Abstract
A novel method of measuring cell proliferation by incorporation of the nucleic acid analog 5-ethynyl 2′deoxyuridine (EdU) into proliferating cells is demonstrated. The copper (I) catalyzed azide-alkyne [3+2] cycloaddition reaction (“click” reaction), is shown to give rapid S-phase signal by flow cytometry and clear differentiation of newly dividing cells by microscopic imaging. Click-based EdU detection of newly synthesized DNA has clear advantages over traditional antibody based 5-bromo 2′deoxyuridine (BrdU) detection. The assay time is reduced to two hours, has equivalent sensitivity, and does not require the harsh and cumbersome fixation and labeling protocols used for BrdU assay. In addition, the EdU based assay can be multiplexed with antibody- based detection and shows co-localization of signal with cell proliferation marker Ki67. Cells grown in the presence of EdU do not require DNAse treatment, heat treatment, or HCl denaturation in order to efficiently react with an azide modified Alexa Fluor® 488 dye conjugate. Bivariant graphs generated by flow cytometry between EdU labeled DNA and Vibrant® Dye Cycle violet show the traditional curve shape generated from BrdU antibody based detection using HCl treatment.
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