title: Use of SNARF-1 to measure murine T cell proliferation in vitro and its application in a novel regulatory T cell suppression assay.
abstract: The green fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) has been used to track the proliferation of T cells in vitro. Such assays often incorporate more than one population of cells, but the paucity of alternative, spectrally distinct dyes suitable for measuring proliferation has hampered the simultaneous tracking of multiple cell populations; furthermore, CFSE is not compatible with green fluorescent protein (GFP), used to identify T cells in various transgenic mice. We have therefore validated the use of the far red dye seminaphthorhodafluor-1 (SNARF)-1 - originally developed to measure intracellular pH - to track murine T cell proliferation in vitro, demonstrating its ability to distinguish multiple cycles of proliferation over three days in a similar fashion to CFSE. The small changes in fluorescence emission attributed to intracellular alkalinisation of proliferating T cells have minimal impact on the ability of SNARF-1 to track cell division and this dye induces minimal cell death at the concentration used in this application. On the basis of these results, we have developed a novel in vitro murine T cell suppression assay, in which the proliferation of both conventional T cells (Tcons) stained with SNARF-1 and regulatory T cells (Tregs) stained with CFSE can be measured simultaneously. We have also demonstrated that SNARF-1 may be used to stain Tcons in assays of suppression involving 'designer' Tregs, generated by the transduction of CD4(+) T cells with constructs encoding the Foxp3(gfp) fusion protein.
authors.affiliation: Regulatory T Cell Laboratory, Infection and Immunity Research Group, Department of Veterinary Clinical Sciences, The Royal Veterinary College, Camden Campus, Royal College Street, London, NW1 0TU, United Kingdom.
authors.forename: Yogesh
authors.initials: Y
authors.lastname: Singh
authors.forename: Julian
authors.initials: J
authors.lastname: Dyson
authors.forename: Oliver A
authors.initials: OA
authors.lastname: Garden
mesh_terms: D000818:Animals; D001578:Benzopyrans / Q000378:metabolism*; D049109:Cell Proliferation; D002469:Cell Separation; D002478:Cells, Cultured; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism*; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D007108:Immune Tolerance; D007118:Immunoassay / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D009284:Naphthols / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism*; D013388:Succinimides / Q000378:metabolism*; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D019076:Transgenes / Q000235:genetics
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D001578:Benzopyrans; D005452:Fluoresceins; D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; D009284:Naphthols; D012235:Rhodamines; D013388:Succinimides; C091666:seminaphthorhodaminefluoride