TUNEL Assay

Deep Red Fluorescence

Cell Meter™ TUNEL apoptosis assay kit provides a robust tool for conveniently detecting apoptosis caused by DNA fragmentation. The assay is non-radioactive and rapid. The TUNEL assay uses the terminal deoxynucleotidyl transferase (TdT) to catalyze the incorporation of TF5-dUTP at the free 3’-hydroxyl ends of the fragmented DNAs. The resulted TF5-labeled DNAs are analyzed by fluorescence microscopy (Cy5 filter set) or flow cytometry with 633 or 640 nm laser with 660/20 nm filter (APC channel). Its red emission can be conveniently multiplexed with GFP labelled targets. Direct incorporation of fluorescent TF5-labeled nucleotides significantly reduces the number of test steps. The kit is optimized to detect apoptosis in fixed cells and formalin-fixed, paraffin-embedded tissue sections.

Example protocol

AT A GLANCE

Protocol summary

Treat samples as desired
Fix cells with 4% formaldehyde solution for 30 minutes on ice
Permeabilize cells with 70% ice-cold ethanol for 60 minutes on ice
Add TdT staining solution to samples and incubate for 60 minutes at 37 °C
Monitor the fluorescence intensity using fluorescence microscopy with Cy5 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 TF5-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.

Treat your samples as desired.
Wash the samples with buffer of your choice such as PBS containing Ca⁺² and Mg⁺².
Fix the samples by adding 100 µL of 4% paraformaldehyde in PBS and incubate the samples for 30 minutes on ice.
Remove fixation solution and wash samples with PBS.
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.
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
Add 50 µL of TdT staining solution to the samples and incubate for 60 to 120 minutes at 37 °C.
Remove TdT working solution and wash samples with PBS.
Resuspend the samples in PBS and monitor the fluorescence intensity with flow cytometer using 660/20 nm filter (APC channel) or fluorescence microscope with Cy5 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

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)
Wash the samples by immersing the slides in 100% ethanol for 5 minutes at room temperature in a Coplin jar.
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
Wash the samples by immersing the slides in 0.85% NaCl for 5 minutes at room temperature.
Wash the samples by immersing the slides in PBS for 5 minutes at room temperature. Repeat one more wash. (Total 2 washes)

Fixation protocol

Fix the tissue sections by immersing slides in 4% paraformaldehyde solution in PBS for 15-20 minutes at room temperature.
Wash the samples by immersing the slides in PBS for 5 minutes at room temperature. Repeat one more wash. (Total 2 washes)
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.
Wash the samples by immersing the slides in PBS for 5 minutes at room temperature.
Fix the tissue sections by immersing slides in 4% paraformaldehyde solution in PBS for 15-20 minutes at room temperature.
Wash the samples by immersing the slides in PBS for 5 minutes at room temperature. Repeat one more wash. (Total 2 washes)

Staining protocol

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.
Add 50 µL of TdT staining solution to the samples and incubate for 60 to 120 minutes at 37 °C.
Remove TdT working solution and wash samples with PBS.
Add mounting medium with DAPI (AAT Bioquest Cat# 20005) and monitor the fluorescence intensity fluorescence microscope with Cy5 filter set.

Spectrum

Open in Advanced Spectrum Viewer

Product family

Name	Excitation (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	411	472	-	0.14	0.12
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit Green Fluorescence	498	517	80000¹	0.32	0.35
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit Red Fluorescence	544	570	100000	0.27	0.34

Citations

View all 8 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 15 references: Citation Explorer

A Novel Theranostic Combination of Near-infrared Fluorescence Imaging and Laser Irradiation Targeting c-KIT for Gastrointestinal Stromal Tumors.
Authors: Fujimoto, Shota and Muguruma, Naoki and Okamoto, Koichi and Kurihara, Takeshi and Sato, Yasushi and Miyamoto, Yoshihiko and Kitamura, Shinji and Miyamoto, Hiroshi and Taguchi, Takahiro and Tsuneyama, Koichi and Takayama, Tetsuji
Journal: Theranostics (2018): 2313-2328

Multicomponent Synthesis of Some Molecular Hybrid Containing Thiazole Pyrazole as Apoptosis Inducer.
Authors: Kumar, Parvin and Duhan, Meenakshi and Kadyan, Kulbir and Bhardwaj, Jitender Kumar and Saraf, Priyanka and Mittal, Meenu
Journal: Drug research (2018): 72-79

[Salvianolate protects H9c2 cells from hypoxia/reoxygenation injury-induced apoptosis by attenuating mitochondrial DNA oxidative damage].
Authors: Yue, R C and Yang, X L and Zhang, R Y and Liu, S and Liu, J and Zeng, J and Liang, H and Wang, W and Hu, H X and Zeng, C Y
Journal: Zhonghua xin xue guan bing za zhi (2017): 57-63

Caspase 3 as a therapeutic target for regulation of intervertebral disc degeneration in rabbits.
Authors: Sudo, Hideki and Minami, Akio
Journal: Arthritis and rheumatism (2011): 1648-57

Induction of apoptosis and inhibition of PI3K/Akt pathway in PC-3 and LNCaP prostate cancer cells by ethanolic neem leaf extract.
Authors: Gunadharini, Dharmalingam N and Elumalai, Perumal and Arunkumar, Ramachandran and Senthilkumar, Kalimuthu and Arunakaran, Jagadeesan
Journal: Journal of ethnopharmacology (2011): 644-50

Page updated on October 8, 2024

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Sample Type	Fixed Cell and Tissue Fixed Cell and Tissue Live Cell
Fluorescence	Deep Red Blue Deep Red Green Red
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Catalog Number	228442284922851228532285522857
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