysis of the proliferations of T-lymphocyte subsets in vitro by EdU incorporation
Multicolor flow cytometry analysis of the proliferations of T-lymphocyte subsets in vitro by EdU incorporation
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EdU Incorporation 对 T 淋巴细胞亚群体外增殖的多色流式细胞术分析
DOI：
10.1002/cyto.a.22113
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发表时间：
2012-10-01
期刊：
影响因子：
3.7
通讯作者：
Li, Jinming
中科院分区：
文献类型：
--
作者：
Sun, Yanli;Sun, Yu;Li, Jinming
EDU(5-乙炔基-2‘-脱氧尿苷)掺入在体内外研究细胞动力学、DNA合成和细胞增殖方面优于[~3H]胸腺嘧啶核苷和BrdU(5-溴-2’-脱氧尿苷)掺入。在这里，我们描述了一种结合EDU掺入和免疫染色与流式细胞术分析相结合的方法来检测体外T淋巴细胞亚群的增殖，并优化了检测条件。我们发现EDU+细胞的数量与EDU浓度、孵育时间和Click反应液的体积有关，标记1×10~6淋巴细胞的EDU最佳浓度为1050mM，孵育时间为812h，Click体积为100mU L为宜。固定宜在通透前进行，不宜与之同时进行。此外，含0.05%皂苷的透透性洗涤剂PBS优于含0.1%Triton X-100的Tris缓冲盐水。此外，用含0.05%皂苷的PBS充分洗涤对EDU+细胞的染色没有影响。此外，含有EDU的淋巴细胞可以在4摄氏度、-80摄氏度和液氮中保存长达21天。本研究有助于优化流式细胞仪检测EDU掺入法检测T细胞亚群增殖及细胞表面抗原标记的方法。(C)2012年国际细胞测量促进会
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