Multiparameter flow cytometric approach for simultaneous evaluation of proliferation and cytokine-secreting activity in T cells responding to allo-stimulation.
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).
Tanaka
Yuka
Y
Programs for Biomedical Research, Department of Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
Ohdan
Hideki
H
Onoe
Takashi
T
Asahara
Toshimasa
T
D000818:Animals; D016207:Cytokines / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D008213:Lymphocyte Activation / Q000276:immunology*; D007959:Lymphocyte Culture Test, Mixed; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D013194:Staining and Labeling / Q000379:methods; D013388:Succinimides / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology*
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D016207:Cytokines; D005452:Fluoresceins; D013388:Succinimides