Fluorescence image- mouse intestine cross section (green stained outer layer, red stained apoptotic cells in center)

Cells undergoing apoptosis exhibit changes in nuclear morphology, including DNA fragmentation, chromatin condensation, degradation of nuclear envelope, nuclear blebbing, and DNA strand breaks. We offer a variety of TUNEL assays and DNA stains for analyzing these changes; many of which are also compatible with other reagents and procedures used to detect 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). 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.

TUNEL assays using click chemistry

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.

Structural formula of Click-iT EdUTP, rendered via Hayworth projection

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).

Schematic of EdUTP incorporation into DNA followed by addition of dye or biotin azide molecule and addition of optional counterstains.
Figure 2. Detection of apoptosis with the Click-iT TUNEL imaging assay.
Bar graph showing highest percentage of positive apoptotic cells detected using the Click-iT EdUTP derivative.

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 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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TUNEL assays for tissue analysis

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.

See selection guide

Tissue sections transgenic mouse expressing GFP in intestinal muscle tunel assay detection.
Figure 4. The Click-iT Plus TUNEL Assay can be multiplexed with a variety of other fluorescent probes. Formalin-fixed, paraffin-embedded (FFPE) tissue from a transgenic mouse expressing GFP in intestinal muscle was treated with DNase I, followed by the Click-iT Plus TUNEL Assay with Alexa Fluor 594 dye, and then stained with Hoechst 33342 dye and Alexa Fluor 647 Phalloidin. (A) Cell nuclei are stained by Hoechst 33342 dye (blue), (B) the GFP signal (green) is detected in the surrounding muscular layer, (C) filamentous actin is stained by Alexa Fluor 647 Phalloidin (purple), and (D) the TUNEL-positive signal resulting from DNase I treatment is clearly defined by the Click-iT Plus TUNEL Assay with Alexa Fluor 594 dye (red). The last panel (E) is the multiplexed image resulting from an overlay of the four fluorescent signals.

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).

See selection guide

Schematic showing incorporation of EdUTP into DNA followed by biotin azide attachment, binding of streptavidin-peroxidase to biotin and development of colored product with DAB addition.
Figure 5. Assay workflow for the Click-iT TUNEL Colorimetric IHC Detection Kit.
Brightfield microscopic view of tissue section with brown-colored nuclei, pink-colored connective tissue and cytoplasm, and blue-colored nonapoptotic cell nuclei.

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.

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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).

Microscopic view of two cells orange fluorescently stained tubulin, green fluorescently stained activated caspase within cytoplasm and magenta colored TUNEL detection in the nuclei.

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.

DNA stains to visualize chromatin condensation

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.

See the selection guide

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Citations

Selection guides for TUNEL assays and DNA stains

  Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection Click-iT TUNEL Colorimetric IHC Detection Kit
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
Sample Type
  • Optimized for tissue samples
  • Can also be used for adherent cells
Readout Fluorescent Colorimetric
Platform
  • Fluorescence imaging microscopy
  • High content analysis
Brightfield imaging microscopy
Fixation  Required (mild fixation and permeabilization)
Compatibility Multiplexing with fluorescent proteins and fluorescent phalloidin dye conjugates   Multiplexing with hematoxylin and methyl green
Label Alexa Fluor 488 Alexa Fluor 594 Alexa Fluor 647 Biotin
Ex/Em (nm) 495/519 590/615 650/670 N/A
Standard filter set FITC filter Texas Red filter Cy5 filter N/A
Cat. No. C10617 C10618 C10619 C10625
  Click-iT TUNEL Alexa Fluor Imaging Assays
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. Detection occurs via mild click reaction conditions, without the need for an antibody.
Sample Type
  • Cultured cells
  • Fixed cells
Platform
  • Imaging microscopy
  • High content analysis
Fixation Required (mild fixation and permeabilization)
Compatibility
  • Multiplexing with standard fluorescent dyes
  • Not recommended for multiplexing with fluorescent proteins or phalloidin
Label Alexa Fluor 488 Alexa Fluor 594 Alexa Fluor 647
Ex/Em (nm) 495/519 590/615 650/670
Standard filter set FITC filter Texas Red filter Cy5 filter
Cat. No. C10245 C10246 C10247
  ReadyProbes assays for imaging microscopy
  NucBlue Live NucRed Live 647 NucBlue Fixed Cell
Application Visualization of chromatin condensation, one of the hallmarks of late stage apoptosis
Basis of assay
  • Membrane-permeable dyes
  • Fluorescently stains nucleic acids, RNA and DNA
  • Membrane impermeable dye
  • Fluorescently stains nucleic acids, RNA and DNA
Sample Type Live Fixed
Platform
  • Imaging microscopy
  • High content analysis
Fixation Fixable Not fixable Not fixable
Compatibility Compatible with other reagents and procedures used to detect additional cell health and viability parameters.
Label Hoechst 33342 NucRed Live DAPI
Ex/Em (nm) 360/460 638/686 360/460
Standard filter set DAPI filter CY5 filter DAPI filter
Cat. No. R37605 R37106 R37606
Protocol Get protocol Get protocol Get protocol
  Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection Click-iT TUNEL Colorimetric IHC Detection Kit
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
Sample Type
  • Optimized for tissue samples
  • Can also be used for adherent cells
Readout Fluorescent Colorimetric
Platform
  • Fluorescence imaging microscopy
  • High content analysis
Brightfield imaging microscopy
Fixation  Required (mild fixation and permeabilization)
Compatibility Multiplexing with fluorescent proteins and fluorescent phalloidin dye conjugates   Multiplexing with hematoxylin and methyl green
Label Alexa Fluor 488 Alexa Fluor 594 Alexa Fluor 647 Biotin
Ex/Em (nm) 495/519 590/615 650/670 N/A
Standard filter set FITC filter Texas Red filter Cy5 filter N/A
Cat. No. C10617 C10618 C10619 C10625
  Click-iT TUNEL Alexa Fluor Imaging Assays
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. Detection occurs via mild click reaction conditions, without the need for an antibody.
Sample Type
  • Cultured cells
  • Fixed cells
Platform
  • Imaging microscopy
  • High content analysis
Fixation Required (mild fixation and permeabilization)
Compatibility
  • Multiplexing with standard fluorescent dyes
  • Not recommended for multiplexing with fluorescent proteins or phalloidin
Label Alexa Fluor 488 Alexa Fluor 594 Alexa Fluor 647
Ex/Em (nm) 495/519 590/615 650/670
Standard filter set FITC filter Texas Red filter Cy5 filter
Cat. No. C10245 C10246 C10247
  ReadyProbes assays for imaging microscopy
  NucBlue Live NucRed Live 647 NucBlue Fixed Cell
Application Visualization of chromatin condensation, one of the hallmarks of late stage apoptosis
Basis of assay
  • Membrane-permeable dyes
  • Fluorescently stains nucleic acids, RNA and DNA
  • Membrane impermeable dye
  • Fluorescently stains nucleic acids, RNA and DNA
Sample Type Live Fixed
Platform
  • Imaging microscopy
  • High content analysis
Fixation Fixable Not fixable Not fixable
Compatibility Compatible with other reagents and procedures used to detect additional cell health and viability parameters.
Label Hoechst 33342 NucRed Live DAPI
Ex/Em (nm) 360/460 638/686 360/460
Standard filter set DAPI filter CY5 filter DAPI filter
Cat. No. R37605 R37106 R37606
Protocol Get protocol Get protocol Get protocol

Resources

Apoptosis Protocols

Tools and resources

Fluorescence SpectraViewer
Online tool for visualization of the excitation and emission of fluorescent reagents. Tool allows for checking spectral compatibility for multiple fluorophores.

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