Cell Meter™ Live Cell TUNEL Apoptosis Assay Kit *Green Fluorescence*
DNA fragmentation represents a characteristic of late stage apoptosis. DNA fragmentation in apoptotic cells can be detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). The TUNEL assay relies on the presence of nicks in the DNA which can be identified by TdT, an enzyme that catalyzes the addition of dUTPs that are secondarily labeled with a marker. All the existing TUNEL assays contain the highly toxic sodium cacodylate which might induces apoptosis and also decrease DNA production and DNA strands. Our Cell Meter™ TUNEL Apoptosis Assay Kit uses a proprietary buffer system free of sodium cacodylate. The kit is based on the incorporation of our proprietary fluorescent dye into the DNA fragments that form during apoptosis. The assay is optimized for the direct detection of apoptosis in either detached or attached cells without using any antibodies. The kit provides all the essential components with an optimized assay protocol. It is suitable for fluorescence microplate reader, fluorescence microscope, or flow cytometer. Its signal can be easily detected at the popular FITC channel.
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-meter-tunel-apoptosis-assay-kit-green-fluorescence%2Ffigure-for-cell-meter-tunel-apoptosis-assay-kit-green-fluorescence_f74qc.jpg&w=640&q=75)
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-meter-tunel-apoptosis-assay-kit-green-fluorescence%2Ffigure-for-cell-meter-tunel-apoptosis-assay-kit-green-fluorescence_f74qc.jpg&w=640&q=75)
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcell-meter-tunel-apoptosis-assay-kit-green-fluorescence%2Ffigure-for-cell-meter-tunel-apoptosis-assay-kit-green-fluorescence_f74qc.jpg&w=128&q=25)
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
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
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
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
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)
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)
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
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
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
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
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
Authors: Hewitson TD, Darby IA.
Journal: Methods Mol Biol (2010): 161