Ethylene-Diamine-Tetra-Acetate (EDTA) mimics the effect of regulatory T cells in suppression assays: a potential pitfall when using AutoMACS-separated cells.
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.
Dige
Anders
A
Gastro-Immuno Research Laboratory, Department of Medicine V (Hepatology and Gastroenterology), Aarhus University Hospital, Denmark. andedige@rm.dk
Hvas
Christian Lodberg
CL
Kelsen
Jens
J
Deleuran
Bent
B
Dahlerup
Jens F
JF
Agnholt
Jørgen
J
D016477:Artifacts; D002021:Buffers; D016022:Case-Control Studies; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D018920:Coculture Techniques; D003424:Crohn Disease / Q000276:immunology*; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology*; D006801:Humans; D018189:Immunomagnetic Separation; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D053645:Interleukin-2 Receptor alpha Subunit / Q000032:analysis; D015203:Reproducibility of Results; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects* / Q000276:immunology
D002021:Buffers; C508017:IL2RA protein, human; D007166:Immunosuppressive Agents; D053645:Interleukin-2 Receptor alpha Subunit; D004492:Edetic Acid