TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays are a widely used technique in the field of molecular biology and cell death research. Cells undergoing apoptosis exhibit changes in nuclear morphology, including DNA fragmentation, chromatin condensation, degradation of nuclear envelope, nuclear blebbing, and DNA strand breaks. Thermo Fisher Scientific offers TUNEL assays that enable researchers to visualize and quantify DNA fragmentation in tissue samples or cultured cells. Additionally, our assays are designed to be compatible with other reagents and procedures allowing for the detection of additional cell health and viability parameters.
What are 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.
TUNEL assays using click chemistry
Click-iT TUNEL 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 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.
Figure 1. Click-iT EdUTP nucleotide structure provided in the Click-iT TUNEL Imaging Assay Kits.
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).
Figure 3. Comparison of modified dUTP type as a function of percentage of apoptotic cells detected. HeLa cells were treated with 0.5 μM staurosporine for 4 hours. Following fixation and permeabilization, TUNEL imaging assays were performed according to the manufacturer's instructions for Click-iT EdUTP, BrdUTP and 2 different fluorescein dUTP products. The percent positives were calculated based upon the corresponding negative control. Imaging and analysis was performed using a Thermo Fisher Scientific ArrayScan II.
Click-iT Plus TUNEL 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.
TUNEL assays using BrdU
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
| TUNEL assays for tissues | TUNEL assays for cultured cells | |||||
|---|---|---|---|---|---|---|---|
| Click-iT Plus TUNEL Assays for In Situ Apoptosis Detection | Click-iT TUNEL Colorimetric IHC Detection Kit | Click-iT TUNEL Alexa Fluor Imaging Assays for Microscopy and HCS | |||||
| Basis of assay | The assays utilize EdUTP (a dUTP modified with a small, bioorthogonal alkyne moiety), which is incorporated at the 3’-OH ends of fragmented DNA by the TdT enzyme. | ||||||
| Application | In situ apoptosis detection | Apoptosis detection occurs via mild click reaction conditions, without the need for an antibody. | |||||
Sample type | Optimized for tissue samples Can also be used for adherent cells | Cultured cells Fixed cells | |||||
| Readout | Fluorescent | Colorimetric | Fluorescent | ||||
Label | Alexa Fluor 488 | Alexa Fluor 594 | Alexa Fluor 647 | Biotin | Alexa Fluor 488 | Alexa Fluor 594 | Alexa Fluor 647 |
| Ex/Em (nm) | 495/519 | 590/615 | 650/670 | N/A | 495/519 | 590/615 | 650/670 |
| Standard filter set | FITC filter | Texas Red filter | Cy5 filter | N/A | FITC filter | Texas Red filter | Cy5 filter |
| Instrument platform | Fluorescence imaging microscopy High content analysis | Brightfield imaging microscopy | Imaging microscopy High content analysis | ||||
| Compatibility | Multiplexing with fluorescent proteins and fluorescent phalloidin dye conjugates | Multiplexing with hematoxylin and methyl green | Multiplexing with standard fluorescent dyes Not recommended for multiplexing with fluorescent proteins or phalloidin | ||||
| Fixation | Required (mild fixation and permeabilization) | ||||||
| See all Click-iT Plus TUNEL Assays | C10625 | See all Click-iT TUNEL Alexa Fluor Assays | ||||
TUNEL assays for tissue analysis
Click-iT Plus TUNEL assay for fluorescent 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.
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).
Figure 5.Assay workflow for the Click-iT TUNEL Colorimetric IHC Detection Kit. Incorporation of EdUTP into DNA is followed by biotin azide attachment, then binding of streptavidin-peroxidase to biotin, and the development of colored product with the addition of DAB.
Figure 6. Mouse tissue section labeling with the colorimetric Click-iT TUNEL apoptosis assay. An 8 μm formalin-fixed, paraffin-embedded (FFPE) section of mouse thymus was labeled with DAB using the Click-iT TUNEL Colorimetric IHC Detection Kit to reveal apoptotic cells (dark brown nuclei). The tissue was subsequently stained with eosin Y (pink) followed by nuclear counterstaining with methyl green (blue), dehydrated, and hard-mounted in Thermo Scientific Cytoseal 60 Mounting Medium. The brightfield image was acquired using a 20x objective on the EVOS FL Auto Imaging System equipped with a color camera.
TUNEL assays for cultured cells
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).
Figure 7. Late-stage apoptosis visualized using the Click-iT TUNEL Imaging Assay. HeLa cells were treated with staurosporine, then fixed and permeabilized. The Click-iT TUNEL Alexa Fluor 647 Imaging Assay was performed. Activated caspase-3 was detected with a rabbit polyclonal primary antibody for cleaved caspase-3 and labeled with Alexa Fluor 488 goat anti–rabbit IgG antibody (green). Tubulin was detected with a mouse monoclonal anti-tubulin antibody and labeled with Alexa Fluor 555 goat anti–mouse IgG (orange). Nuclei were stained with Hoechst 33342 (blue). The light blue color represents an overlay of caspase (green), Hoechst (blue), and TUNEL (magenta) signals.
| APO-BrdU TUNEL Assay Kit, with Alexa Fluor 488 Anti-BrdU |
|---|---|
| Basis of assay | The assay utilizes BrdU, which is incorporated at the 3’-OH ends of fragmented DNA by the TdT enzyme. |
| Application | Apoptosis detection occurs via an Alexa Fluor 488 dye-labeled anti-BrdU antibody |
| Sample type | Fixed cells Can also be used for adherent cells |
| Readout | Fluorescent |
| Label | Alexa Fluor 488 |
| Ex/Em (nm) | 490/525 |
| Standard filter set | FITC filter |
| Nuclear counterstains | Propidium iodide |
| Instrument platform | Flow cytometry Fluorescence imaging microscopy |
| Fixation | Required |
| A23210 |
TUNEL assay for flow cytometry
APO-BrdU TUNEL assay for fluorescent detection
The Invitrogen APO-BrdU TUNEL Assay Kit is a two-color assay for labeling DNA breaks. It has been optimized for flow cytometry of cultured cells, although it can be used with adherent cells that are trypsinized after apoptosis induction. The kit includes propidium iodide to analyze total DNA content and fixed samples of positive and negative control cells to assess assay performance. The protocol can also be adapted for fluorescence microscopy but it is recommended that cells be placed onto slides after antibody staining but before the propidium iodide/RNase treatment.
What are 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.
TUNEL assays using click chemistry
Click-iT TUNEL 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 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.
Figure 1. Click-iT EdUTP nucleotide structure provided in the Click-iT TUNEL Imaging Assay Kits.
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).
Figure 3. Comparison of modified dUTP type as a function of percentage of apoptotic cells detected. HeLa cells were treated with 0.5 μM staurosporine for 4 hours. Following fixation and permeabilization, TUNEL imaging assays were performed according to the manufacturer's instructions for Click-iT EdUTP, BrdUTP and 2 different fluorescein dUTP products. The percent positives were calculated based upon the corresponding negative control. Imaging and analysis was performed using a Thermo Fisher Scientific ArrayScan II.
Click-iT Plus TUNEL 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.
TUNEL assays using BrdU
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
| TUNEL assays for tissues | TUNEL assays for cultured cells | |||||
|---|---|---|---|---|---|---|---|
| Click-iT Plus TUNEL Assays for In Situ Apoptosis Detection | Click-iT TUNEL Colorimetric IHC Detection Kit | Click-iT TUNEL Alexa Fluor Imaging Assays for Microscopy and HCS | |||||
| Basis of assay | The assays utilize EdUTP (a dUTP modified with a small, bioorthogonal alkyne moiety), which is incorporated at the 3’-OH ends of fragmented DNA by the TdT enzyme. | ||||||
| Application | In situ apoptosis detection | Apoptosis detection occurs via mild click reaction conditions, without the need for an antibody. | |||||
Sample type | Optimized for tissue samples Can also be used for adherent cells | Cultured cells Fixed cells | |||||
| Readout | Fluorescent | Colorimetric | Fluorescent | ||||
Label | Alexa Fluor 488 | Alexa Fluor 594 | Alexa Fluor 647 | Biotin | Alexa Fluor 488 | Alexa Fluor 594 | Alexa Fluor 647 |
| Ex/Em (nm) | 495/519 | 590/615 | 650/670 | N/A | 495/519 | 590/615 | 650/670 |
| Standard filter set | FITC filter | Texas Red filter | Cy5 filter | N/A | FITC filter | Texas Red filter | Cy5 filter |
| Instrument platform | Fluorescence imaging microscopy High content analysis | Brightfield imaging microscopy | Imaging microscopy High content analysis | ||||
| Compatibility | Multiplexing with fluorescent proteins and fluorescent phalloidin dye conjugates | Multiplexing with hematoxylin and methyl green | Multiplexing with standard fluorescent dyes Not recommended for multiplexing with fluorescent proteins or phalloidin | ||||
| Fixation | Required (mild fixation and permeabilization) | ||||||
| See all Click-iT Plus TUNEL Assays | C10625 | See all Click-iT TUNEL Alexa Fluor Assays | ||||
TUNEL assays for tissue analysis
Click-iT Plus TUNEL assay for fluorescent 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.
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).
Figure 5.Assay workflow for the Click-iT TUNEL Colorimetric IHC Detection Kit. Incorporation of EdUTP into DNA is followed by biotin azide attachment, then binding of streptavidin-peroxidase to biotin, and the development of colored product with the addition of DAB.
Figure 6. Mouse tissue section labeling with the colorimetric Click-iT TUNEL apoptosis assay. An 8 μm formalin-fixed, paraffin-embedded (FFPE) section of mouse thymus was labeled with DAB using the Click-iT TUNEL Colorimetric IHC Detection Kit to reveal apoptotic cells (dark brown nuclei). The tissue was subsequently stained with eosin Y (pink) followed by nuclear counterstaining with methyl green (blue), dehydrated, and hard-mounted in Thermo Scientific Cytoseal 60 Mounting Medium. The brightfield image was acquired using a 20x objective on the EVOS FL Auto Imaging System equipped with a color camera.
TUNEL assays for cultured cells
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).
Figure 7. Late-stage apoptosis visualized using the Click-iT TUNEL Imaging Assay. HeLa cells were treated with staurosporine, then fixed and permeabilized. The Click-iT TUNEL Alexa Fluor 647 Imaging Assay was performed. Activated caspase-3 was detected with a rabbit polyclonal primary antibody for cleaved caspase-3 and labeled with Alexa Fluor 488 goat anti–rabbit IgG antibody (green). Tubulin was detected with a mouse monoclonal anti-tubulin antibody and labeled with Alexa Fluor 555 goat anti–mouse IgG (orange). Nuclei were stained with Hoechst 33342 (blue). The light blue color represents an overlay of caspase (green), Hoechst (blue), and TUNEL (magenta) signals.
| APO-BrdU TUNEL Assay Kit, with Alexa Fluor 488 Anti-BrdU |
|---|---|
| Basis of assay | The assay utilizes BrdU, which is incorporated at the 3’-OH ends of fragmented DNA by the TdT enzyme. |
| Application | Apoptosis detection occurs via an Alexa Fluor 488 dye-labeled anti-BrdU antibody |
| Sample type | Fixed cells Can also be used for adherent cells |
| Readout | Fluorescent |
| Label | Alexa Fluor 488 |
| Ex/Em (nm) | 490/525 |
| Standard filter set | FITC filter |
| Nuclear counterstains | Propidium iodide |
| Instrument platform | Flow cytometry Fluorescence imaging microscopy |
| Fixation | Required |
| A23210 |
TUNEL assay for flow cytometry
APO-BrdU TUNEL assay for fluorescent detection
The Invitrogen APO-BrdU TUNEL Assay Kit is a two-color assay for labeling DNA breaks. It has been optimized for flow cytometry of cultured cells, although it can be used with adherent cells that are trypsinized after apoptosis induction. The kit includes propidium iodide to analyze total DNA content and fixed samples of positive and negative control cells to assess assay performance. The protocol can also be adapted for fluorescence microscopy but it is recommended that cells be placed onto slides after antibody staining but before the propidium iodide/RNase treatment.
TUNEL assays ordering information
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