TUNEL Assays

During the later stages of programmed cell death, or apoptosis, DNA becomes highly fragmented. This fragmentation provides an opportunity to attach a modified dUTP to the 3’-OH end of the damaged DNA using the enzyme terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction. The modified dUTP, such as BrdUTP or EdUTP, can then be detected using a range of different detection strategies. Antibodies against BrdUTP have been used for indirect detection whereas direct detection can be achieved by incorporating biotin- or fluorescently-modified nucleotide (i.e., biotin-dUTP or fluorescein-dUTP). The dUTP modification, EdUTP allows even more flexibility in the detection strategies using the “click” chemistry reaction. The TUNEL assay has become a widely used in situ assay for the detection of apoptosis.

Classically, TUNEL assays have employed incorporation of BrdUTP into the DNA with subsequent detection by a variety of means. Thermo Fisher Scientific has developed a modified TUNEL assay that incorporates an alkyne-modified dUTP, EdUTP (Figure 1) at the 3'-OH ends of fragmented DNA using terminal deoxynucleotidyl transferase (TdT). In the Invitrogen Click-iT TUNEL assays the detection of this incorporated nucleotide is executed using click chemistry which is a reaction is based on a copper-catalyzed azide–alkyne cycloaddition and derives its high degree of specificity from the fact that the azide and alkyne reaction partners have no endogenous representation in biological molecules, cells, tissues, or model organisms. The detection of the incorporated EdUTP can be achieved using fluorescence- or colorimetric-based strategies.

The basic steps in the Click-iT TUNEL Imaging assay are shown in Figure 2. After samples are treated, cells or tissue are fixed and permeabilized to preserve late-stage apoptotic cells, thereby lessening the possibility of false-negative results due to cell detachment and subsequent loss. The EdUTP nucleotide is then rapidly incorporated into the fragmented DNA by TdT. The next step is to attach the dye or biotin azide, followed by addition of an optional counterstain. When compared with assays that use one-step incorporation of dye- or biotin modified nucleotides, this two-step method used in the Click-iT TUNEL imaging assay results in detection of a higher percentage of apoptotic cells under identical conditions in two hours or less (Figure 3).

The detection chemistry involved in the original Click-iT TUNEL imaging assay relies on the use of copper, and we have found that biomolecules exhibit varying sensitivities to copper concentration. For example, the high copper concentration used to catalyze the click chemistry reaction impacts the ability to multiplex with fluorescent proteins (e.g. GFP) due to losses in the protein’s fluorescence or to use the actin-specific compound, phalloidin, which exhibits a loss of binding. To overcome these challenges we have developed the Click-iT Plus TUNEL assay in which the copper concentration is optimized to preserve both the signal of fluorescent proteins and compatibility with phalloidin binding while also driving the detection reaction.

The APO-BrdU TUNEL assay is a two-color assay for labeling DNA breaks and total cellular DNA to detect apoptotic cells by imaging or flow cytometry. The Invitrogen APO-BrdU assays labels the 3’-OH end of fragmented DNA with BrdU and TdT, similar to the TUNEL technique described in Figure 2. BrdU is then detected using Alexa Fluor 488 labeled anti-BrdU monoclonal antibody. This assay also includes propidium iodide for determining total cellular DNA content and fixed positive and negative control cells for assessing assay performance.

Learn about alternative apoptosis assays