Apoptosis Assays-TUNEL Assay Detect in Situ DNA Strand Breaks

Activation of endonucleases that cleave chromosomal DNA preferentially at internucleosomal sections is a hallmark of apoptosis. DNA fragmentation revealed by the presence of a multitude of DNA strand breaks, therefore, is considered to be the gold standard for identification apoptotic cells. Different methods have been developed to accurately assess DNA damage and fragmentation in cells and tissues, which represents a characteristic of late stage apoptosis. Two widely-used assays to detect DNA fragmentation are Single Cell Gel Electrophoresis–also known as Comet assay (Part 1), and TUNEL [Terminal deoxynucleotidyl Transferase (TdT)-mediated dUTP Nick-End Labeling] assay.

Part2: TUNEL assay detect in situ DNA strand breaks

In 1992, three Israeli scientists proposed a method for in situ detection of segmented DNA for the first time. However, for years there has been a debate about its accuracy, due to problems in the assay which caused necrotic cells to be inappropriately labeled as apoptotic cells. The method has subsequently been improved dramatically, based on labeled (X) deoxyuridine triphosphate nucleotides (X-dUTPs) to 3'-OH termini of DNA strand breaks in situ with the use of exogenous terminal deoxynucleotidyl transferase (TdT), and if performed correctly should only identify cells in the last phase of apoptosis. New methods incorporate the dUTPs modified by fluorophores or haptens, including biotin or bromine, which can be detected directly in the case of a fluorescently-modified nucleotide (i.e., fluorescein-dUTP), or indirectly with streptavidin or antibodies, if biotin-dUTP or BrdUTP are used, respectively.

Figure 1. Schematic illustration of DNA strand breaks labeling with Br-dUTP utilizing exogenous terminal deoxynucleotidyl transferase (TdT)

The assay sensitivity strongly depends on the incorporation efficiency of the modified dUTP that is influenced by size/bulkiness of the attached label. A number of fluorescently labeled dUTPs , biotinylated dUTPs or digoxigenylated dUTPs are generally suitable substrates for TdT, but dUTP labeled with smaller labels such as bromine (BrdUTP) or an alkyne group (EdUTP) have been demonstrated to exhibit a higher incorporation efficiency and thus higher sensitivity in TUNEL assays probably due to minimal sterical hindrance.

However, TUNEL false positivity may result from necrotic cell death (at least in some cases), as well as from inappropriate processing of samples, which may occur, for example, during sectioning. For these reasons, although in many cases (and in particular in some disease models), TUNEL remains the only method for investigating apoptosis in situ, whenever possible, researchers should include appropriate positive and negative controls and should corroborate the results of TUNEL by at least one independent experimental approach.