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TUNEL Assay
TUNEL assays detect DNA fragmentation in live and fixed cells, which often occur in late-stage apoptosis.
Internucleosomal DNA fragmentation caused by activated endonucleases is universally regarded as the biochemical hallmark of apoptosis. Cells containing these DNA strand breaks can be identified with the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. This well-established in situ staining method relies on the enzyme terminal deoxynucleotidyl transferase (TdT) to catalyze the incorporation of modified dUTPs to the 3'-hydroxyl termini of fragmented DNA. Depending upon the choice of modified dUTP - BrdUTP, biotin-dUTP, or a fluorescein-dUTP - apoptotic cells can be identified and measured using various detection strategies and systems, including fluorescence microscopy, flow cytometry, and fluorescence-based microplate studies.
AAT Bioquest offers several fluorimetric TUNEL assays optimized for in situ apoptosis detection in live cells or fixed cells and tissue samples. These products can be combined with other cell-based viability and apoptosis assays to provide a comprehensive assessment of cell health, assess toxicity and safety of drug candidates, or analyze cancer and disease-related cellular changes.
Principles and mechanism
During the later stages of apoptosis, caspase-activated endonucleases cleave genomic DNA into oligonucleosomal fragments (~180-200 base pairs long). Using the TUNEL assay, the exposed 3'-OH termini of these breaks can be marked with modified dUTPs for subsequent visualization and quantification of apoptotic cells in situ. This is generally done either directly using dye-modified dUTP or indirectly using BrdUTP and antibody conjugates against BrdUTP. Regardless of the labeling strategy, both require the enzyme terminal deoxynucleotidyl transferase (TdT) to catalyze the incorporation of the modified dUTPs to the 3'-OH termini.
Detection of DNA fragments by this assay requires sample fixation using a crosslinking reagent such as 4% paraformaldehyde (avoid ethanol-based fixatives as it hinders the extraction of small DNA fragments) and sample permeabilization. Both steps are critical to the TUNEL assay as it facilitates the entry of exogenous TdT and antibody conjugates against BrdUTP. Keep in mind that several variables influence the staining kinetics of the TUNEL assay. Some of these variables, including reagent concentration, fixation of the sample, and accessibility of DNA strand breaks, may vary between tissue or cell sample types. Standardizing the TUNEL assay using samples with positive and negative apoptosis controls can alleviate these potential influences and reduce false positives or negative results.
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells with test compounds.
- Incubate with TUNEL working solution for 30 min to 1 hour at 37°C.
- Wash the cells.
- Fix cells with 4% formaldehyde (optional).
- Read fluorescence intensity at Ex/Em = 490/525 nm (Cutoff = 515 nm), fluorescence microscope with FITC filter or flow cytometer with FITC channel.
Important Thaw all the components at room temperature before starting the experiment.
PREPARATION OF WORKING SOLUTION
Add 0.5 μL of 100X Tunnelyte™ Green (Component A) into 50 μL of Reaction Buffer (Component B) to make a total volume of 50.5 μL of TUNEL working solution. Protect from light. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
- Culture cells to an optimal density for apoptosis induction according to your specific protocol. We recommend about 30,000 to 50,000 cells/well for adherent cells grown in a 96-well microplate culture, or about 1 to 2 x 106 cells/mL for non-adherent cells. At the same time, culture a non-induced negative control cell population at the same density as the induced population for every labeling condition. Note: We treated HeLa cells with 100 nM - 1 µM staurosporine for 4 hours to induce cell apoptosis. See Figure 1 for details.
Stain and Fixation:
- Remove cell media.
- Add 50 µL of TUNEL working solution to each sample.
- Incubate at 37°C for 30-60 minutes.
- Remove TUNEL working solution, and wash the cells 1 - 2 times with 200 µL/well of PBS.
- Add 100 uL Reaction buffer (Component B) to each sample.
- Monitor the fluorescence intensity with a fluorescence microplate reader at Ex/Em = 490/525 nm (Cutoff = 515 nm), a fluorescence microscope with FITC filter set or a flow cytometer with FITC channel.
- Optional: Remove the reaction buffer from Step 5, and add 100 µL/well/96-well plate of 4% formaldehyde fixative buffer (not supplied) to each well. Note: For non-adherent cells, add desired amount (such as 2X106 cells/mL) of 4% formaldehyde fixative buffer.
- Incubate plates for 20 to 30 minutes at room temperature.
- Remove fixative.
- Wash the cells with PBS 2-3 times, and replace with 100 µL PBS/well/96-well plate.
- Monitor the fluorescence intensity with a fluorescence microplate reader at Ex/Em = 490/525 nm (Cutoff = 515 nm), a fluorescence microscope with FITC filter set or a flow cytometer with FITC channel.
- Optional: Stain the nucleus with 1X Hoechst (Component C) at Ex/Em = 350/460 nm for image analysis
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) |
| Cell Meter™ Live Cell TUNEL Apoptosis Assay Kit *Red Fluorescence* | 549 | 648 | 27500 |
Citations
Authors: Wang, Yuqian and Huang, Renqi and Lu, Yougong and Liu, Mingqi and Mo, Ran
Journal: Nature Communications (2024): 1--13
Authors: Y{\i}ld{\i}r{\i}m, Onurcan and Se{\c{c}}me, M{\"u}cahit and Dodurga, Yavuz and Mete, G{\"u}l{\c{c}}in Abban and Fenkci, Semin Melahat
Journal: Biological Trace Element Research (2024): 1--11
Authors: Hsiao, Wen-Wei and Lau, Ka-Man and Chien, Shih-Chang and Chu, Fang-Hua and Chung, Wen-Hsin and Wang, Sheng-Yang
Journal: Plants (2024): 321
Authors: Y{\i}ld{\i}r{\i}m, Onurcan and Se{\c{c}}me, M{\"u}cahit and Dodurga, Yavuz and Mete, G{\"u}l{\c{c}}in Abban and Fenkci, Semin Melahat
Journal: (2023)
Authors: Qiu, Shi and Qi, Wei and Wu, Wen and Qiu, Qian and Ma, Jiali and Li, Yingjun and Fan, Wenhui and Li, Junli and Xu, Yang and Chen, Hai and others,
Journal: iLABMED (2023)
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Journal: Zhongguo Zhong Yao Za Zhi (2010): 1060
Authors: Hewitson TD, Darby IA.
Journal: Methods Mol Biol (2010): 161
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