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

Immunol Invest. 2004 Aug;33(3):309-24. doi: 10.1081/imm-120038079.


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).

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cytokines / metabolism*
  • Female
  • Flow Cytometry / methods*
  • Fluoresceins / metabolism
  • Fluorescent Antibody Technique
  • Lymphocyte Activation / immunology*
  • Lymphocyte Culture Test, Mixed
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Staining and Labeling / methods
  • Succinimides / metabolism
  • T-Lymphocytes / immunology*


  • 5-(6)-carboxyfluorescein diacetate succinimidyl ester
  • Cytokines
  • Fluoresceins
  • Succinimides