Gastro-Immuno Research Laboratory, Department of Medicine V (Hepatology and Gastroenterology), Aarhus University Hospital, Denmark.
J Immunol Methods. 2010 Feb 28;353(1-2):141-4. doi: 10.1016/j.jim.2009.11.019. Epub 2009 Dec 1.
CD4+CD25+ regulatory T cells (Tregs) mediate tolerance towards self antigens and prevent the development of autoimmunity. Treg function is typically evaluated by the ability to suppress proliferation and cytokine production of co-cultured CD4+CD25- T cells in Treg suppression assays. Purified Tregs are often obtained using the "Regulatory T Cell isolation kit" from Miltenyi Biotech. Separation can be performed manually using single columns or automated using the AutoMACS Cell Separator. In this Technical Note we present a serious pitfall in Treg suppression assays when evaluating magnetically separated CD4+CD25+ T cells obtained by the "Regulatory T Cell isolation kit" and AutoMACS Cell Separator. The AutoMACS Running Buffer recommended by the manufacturer for separation contains Ethylene-Diamine-Tetra-Acetate (EDTA). Here we show that even minute traces of EDTA in the CD4+CD25+ T cell fraction mediate significant suppression of CD4+CD25- T cell proliferation. The suppressive effect of EDTA is dose-dependent and mimics Treg mediated suppression of CD4+CD25- T cell proliferation. The influence of EDTA can be eliminated by thorough washing of the CD4+CD25+ T cell fraction following the separation. Our observation may have implications for other cell separation methods using EDTA-containing buffers.
CD4+CD25+ 调节性 T 细胞 (Tregs) 介导对自身抗原的耐受性，并防止自身免疫的发展。Treg 功能通常通过在 Treg 抑制测定中抑制共培养的 CD4+CD25-T 细胞的增殖和细胞因子产生的能力来评估。通常使用 Miltenyi Biotech 的“调节性 T 细胞分离试剂盒”来获得纯化的 Tregs。分离可以手动使用单个柱子进行，也可以使用 AutoMACS 细胞分离器自动进行。在本技术说明中，我们提出了当评估使用“调节性 T 细胞分离试剂盒”和 AutoMACS 细胞分离器获得的磁性分离的 CD4+CD25+T 细胞时，Treg 抑制测定中的一个严重缺陷。制造商推荐用于分离的 AutoMACS 运行缓冲液含有乙二胺四乙酸 (EDTA)。在这里，我们表明 CD4+CD25+T 细胞群中即使微量的 EDTA 也会介导对 CD4+CD25-T 细胞增殖的显著抑制。EDTA 的抑制作用呈剂量依赖性，并模拟 Treg 介导的 CD4+CD25-T 细胞增殖抑制。通过在分离后彻底洗涤 CD4+CD25+T 细胞群，可以消除 EDTA 的影响。我们的观察结果可能对使用含 EDTA 缓冲液的其他细胞分离方法具有重要意义。
