EdU incorporation is an alternative non-radioactive assay to [(3)H]thymidine uptake for in vitro measurement of mice T-cell proliferations.
BACKGROUND
T lymphocyte proliferations can be measured by [(3)H]thymidine incorporation. However, many labs avoid this technique because of the need to use radioactive substrates. In addition, [(3)H]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 [(3)H]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 [(3)H]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 [(3)H]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.


CONCLUSIONS
EdU-Click technology accurately measures proliferation of murine T lymphocyte and can be used as an alternative to [(3)H]thymidine assays. The EdU-Click technology also allows identification of proliferating cells.
Yu
Yongmao
Y
The Division of Allergy and Clinical Immunology and Developmental and Stem Cell Biology Program, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada.
Arora
Alka
A
Min
Weixian
W
Roifman
Chaim M
CM
Grunebaum
Eyal
E
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000494:pharmacology; D017252:CD3 Complex / Q000276:immunology; D049109:Cell Proliferation; D003300:Copper / Q000737:chemistry* / Q000494:pharmacology; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D051379:Mice; D018345:Mice, Knockout; D011683:Purine-Nucleoside Phosphorylase / Q000235:genetics / Q000276:immunology; D013154:Spleen / Q000166:cytology / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D013936:Thymidine / Q000737:chemistry* / Q000494:pharmacology; D014316:Tritium / Q000737:chemistry / Q000494:pharmacology
D000906:Antibodies; D017252:CD3 Complex; D014316:Tritium; D003300:Copper; D011683:Purine-Nucleoside Phosphorylase; C031086:5-ethynyl-2'-deoxyuridine; D013936:Thymidine; D003857:Deoxyuridine