Chromatin Condensation and TUNEL Assays for Imaging

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). A relatively newer dUTP modification (EdUTP) now 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.

Click-iT TUNEL imaging assays

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 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).

Click-iT Plus TUNEL imaging assays

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.

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Click-iT Plus TUNEL assay for fluorescence detection

The Invitrogen Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection kit has been optimized for tissue sections, although it can be used with adherent cells. The fluorescence-based assay is compatible with GFP and other fluorescent proteins and can be multiplexed with phalloidin as shown in Figure 4. The Click-iT Plus TUNEL assay generates a bright, photostable fluorescent signal and is available with Alexa Fluor 488, Alexa Fluor 594 or Alexa Fluor 647 azide reagents.

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Click-iT TUNEL assay for colorimetric detection

The Invitrogen Click-iT TUNEL Colorimetric IHC Detection Kit has been optimized to identify apoptotic cells in tissue through the use of biotin azide and streptavidin-peroxidase conjugation, although it can be used for adherent cells. After incorporation of the EdUTP into sites of DNA fragmentation (Figure 5), the EdUTP is labeled with biotin azide. The subsequent addition of a streptavidin-peroxidase and DAB (peroxidase substrate) results in a dark brown signal that can be detected with light microscopy and stored for future analysis. The assay is also compatible with multiplexing with other colorimetric tissue stains (Figure 6).

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The Invitrogen Click-iT TUNEL Alexa Fluor Imaging Assay Kits have been optimized for HCS and microscope imaging of cultured cells. The small and specific labeling moiety enables a fast and efficient experimental protocol. The assay is complete within 2 hours, and provides efficient discrimination of even high levels of apoptotic cells in a population. With a choice of fluorescent labels, the Click-iT TUNEL assay allows multiplexing with surface and intracellular biomarker detection. For detection of cytoskeleton, however, we do recommend that you use a primary antibody instead of phalloidin (Figure 7). We also do not recommend use with fluorescent proteins. The assay has been tested in HeLa, A549 and CHO K1 cells with a variety of reagents that induce apoptosis, including staurosporine, and multiplexed with antibody-based detection of other apoptosis biomarkers such as cleaved poly (ADP-ribose) polymerase (PARP), cleaved caspase-3 and phosphohistone 2B. It has also proven effective for detection of apoptosis induced by siRNA knockdown of the DEC2 transcription factor in human MCF-7 breast cancer cells (1).

DNA stains provide the fastest and most convenient route to distinguish the condensed nuclei in apoptotic cells. Nuclear staining can be performed on live or fixed cells, and we offer a choice of wavelengths for multiplexing with other cellular labels. The ReadyProbes formulations are stable at room temperature and provided ready-to-use DNA stains in convenient dropper bottles.

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• Multiplex TUNEL assays for in situ apoptosis detection in tissue.
• Colorimetric TUNEL assays for IHC. Proliferating and apoptotic cells revealed.
• High-content screening. Assays for multiparametric analysis.

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• Liu Y1, Sato F, Kawamoto T et al. (2010) Genes Cells 15:315-25.