title: Enhancing the sensitivity of assays to detect T cell reactivity: the effect of cell separation and cryopreservation media.
abstract: The identification of sensitive assay formats capable of distinguishing islet autoreactive T cells directly ex vivo in blood is a major goal in type 1 diabetes research. Whereas much effort has been made to identify suitable assay formats, relatively little attention has been paid to optimizing the quality of cell preparations used. To address this issue we investigated the role of two key variables in the preparation of peripheral blood mononuclear cells (PBMCs): the separation media used in density gradient centrifugation and the method of cryopreservation. PBMCs were prepared from a single individual using different protocols and tested for responses to suboptimal concentrations of tetanus toxoid, representing a low frequency recall response. Significant differences were observed in T cell responses dependent upon the selection of the separation media and cryopreservation methods used, indicating that relatively small differences in preparation of PBMCs can have measurable effects on assay sensitivity.
authors.affiliation: Department of Immunobiology, Guy's, King's & St Thomas' School of Medicine, 2nd Floor, New Guy's House, Guy's Hospital London SE1 9RT, UK. timothy.tree@kcl.ac.uk
authors.forename: Timothy I M
authors.initials: TI
authors.lastname: Tree
authors.forename: Bart O
authors.initials: BO
authors.lastname: Roep
authors.forename: Mark
authors.initials: M
authors.lastname: Peakman
mesh_terms: D049109:Cell Proliferation; D002469:Cell Separation; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D015925:Cryopreservation; D016207:Cytokines / Q000378:metabolism; D005434:Flow Cytometry; D019085:Fluorescent Antibody Technique, Direct; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007371:Interferon-gamma / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D008213:Lymphocyte Activation; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D013696:Temperature; D013745:Tetanus Toxoid / Q000276:immunology; D013997:Time Factors
chemical_list: D016207:Cytokines; D013745:Tetanus Toxoid; D007371:Interferon-gamma