Multiparameter flow cytometric approach for simultaneous evaluation of proliferation and cytokine-secreting activity in T cells responding to allo-stimulation.

Abstract:

:We report a method combining mixed lymphocyte reaction (MLR) using a carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeling technique, intracellular cytokine immunofluorescence staining (ICIS), and multiparameter flow cytometry for simultaneous determination of proliferation and cytokine-secreting activity in T cells responding to allo-stimulation. C57BL/6 (B6) mice and Balb/c mice were used in the experiments. CFSE-labeled responder splenocytes were cultured with irradiated stimulator splenocytes, followed by ICIS. In both the Balb/c stimulator-versus-B6 responder (Balb/c-vs.-B6) and the B6-vs.-Balb/c allogeneic combinations, interleukin (IL)-2 secreting cells and interferon (IFN)-gamma secreting cells were identified predominantly in proliferating CD4+ and CD8+ T cell fractions, respectively. The suitability of this method was proven by demonstrating a close relationship between the values of cytokines in culture supernatants (that were determined by Cytometric Bead Array assay) and indexes for cytokine-production (that were obtained by multiplying the percentage of cytokine-producing cells in T cells and mean fluorescence intensity of cytokine-staining determined by the combined MLR and ICIS).

journal_name

Immunol Invest

authors

Tanaka Y,Ohdan H,Onoe T,Asahara T

doi

10.1081/imm-120038079

subject

Has Abstract

pub_date

2004-08-01 00:00:00

pages

309-24

issue

3

eissn

0882-0139

issn

1532-4311

journal_volume

33

pub_type

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