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

Abstract

Rationale T lymphocyte proliferations can be measured by [ 3H]thymidine incorporation. However, many labs avoid this technique because of the need to use radioactive substrates. In addition, [ 3H]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 [ 3H]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 [ 3H]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 [ 3H]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 [ 3H]thymidine assays. The EdU-Click technology also allows identification of proliferating cells.