Click-iT® TUNEL Assay for Apoptosis
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Click-it® TUNEL is a break-through technology for detection of DNA fragmentation. For TUNEL assays to yield meaningful results, it is necessary not only that the modified nucleotide is an acceptable substrate for TdT, but also that the detection method is sensitive and avoids detrimental loss of cells from the sample.
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How the assay works
The Click-iT® TUNEL Imaging Assays employ dUTP modified with an alkyne (a small, bio-orthogonal functional group). The incorporated nucleotide is detected through “click” chemistry-a copper-catalyzed reaction between an azide and an alkyne (Figure 1).
Click chemistry fills the void when methods such as direct labeling or the use of antibodies are not sufficient. The small size of the Alexa Fluor® azide (MW ~1,000) compared to that of an antibody (MW ~150,000) enables effortless penetration of complex samples with only mild fixation and permeabilization needed.
When compared with assays using one of the other modified nucleotides, the Click-iT® TUNEL assay is able to detect a higher percentage of apoptotic cells under identical conditions (Figure 2) within 2 hours.
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| (click image to enlarge) Figure 1. Detection of apoptosis with the Click-iT® TUNEL assay. |
Click chemistry fills the void when methods such as direct labeling or the use of antibodies are not sufficient. The small size of the Alexa Fluor® azide (MW ~1,000) compared to that of an antibody (MW ~150,000) enables effortless penetration of complex samples with only mild fixation and permeabilization needed.
When compared with assays using one of the other modified nucleotides, the Click-iT® TUNEL assay is able to detect a higher percentage of apoptotic cells under identical conditions (Figure 2) within 2 hours.
 | Figure 2. TUNEL assay time course comparison showing the percentage of apoptotic cells detected. HeLa cells were treated with 0.5 μM staurosporine for the lengths of time indicated. Following fixation and permeabilization, TUNEL assays using either Click-iT® EdUTP (from the Click-iT® TUNEL Alexa Fluor® 488 Imaging Assay) or fluorescein dUTP (DeadEnd™ Colorimetric TUNEL System, Promega) were performed according to the manufacturer’s instructions. The percentage of apoptotic cells was calculated based upon the corresponding negative control. Imaging and analysis was performed using a Thermo Scientific Cellomics® ArrayScan® II (Thermo Fisher Scientific). |
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Click-iT® TUNEL in Action
To unequivocally establish programmed cell death in any given cell or tissue model, two or more biomarkers of apoptosis must be observed. The Click-iT® TUNEL Imaging Assays are available in three different bright and photostable Alexa Fluor® dyes, ranging from green to far-red fluorescence, providing flexibility when working with other apoptosis detection reagents that are typically available only in green- or red-fluorescent colors.
The Click-iT® TUNEL assays have 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, or phosphohistone 2B (Figure 3).
Each Click-iT® TUNEL kit contains all of the components necessary to accurately and reliably detect DNA fragmentation in adher¬ent cells grown on coverslips or in 96-well microplates.
Figure 3. Straightforward multiplexed analysis of programmed cell death. HeLa cells were treated with 0.5 μM staurosporine for the lengths of time indicated. The cells were fixed and permeabilized, followed by treatment with the Click-iT® TUNEL Alexa Fluor® 594 Imaging Assay. Cleaved PARP was detected with a rabbit anti–cleaved PARP antibody and visualized using an Alexa Fluor® 488 goat anti–rabbit IgG antibody. Cells exhibiting yellow fluorescence are positive for both cleaved PARP (green fluorescence) and DNA strand breaks (red fluorescence). The images were quantitated using the Thermo Scientific Cellomics® ArrayScan® VTI platform (Thermo Fisher Scientific).
The Click-iT® TUNEL assays have 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, or phosphohistone 2B (Figure 3).
Each Click-iT® TUNEL kit contains all of the components necessary to accurately and reliably detect DNA fragmentation in adher¬ent cells grown on coverslips or in 96-well microplates.
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(click image to enlarge)
Figure 3. Straightforward multiplexed analysis of programmed cell death. HeLa cells were treated with 0.5 μM staurosporine for the lengths of time indicated. The cells were fixed and permeabilized, followed by treatment with the Click-iT® TUNEL Alexa Fluor® 594 Imaging Assay. Cleaved PARP was detected with a rabbit anti–cleaved PARP antibody and visualized using an Alexa Fluor® 488 goat anti–rabbit IgG antibody. Cells exhibiting yellow fluorescence are positive for both cleaved PARP (green fluorescence) and DNA strand breaks (red fluorescence). The images were quantitated using the Thermo Scientific Cellomics® ArrayScan® VTI platform (Thermo Fisher Scientific).
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Find Your Click-iT® Detection Assay
With its equally small “footprint,” the Click-iT® detection molecule can easily penetrate complex samples for in-depth analyses of key biological processes, including assays for:
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Nascent DNA Synthesis/Cell Proliferation
| Nascent RNA Synthesis
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| Nascent Protein Synthesis | Click Chemistry Technology
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Cutting-edge Click-iT® Citations
- EdU, a new Thymidine Analogue for Labeling Proliferating Cells in the Nervous System—J. Neurosci Meth. 177, 122-130 (2009)
- Exploring RNA Transcription and turnover in-vivo by using Click Chemistry—PNAS 105, 15779-15784 (2008)
- Protein Synthesis in Distal Axons is not required for Growth Cone Responses to Guidance Cues—J. Neurosci. 29, 638-652 (2009)