FTUNEL

FTUNEL, or fluorescent TUNEL, is a fluorescence-based adaptation of the TUNEL assay used to detect DNA fragmentation as an indicator of apoptosis. The technique relies on terminal deoxynucleotidyl transferase (TdT) to catalyze the addition of fluorescently labeled dUTP to exposed 3'-OH ends of fragmented DNA, producing a signal visible by fluorescence microscopy and, in some setups, quantifiable by flow cytometry or image analysis. FTUNEL enables localization of apoptotic cells within tissue sections or cell populations and can be paired with additional markers for multiparametric analysis.

Overview and development: The original TUNEL method was introduced in the early 1990s to label DNA breaks in apoptotic cells. Fluorescent variants of TUNEL, collectively referred to as FTUNEL, were developed to improve sensitivity, enable easier visualization, and facilitate compatibility with modern fluorescence imaging techniques. FTUNEL procedures are now widely used in basic and translational research to study cell death in diverse biological contexts.

Method and workflow: Typical FTUNEL workflows involve fixing and permeabilizing samples, conducting the TdT-mediated reaction with a fluorescent dUTP, and washing to remove unincorporated nucleotides. Detection is performed with fluorescence microscopy, and in some cases, flow cytometry or high-content imaging is employed. Appropriate controls are essential, including positive controls (DNA-damaged samples) and negative controls (omitting TdT or using DNase-treated tissues).

Applications and limitations: FTUNEL is used to assess apoptosis in cancer, neuroscience, cardiology, developmental biology, and toxicology, among other fields. While sensitive and capable of providing spatial context, FTUNEL can produce false positives in necrotic or highly damaged tissues and may reflect DNA fragmentation from processes other than apoptosis. Quantification requires standardized protocols, careful optimization of imaging settings, and proper controls.

See also: TUNEL assay, apoptosis, DNA fragmentation, TdT.