Novel Click-iT® TUNEL Assay for Detection of Cell Death

Abstract

The detection of cytotoxicity and cell death is a critical aspect of toxicological profiling and the drug discovery process. Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling (TUNEL) is by far the most popular method of assessing DNA fragmentation and detection of cell death. In this communication, we demonstrate the application of a novel click chemistry based TUNEL assay for high content imaging and analysis. This assay involves the incorporation of a modified “clickable” nucleotide, ethynyl dUTP (EdUTP), by the enzyme Terminal deoxynucleotidyl Transferase (TdT) at the 3’OH end of DNA strand breaks. The single strand tail of nucleotides is then covalently conjugated to an azide modified Alexa Fluor® 488, 594, or 647 fluorescent dye using a copper (I) catalyzed azide-alkyne reaction. Using HPLC and polyacrylamide gel based assays, we observed that the incorporation of EdUTP was more efficient than other modified nucleotides such as BrdUTP, fluorescein-, BODIPY® and biotin- conjugated dUTP. The TUNEL assay was also multiplexed with antibody-based detection of other apoptosis biomarkers such as cleaved poly ADP ribosylation protein (PARP), cleaved caspase 3, and phospho-histone 2B. Multiplexing two or more biomarkers of apoptosis is necessary to unequivocally establish programmed cell death in any given cell or tissue model and this approach underscores the value of both spatial resolution and quantitative analysis possible when imaging heterogeneous populations of cells.