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Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Green Fluorescence*

HeLa cells were fixed and treated with or without DNAse for 60 mins at 37 °C. The cells were then stained with Cell Meter™ TUNEL apoptosis assay kit. DNA strand breaks showed intense fluorescent staining in DNAse treated cells. The signal was acquired with fluorescence microscope using a FITC filter set.
HeLa cells were fixed and treated with or without DNAse for 60 mins at 37 °C. The cells were then stained with Cell Meter™ TUNEL apoptosis assay kit. DNA strand breaks showed intense fluorescent staining in DNAse treated cells. The signal was acquired with fluorescence microscope using a FITC filter set.
TUNEL assay of formalin-fixed paraffin-embedded (FFPE) human lung adenocarcinoma sections with Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Green Fluorescence* (Cat#22851). DNA strand breaks showed intense fluorescent staining in DNAse treated tissue section.  Cell nucleus was stained with Nuclear Blue™ DCS1 (Cat#17548).
Ordering information
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Catalog Number22851
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Absorbance (nm)487
Correction Factor (260 nm)0.32
Correction Factor (280 nm)0.275
Extinction coefficient (cm -1 M -1)800001
Excitation (nm)498
Emission (nm)517
Quantum yield0.79001, 0.952
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501
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OverviewpdfSDSpdfProtocol


Absorbance (nm)
487
Correction Factor (260 nm)
0.32
Correction Factor (280 nm)
0.275
Extinction coefficient (cm -1 M -1)
800001
Excitation (nm)
498
Emission (nm)
517
Quantum yield
0.79001, 0.952
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit provides a robust tool for conveniently detecting DNA fragmentation caused by apoptosis. The assay is a non-radioactive, simple, accurate and rapid method for monitoring apoptosis in. fixed cells and tissues via imaging DNA fragmentation. The TUNEL assay uses terminal deoxynucleotidyl transferase (TdT) to catalyze the incorporation of fluorescein-12-dUTP at the 3’-hydroxyl ends of the fragmented DNA. The fluorescein-labeled DNA is analyzed by fluorescence microscopy or flow cytometry (excitation at 488 nm with 530/30 nm emission filter). The kit can be used to detect apoptosis in fixed cells and formalin-fixed, paraffin-embedded tissue sections.

Platform


Flow cytometer

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

Fluorescence microscope

ExcitationFITC filter set
EmissionFITC filter set
Recommended plateBlack wall/clear bottom

Components


Component A: TdT enzyme1 vial (12.5 µL)
Component B: Fluorescein 12-dUTP1 vial (12.5 µL)
Component C: CoCl2 solution1 vial (125 µL)
Component D: TdT Reaction Buffer1 vial (1.2 mL)

Example protocol


AT A GLANCE

Protocol summary
  1. Treat samples as desired
  2. Fix cells with 4% formaldehyde solution for 30 minutes on ice
  3. Permeabilize cells with 70% ice-cold ethanol for 60 minutes on ice
  4. Add TdT staining solution to samples and incubate for 60 minutes at 37 °C
  5. Monitor the fluorescence intensity using fluorescence microscopy with FITC filter set 

Important
Bring all the kit components at room temperature before starting the experiment.

PREPARATION OF WORKING SOLUTION

TdT staining solution
For one test, Mix the following to make a total volume of 51 µL;
45 µL TdT Reaction Buffer (Component D)
5 µL CoCl2 (Component C)
0.5 µL Fluorescein 12-dUTP (Component B)
0.5 µL TdT enzyme (Component A).
Note     TdT staining solution should be used promptly.

SAMPLE EXPERIMENTAL PROTOCOL

Protocol for cells staining
The following protocol can be used as a guideline and should be optimized according to the needs.
  1. Treat your samples as desired.
  2. Wash the samples with buffer of your choice such as PBS containing Ca+2 and Mg+2.
  3. Fix the samples by adding 100 µL of 4% paraformaldehyde in PBS and incubate the samples for 30 minutes on ice.
  4. Remove fixation solution and wash samples with PBS.
  5. Add 100 µL of 70% of ice cold ethanol to samples and incubate the samples for 60 minutes on ice.
    Note     Samples can be stored at -20 °C at this step for several days before use.
  6. Remove alcohol and wash cells with PBS.
    Note     For a positive control, incubate fixed samples with 2-5 µg/mL of DNAse in PBS containing Ca+2 and Mg+2 for 60 minutes at 37 °C. Remove the DNAse and wash cells thoroughly and continue with the rest of the protocol
  7. Add 50 µL of TdT staining solution to the samples and incubate for 60 to 120 minutes at 37 °C.
  8. Remove TdT working solution and wash samples with PBS.
  9. Resuspend the samples in PBS and monitor the fluorescence intensity with flow cytometer using 530 /30 nm filter (FITC channel) or fluorescence microscope with FITC filter set. 

Protocol for tissue staining
The following protocol can be used as a guideline and should be optimized according to the needs.
Deparaffinization and rehydration protocol
  1. Deparaffinize tissue sections (attached to the microscopic slides) by immersing slides in fresh xylene in a Coplin jar for 5 minutes at room temperature. Repeat one more time. (Total 2 washes)
  2. Wash the samples by immersing the slides in 100% ethanol for 5 minutes at room temperature in a Coplin jar.
  3. Rehydrate the samples by immersing the slides through various concentrations of alcohol subsequently (100, 95, 85, 70, 50%) for 5 minutes each at room temperature
  4. Wash the samples by immersing the slides in 0.85% NaCl for 5 minutes at room temperature.
  5. Wash the samples by immersing the slides in PBS for 5 minutes at room temperature. Repeat one more wash. (Total 2 washes) 
Fixation protocol
  1. Fix the tissue sections by immersing slides in 4% paraformaldehyde solution in PBS for 15-20 minutes at room temperature.
  2. Wash the samples by immersing the slides in PBS for 5 minutes at room temperature. Repeat one more wash. (Total 2 washes)
  3. Remove the liquid and place the slides on a flat surface. Treat tissue sections with 100 µL of 20 µg/mL Proteinase K solution. Add enough to cover the entire tissue surface. Incubate slides for 10 minutes at room temperature.
  4. Wash the samples by immersing the slides in PBS for 5 minutes at room temperature.
  5. Fix the tissue sections by immersing slides in 4% paraformaldehyde solution in PBS for 15-20 minutes at room temperature.
  6. Wash the samples by immersing the slides in PBS for 5 minutes at room temperature. Repeat one more wash. (Total 2 washes) 
Staining protocol
  1. Optional: For a positive control, incubate fixed samples with 2-5 µg/mL of DNAse in PBS containing Ca+2 and Mg+2 for 60 minutes at 37 °C. Remove the DNAse and wash cells thoroughly with PBS and continue with the rest of the protocol.
  2. Add 50 µL of TdT staining solution to the samples and incubate for 60 to 120 minutes at 37 °C.
  3. Remove TdT working solution and wash samples with PBS.
  4. Add mounting medium with DAPI (AAT Bioquest Cat# 20005) and monitor the fluorescence intensity fluorescence microscope with FITC filter set. 

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Absorbance (nm)487
Correction Factor (260 nm)0.32
Correction Factor (280 nm)0.275
Extinction coefficient (cm -1 M -1)800001
Excitation (nm)498
Emission (nm)517
Quantum yield0.79001, 0.952

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Correction Factor (260 nm)Correction Factor (280 nm)
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Blue Fluorescence*411472-0.140.12
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Red Fluorescence*5445701000000.270.34
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Deep Red Fluorescence*649664250000-0.027

Citations


View all 4 citations: Citation Explorer
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
Effects of Boric Acid on Invasion, Migration, Proliferation, Apoptosis, Cell Cycle 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: (2021)
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 50 references: Citation Explorer
Improvement of in situ Follicular Activation and Early Development in Cryopreserved Human Ovarian Cortical Tissue by Co-Culturing with Mesenchymal Stem Cells.
Authors: Hosseini, Marzieh and Salehpour, Saghar and Ghaffari Novin, Marefat and Shams Mofarahe, Zahra and Abdollahifar, Mohammad-Amin and Piryaei, Abbas
Journal: Cells, tissues, organs (2020): 1-11
Exosomes derived from adipose tissue, bone marrow, and umbilical cord blood for cardioprotection after myocardial infarction.
Authors: Xu, Huiyu and Wang, Zhongchao and Liu, Longmei and Zhang, Baoxia and Li, Bao
Journal: Journal of cellular biochemistry (2020): 2089-2102
RHEB gene therapy maintains the chondrogenic characteristics and protects cartilage tissue from degenerative damage during experimental murine osteoarthritis.
Authors: Ashraf, S and Kim, B J and Park, S and Park, H and Lee, S-H
Journal: Osteoarthritis and cartilage (2019): 1508-1517
Tissue kallikrein-related peptidase8 protects rat heart against acute ischemia reperfusion injury.
Authors: Huang, Meineng and Du, Jiankui and Wang, Yifei and Ma, Shiyu and Hu, Ting and Shang, Jing and Yu, Qing and Zhu, Xiaoyan and Zhang, Guanxin and Cong, Binhai
Journal: International journal of biological macromolecules (2019): 1126-1133
The role of Toll-like receptor 4 in apoptosis of brain tissue after induction of intracerebral hemorrhage.
Authors: Fei, Xiaowei and He, Yeting and Chen, Jia and Man, Weitao and Chen, Chen and Sun, Kai and Ding, Boyun and Wang, Chongwu and Xu, Ruxiang
Journal: Journal of neuroinflammation (2019): 234
Vitrification freezing of large ovarian tissue in the human body.
Authors: Zhao, Qian and Zhang, Ying and Su, Ke and Wang, Xiao-Wan and Hai, Pan-Pan and Han, Bing and Bian, Ai-Ping and Guo, Rui-Xia
Journal: Journal of ovarian research (2019): 77
Correlation of MiR-152 expression with VEGF expression in placental tissue of preeclampsia rat and its influence on apoptosis of trophoblast cells.
Authors: Zhang, L and Yuan, J-M and Zhao, R-H and Wang, L-M and Tu, Z-B
Journal: European review for medical and pharmacological sciences (2019): 3553-3560
IL-10 inhibits apoptosis in brain tissue around the hematoma after ICH by inhibiting proNGF.
Authors: Song, L and Xu, L-F and Pu, Z-X and Wang, H-H
Journal: European review for medical and pharmacological sciences (2019): 3005-3011
Comparison between Slow Freezing and Vitrification for Human Ovarian Tissue Cryopreservation and Xenotransplantation.
Authors: Lee, Sanghoon and Ryu, Ki-Jin and Kim, Boram and Kang, Dahyeon and Kim, Yoon Young and Kim, Tak
Journal: International journal of molecular sciences (2019)
Exosomal miR-320d derived from adipose tissue-derived MSCs inhibits apoptosis in cardiomyocytes with atrial fibrillation (AF).
Authors: Liu, Lina and Zhang, Haoran and Mao, Hongyu and Li, Xiaohong and Hu, Yamin
Journal: Artificial cells, nanomedicine, and biotechnology (2019): 3976-3984