TUNELAssay

The TUNEL assay, short for terminal deoxynucleotidyl transferase dUTP nick end labeling, is a method used to detect DNA fragmentation resulting from apoptotic signaling cascades. It is widely used in histology and cytometry to identify apoptotic cells in tissue sections or cell populations.

Principle: In cells undergoing apoptosis, endonucleases cleave genomic DNA, creating numerous 3'-OH ends. The assay exploits the enzyme terminal deoxynucleotidyl transferase (TdT), which catalyzes the addition of labeled nucleotides to these ends. The incorporated labels can be detected with fluorescence, enzymes, or chromogenic reporters, enabling visualization by microscopy or quantification by flow cytometry or image analysis.

Procedure overview: Samples are fixed and permeabilized to allow enzyme access. TdT is applied with labeled dUTP or analogs; after incubation, unincorporated label is washed away, and labeled cells are detected. Variants include in situ TUNEL on tissue sections and flow cytometry–adapted TUNEL. Labels may be fluorescent dyes, biotin, or digoxigenin, detected by appropriate reporters.

Applications: TUNEL is commonly used to assess apoptosis in tissues and cultured cells, study development, cancer biology, neurodegenerative diseases, stroke, myocardial infarction, and treatment responses. It can be combined with counterstaining or other markers to identify cell types or cell cycle phase.

Limitations: DNA fragmentation can arise from necrosis, mechanical injury, or fixation; thus TUNEL is not absolutely specific for apoptosis. False positives may occur with aggressive tissue processing or non-apoptotic DNA breaks; proper controls and complementary assays are recommended. Autofluorescence and labeling efficiency can affect results; interpretation should consider experimental context.