TUNEL assays detect DNA fragmentation in live and fixed cells, which often occur in late-stage apoptosis.  Internucleosomal DNA fragmentation caused by activated endonucleases is universally regarded as the biochemical hallmark of apoptosis. Cells containing these DNA strand breaks can be identified with the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. This well-established in situ staining method relies on the enzyme terminal deoxynucleotidyl transferase (TdT) to catalyze the incorporation of modified dUTPs to the 3'-hydroxyl termini of fragmented DNA. Depending upon the choice of modified dUTP - BrdUTP, biotin-dUTP, or a fluorescein-dUTP - apoptotic cells can be identified and measured using various detection strategies and systems, including fluorescence microscopy, flow cytometry, and fluorescence-based microplate studies.

AAT Bioquest offers several fluorimetric TUNEL assays optimized for in situ apoptosis detection in live cells or fixed cells and tissue samples. These products can be combined with other cell-based viability and apoptosis assays to provide a comprehensive assessment of cell health, assess toxicity and safety of drug candidates, or analyze cancer and disease-related cellular changes.  

Principles and mechanism

During the later stages of apoptosis, caspase-activated endonucleases cleave genomic DNA into oligonucleosomal fragments (~180-200 base pairs long). Using the TUNEL assay, the exposed 3'-OH termini of these breaks can be marked with modified dUTPs for subsequent visualization and quantification of apoptotic cells in situ. This is generally done either directly using dye-modified dUTP or indirectly using BrdUTP and antibody conjugates against BrdUTP. Regardless of the labeling strategy, both require the enzyme terminal deoxynucleotidyl transferase (TdT) to catalyze the incorporation of the modified dUTPs to the 3'-OH termini.

Detection of DNA fragments by this assay requires sample fixation using a crosslinking reagent such as 4% paraformaldehyde (avoid ethanol-based fixatives as it hinders the extraction of small DNA fragments) and sample permeabilization. Both steps are critical to the TUNEL assay as it facilitates the entry of exogenous TdT and antibody conjugates against BrdUTP. Keep in mind that several variables influence the staining kinetics of the TUNEL assay. Some of these variables, including reagent concentration, fixation of the sample, and accessibility of DNA strand breaks, may vary between tissue or cell sample types. Standardizing the TUNEL assay using samples with positive and negative apoptosis controls can alleviate these potential influences and reduce false positives or negative results.