Nine-color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part I: Panel design by an empiric approach.

Polychromatic flow cytometry offers the unprecedented ability to investigate multiple antigens per cell. Unfortunately, unwanted spectral overlaps and compensation problems increase when more than four colors are used, but these problems can be minimized if staining combinations are chosen carefully. We used an empiric approach to design, test and identify six-color T cell immunophenotyping reagent panels that can be expanded to include three or more functional or other markers in the FITC, PE, and APC channels without significant spectral limitations. Thirty different six-color T cell surface antigen reagent panels were constructed to identify major T cell subsets and maturational subtypes as defined by CCR7 and CD45RA expression, while excluding monocytes, B and non-viable cells. Staining performance of each panel was compared on cryopreserved cells from a single healthy donor recorded on a multiparameter cell sorter. Ten of the thirty reagent panels offered reliable resolution of T cell major and maturational surface markers. Of these, two panels were selected that showed the least spectral overlap and resulting background increase in the FITC, PE, and APC channels. These channels were left unoccupied for inclusion of additional phenotypic or functional markers, such as cytokines. Careful reagent titration and testing of multiple candidate panels are necessary to ensure quality results in multiparametric measurements

Nine-color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part II: Panel performance across different instrument platforms.

Cellular immune responses elicited by vaccination are complex and require polychromatic analysis to accurately characterize the phenotype and function of rare, responding cells. Technical challenges and a lack of instrument standardization between research sites have limited the application of polychromatic cytometry in multicenter clinical trials. Two previously developed six-color T cell subset immunophenotyping reagent panels deliberately designed to accommodate three additional low frequency functional measurements were compared for their reproducibility of staining across three different flow cytometers. We repeatedly measured similar T cell subset frequencies between the two reagent panels and across the three different cytometers. Spectral overlap reduced sensitivity in two of the three open measurement channels (PE [IL-2] and APC [IFN gamma]) for one reagent combination, particularly in subsets with low cytokine expression. There was no significant interassay variation for measurements across instrument platforms. Careful panel design will identify reagent combinations that minimize spectral spillover into channels reserved for cytokine measurement and comparable results can be achieved using different cytometers, however, it is important to establish standardized quality control procedures for each instrument to minimize variation between cytometers

Search for a low mass neutral Higgs boson in Z0 decay

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