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

TitleNine-color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part I: Panel design by an empiric approach.
Publication TypeJournal Article
Year of Publication2008
AuthorsMcLaughlin BE, Baumgarth N, Bigos M, Roederer M, De Rosa SC, Altman JD, Nixon DF, Ottinger J, Oxford C, Evans TG, Asmuth DM
JournalCytometry A
Volume73
Issue5
Pagination400-10
Date Published2008 May
ISSN1552-4930
KeywordsAntibodies, Monoclonal, Antigens, Differentiation, T-Lymphocyte, Cytokines, Flow Cytometry, Fluorescent Dyes, Humans, Immunophenotyping, Indicators and Reagents, Reproducibility of Results, Sensitivity and Specificity, Staining and Labeling, T-Lymphocyte Subsets
Abstract

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.

DOI10.1002/cyto.a.20555
Alternate JournalCytometry A
PubMed ID18383316
Grant ListAI051999 / AI / NIAID NIH HHS / United States
C06RR-12088-01 / RR / NCRR NIH HHS / United States
R01 AI051999-03 / AI / NIAID NIH HHS / United States