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Cell Meter™ Live Cell TUNEL Apoptosis Assay Kit *Red Fluorescence*

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 red fluorescence signal was analyzed using fluorescence microscope with a TRITC 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 red fluorescence signal was analyzed using fluorescence microscope with a TRITC filter set. Fluorescently labeled DNA strand breaks shows intense fluorescent staining in SS treated cells.
Apoptotic PCa cells in BM defined by TUNEL staining. PCa cells were recognized by pan cytokeratin with green color, and TUNEL signals were shown with red color. Scale bar, 20 μm. Source: <strong>Axl is required for TGF-β2-induced dormancy of prostate cancer cells in the bone marrow </strong>by Yumoto et al., <em>Scientific Reports</em>, Nov. 2016.
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Catalog Number22844
Unit Size
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Extinction coefficient (cm -1 M -1)27500
Excitation (nm)549
Emission (nm)648
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


Extinction coefficient (cm -1 M -1)
27500
Excitation (nm)
549
Emission (nm)
648
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 proprietary buffer system free of sodium cacodylate. The kit is based on the incorporation of our unique 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 antibody. The kit provides all the essential components with an optimized assay protocol. It is suitable for fluorescence microplate reader, fluorescence microscope, or flow cytometer.

Platform


Flow cytometer

Excitation488 nm laser
Emission660/20 nm filter
Instrument specification(s)PE-Cy5 channel

Fluorescence microscope

ExcitationTRITC filter
EmissionTRITC filter
Recommended plateBlack wall/clear bottom

Components


Component A: 100X Tunnelyte™ Red1 Vial (25 µL)
Component B: Reaction Buffer1 Bottle (10 mL)
Component C: 1000X Hoechst1 Vial (50 µL)

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 using fluorescence microscope with TRITC filter or flow cytometer with FL3 channel

Important  Thaw all the components at room temperature before starting the experiment.

CELL PREPARATION

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.

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

PREPARATION OF WORKING SOLUTION

Add 0.5 μL of 100X Tunnelyte™ Red (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.

SAMPLE EXPERIMENTAL PROTOCOL

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 using a fluorescence microscope with TRITC filter or flow cytometer with FL3 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 using a fluorescence microscope with TRITC filter or flow cytometer with FL3 channel.

  12. Optional: Stain the nucleus with 1X Hoechst (Component C) at Ex/Em = 350/460 nm for image analysis

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Extinction coefficient (cm -1 M -1)27500
Excitation (nm)549
Emission (nm)648

Product family


NameExcitation (nm)Emission (nm)
Cell Meter™ Live Cell TUNEL Apoptosis Assay Kit *Green Fluorescence*498522

Citations


View all 31 citations: Citation Explorer
Coacervate-mediated novel pancreatic cancer drug Aleuria Aurantia lectin delivery for augmented anticancer therapy
Authors: Kim, Sungjun and Choi, Yunyoung and Kim, Kyobum
Journal: Biomaterials Research (2022): 1--12
ARL6IP5 reduces cisplatin-resistance by suppressing DNA repair and promoting apoptosis pathways in ovarian carcinoma
Authors: Kim, Ji-Ye and Bahar, Entaz and Lee, Jung-Yun and Chang, Sunhee and Kim, Se Hoon and Park, Eun Young and Do, Sung-Im and Yoon, Hyonok and Kim, Hyun-Soo
Journal: Cell death \& disease (2022): 1--13
Inhibition of lung microbiota-derived proapoptotic peptides ameliorates acute exacerbation of pulmonary fibrosis
Authors: D’Alessandro-Gabazza, Corina N and Yasuma, Taro and Kobayashi, Tetsu and Toda, Masaaki and Abdel-Hamid, Ahmed M and Fujimoto, Hajime and Hataji, Osamu and Nakahara, Hiroki and Takeshita, Atsuro and Nishihama, Kota and others,
Journal: Nature communications (2022): 1--23
Protection of propofol on liver ischemia reperfusion injury by regulating Cyp2b10/Cyp3a25 pathway
Authors: Wu, Jinli and Yu, Chao and Zeng, Xianggang and Xu, Yini and Sun, Chengyi
Journal: Tissue and Cell (2022): 101891
Invitro anticancer activity of hydrogen sulfide and nitric oxide alongside nickel nanoparticle and novel mutations in their genes in CRC patients
Authors: Housein, Zjwan and Kareem, Tayeb Sabir and Salihi, Abbas
Journal: Scientific Reports (2021): 1-11
Human VAMP3 Suppresses or Negatively Regulates Bax Induced Apoptosis in Yeast
Authors: Akintade, Damilare D and Chaudhuri, Bhabatosh
Journal: Biomedicines (2021): 95
In vitro anticancer activity of hydrogen sulfide and nitric oxide alongside nickel nanoparticle and novel mutations in their genes in CRC patients
Authors: Housein, Zjwan and Kareem, Tayeb Sabir and Salihi, Abbas
Journal: Scientific Reports (2021): 1--11
Exosomes Derived from M2 Macrophages Exert a Therapeutic Effect via Inhibition of the PI3K/AKT/mTOR Pathway in Rats with Knee Osteoarthritic
Authors: Da-Wa, Zha Xi and Jun, Ma and Chao-Zheng, Liu and Sen-Lin, Yang and Chuan, Lu and De-Chun, Li and Zu-Nan, Dong and Hong-Tao, Zhao and Shu-Qing, Wei and Xian-Wei, Pei and others,
Journal: BioMed Research International (2021)

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
In situ localization of apoptosis using TUNEL
Authors: Hewitson TD, Darby IA.
Journal: Methods Mol Biol (2010): 161
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
Testicular apoptosis in feral Clarias gariepinus using TUNEL and cleaved caspase-3 immunohistochemistry
Authors: McClusky LM, Barnhoorn IE, van Dyk JC, Bornman MS.
Journal: Ecotoxicol Environ Saf (2008): 41
Analysis of apoptosis by cytometry using TUNEL assay
Authors: Darzynkiewicz Z, Galkowski D, Zhao H.
Journal: Methods (2008): 250
The effects of benzene exposure on apoptosis in epithelial lung cells: localization by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and the immunocytochemical localization of apoptosis-related gene products
Authors: Weaver CV, Liu SP, Lu JF, Lin BS.
Journal: Cell Biol Toxicol (2007): 201
Simultaneous detection of a cell surface antigen and apoptosis by microwave-sensitized TUNEL assay on paraffin sections
Authors: Christina M, Angelika HL, Bernd P, Martina P.
Journal: J Immunol Methods (2006): 163
Apoptosis in the antral follicles of swamp buffalo and cattle ovary: TUNEL and caspase-3 histochemistry
Authors: Feranil J, Isobe N, Nakao T.
Journal: Reprod Domest Anim (2005): 111