EdU incorporation is an alternative non-radioactive assay to [3H]thymidine uptake for in vitro measurement of mice T-cell proliferations

Rationale: T lymphocyte proliferations can be measured by [H-3]thymidine incorporation. However, many labs avoid this technique because of the need to use radioactive substrates. In addition, [H-3]thymidine incorporation method does not permit simultaneous characterization of the proliferating cells. We developed the 5-ethynyl-2'-deoxyuridine (EdU) and Cu(I)-catalyzed cycloaddition click reaction assay to measure T-cell responses by flow cytometry. Methods: Spleen cells from normal, immune-deficient purine nucleoside phosphorylase (PNP) defective (PNP-/-) mice or PNP-/- mice with partial immune reconstitution were stimulated with anti-CD3 antibodies. The correlation (r) between [H-3]thymidine and EdU incorporations into stimulated T cells was measured and the stimulation index (SI), the ratio between stimulated and non-stimulated cells, was calculated. Flow cytometry was used to characterize the proliferating cells. Results: EdU and [H-3]thymidine incorporation into normal spleen cells were strongly correlated (r = 0.89). Following stimulation, EdU incorporation into spleen cells from normal and immune-reconstituted PNP-/- mice was significantly increased compared to PNP-/- immune-deficient mice. Immune-deficient PNP-/- mice had increased [H-3]thymidine and EdU incorporation into non-stimulated spleen cells, indicative of spontaneous proliferation. Analysis of EdU incorporation showed that the increased proliferation was due primarily to cells expressing CD3, CD4 and IgM. Conclusion: EdU-Click, technology accurately measures proliferation of murine T lymphocyte and can be used as an alternative to [H-3]thymidine assays. The EdU-Click technology also allows identification of proliferating cells. (C) 2009 Elsevier B.V. All rights reserved.