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AAT Bioquest

TUNEL Assay

Green Fluorescence

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 apoptosis with the Cell Meter™ Fixed Cells and Tissue TUNEL Apoptosis imaging assay.

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

  1. Prepare cells with test compounds.
  2. Incubate with TUNEL working solution for 30 min to 1 hour at 37°C.
  3. Wash the cells.
  4. Fix cells with 4% formaldehyde (optional).
  5. 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

  1. 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:

  1. Remove cell media.
  2. Add 50 µL of TUNEL working solution to each sample.
  3. Incubate at 37°C for 30-60 minutes.
  4. Remove TUNEL working solution, and wash the cells 1 - 2 times with 200 µL/well of PBS.
  5. Add 100 uL Reaction buffer (Component B) to each sample.
  6. 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.
  7. 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.
  8. Incubate plates for 20 to 30 minutes at room temperature.
  9. Remove fixative.
  10. Wash the cells with PBS 2-3 times, and replace with 100 µL PBS/well/96-well plate.
  11. 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.
  12. Optional: Stain the nucleus with 1X Hoechst (Component C) at Ex/Em = 350/460 nm for image analysis

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)
Cell Meter™ Live Cell TUNEL Apoptosis Assay Kit *Red Fluorescence*54964827500

Citations

View all 32 citations: Citation Explorer
Immuno-protective vesicle-crosslinked hydrogel for allogenic transplantation
Authors: Wang, Yuqian and Huang, Renqi and Lu, Yougong and Liu, Mingqi and Mo, Ran
Journal: Nature Communications (2024): 1--13
In Vitro Effects of Boric Acid on Cell Cycle, Apoptosis, and miRNAs in Medullary Thyroid Cancer Cells
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
Antifungal Activity of Cedrol from Cunninghamia lanceolate var. konishii against Phellinus noxius and Its Mechanism
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
Effects of boric acid on invasion, migration, proliferation, apoptosis and miRNAs in medullary thyroid cancer cells
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)
An iASPP-derived short peptide restores p53-mediated cell death in cancers with wild-type p53
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)

References

View all 82 references: Citation Explorer
In situ detection of apoptosis by the TUNEL assay: an overview of techniques
Authors: Loo DT., undefined
Journal: Methods Mol Biol (2011): 3
Testicular apoptosis after dietary zinc deficiency: ultrastructural and TUNEL studies
Authors: Kumari D, Nair N, Bedwal RS.
Journal: Syst Biol Reprod Med (2011): 233
Simultaneous PCNA and TUNEL labeling for testicular toxicity evaluation suggests that detection of apoptosis may be more sensitive than proliferation
Authors: D'Andrea MR, Alicknavitch M, Nagele RG, Damiano BP.
Journal: Biotech Histochem (2010): 195
Ultrastructure and TUNEL staining on inhibition of Rubus alceaefolius total alkaloids for apoptosis of liver in rat models of acute hepatitis
Authors: Chen W, Hong Z, Li T, Zhao J, Lin J, Zhou J, Huang M.
Journal: Zhongguo Zhong Yao Za Zhi (2010): 1060
In situ localization of apoptosis using TUNEL
Authors: Hewitson TD, Darby IA.
Journal: Methods Mol Biol (2010): 161
Page updated on October 8, 2024

Ordering information

Price
Sample TypeLive Cell
Fixed Cell and Tissue
Live Cell
FluorescenceGreen
Green
Red
Unit size
Catalog Number
228442284922851228532285522857
Quantity
Add to cart

Additional ordering information

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Spectral properties

Excitation (nm)

498

Emission (nm)

522

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Flow cytometer

Excitation488 nm laser
Emission530, 30 nm filter
Instrument specification(s)FITC channel

Fluorescence microscope

ExcitationFITC filter
EmissionFITC filter
Recommended plateBlack wall, clear bottom

Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateSolid black

Components

Fluorescence images of TUNEL reaction in HeLa cells with the treatment of 100 nM or 1 μM staurosporine (SS) for 4 hours as compare to untreated control. Cells were incubated with TUNEL working solution for 1 hour at 37ºC. The green fluorescence signal was analyzed using fluorescence microscope with a FITC filter set. Fluorescently labeled DNA strand breaks shows intense fluorescent staining in SS treated cells.
Fluorescence images of TUNEL reaction in HeLa cells with the treatment of 100 nM or 1 μM staurosporine (SS) for 4 hours as compare to untreated control. Cells were incubated with TUNEL working solution for 1 hour at 37ºC. The green fluorescence signal was analyzed using fluorescence microscope with a FITC filter set. Fluorescently labeled DNA strand breaks shows intense fluorescent staining in SS treated cells.
Fluorescence images of TUNEL reaction in HeLa cells with the treatment of 100 nM or 1 μM staurosporine (SS) for 4 hours as compare to untreated control. Cells were incubated with TUNEL working solution for 1 hour at 37ºC. The green fluorescence signal was analyzed using fluorescence microscope with a FITC filter set. Fluorescently labeled DNA strand breaks shows intense fluorescent staining in SS treated cells.