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TUNEL Assay

Green Fluorescence
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.

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

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*649663250000-0.027

Citations

View all 9 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
Antiapoptotic and antioxidant effects of melatonin on cat vitrified oocytes
Authors: Colombo, M and Mascaro, A and Pecile, A and Fusi, J and Luvoni, GC and others,
Journal: REPRODUCTION IN DOMESTIC ANIMALS (2023): 192--192
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)

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
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
Systemic LPS induces toll-like receptor 3 (TLR3) expression and apoptosis in testicular mouse tissue.
Authors: Nejsum, Lene N and Piec, Adrian and Fijak, Monika and Ernstsen, Christina V and Fischer, Dania and Maier, Thorsten J and Kinscherf, Ralf and Hofmann, Rainer and Urbschat, Anja
Journal: Cell and tissue research (2019): 143-154
Classification, Scoring, and Quantification of Cell Death in Tissue Sections.
Authors: Janke, Laura J and Ward, Jerrold M and Vogel, Peter
Journal: Veterinary pathology (2019): 33-38
Page updated on October 8, 2024

Ordering information

Price
Sample TypeFixed Cell and Tissue
Fixed Cell and Tissue
Live Cell
FluorescenceGreen
Blue
Deep Red
Green
Red
Unit size
Catalog Number
Quantity
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Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
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Custom sizeInquire
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Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Spectral properties

Absorbance (nm)

487

Correction Factor (260 nm)

0.32

Correction Factor (280 nm)

0.35

Extinction coefficient (cm -1 M -1)

800001

Excitation (nm)

498

Emission (nm)

517

Quantum yield

0.79001, 0.952

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

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

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.
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).

Documents

pdfSafety data sheet
pdfProduct protocol
pdfCertificate of analysis
DNA Fragmentation
TUNEL Assays

Alternative formats

Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Blue Fluorescence*
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Red Fluorescence*
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Deep Red Fluorescence*
Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Green Fluorescence*
Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Blue Fluorescence*
Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Blue Fluorescence Optimized for Flow Cytometry*