TUNEL Assay

Blue 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 DEAC-dUTP at the free 3’-hydroxyl ends of the fragmented DNAs. The resulted DEAC-labeled DNAs are analyzed by fluorescence microscopy (AMC filter set). Its blue emission can be conveniently multiplexed with GFP labelled targets. Direct incorporation of fluorescent DEAC-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 Violet 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 DEAC-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 525/50 nm filter (Pacific Orange channel) or fluorescence microscope with Violet 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 and monitor the fluorescence intensity fluorescence microscope with Violet filter set.

Spectrum

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Product family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit Green Fluorescence	498	517	80000¹	0.7900¹, 0.95²	0.32	0.35
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit Red Fluorescence	544	570	100000	-	0.27	0.34
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit Deep Red Fluorescence	649	663	250000	0.27¹	-	0.027

Citations

View all 9 citations: Citation Explorer

Regulation of YAP Translocation by Myeloid Pten Deficiency Alleviates Acute Lung Injury via Inhibition of Oxidative Stress and Inflammation
Authors: Liu, Yang and Zhou, Wenqin and Zhao, Jiaying and Chu, Mingqiang and Xu, Mingcui and Wang, Xiao and Xie, Liangjie and Zhou, Ying and Song, Lijia and Wang, Jian and others,
Journal: Free Radical Biology and Medicine (2024)

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

References

View all 4 references: Citation Explorer

Complement C3 participates in the function and mechanism of traumatic brain injury at simulated high altitude.
Authors: Wei, Linjie and Zhang, Jianbo and Zhang, Bo and Geng, Junjun and Tan, Qiang and Wang, Ling and Chen, Zhi and Feng, Hua and Zhu, Gang
Journal: Brain research (2020): 146423

[Effect of qihong capsule in inhibiting cell apoptosis induced by Coxsackie virus B].
Authors: Song, Xiao-dong and Wang, Lun and Ji, Bo
Journal: Zhongguo Zhong xi yi jie he za zhi Zhongguo Zhongxiyi jiehe zazhi = Chinese journal of integrated traditional and Western medici (2005): 511-5

Dual excitation multi- fluorescence flow cytometry for detailed analyses of viability and apoptotic cell transition.
Authors: Mazzini, G and Ferrari, C and Erba, E
Journal: European journal of histochemistry : EJH (2003): 289-98

Combined TUNEL and double immunofluorescent labeling for detection of apoptotic mononuclear phagocytes in autoimmune demyelinating disease.
Authors: Ray, S K and Schaecher, K E and Shields, D C and Hogan, E L and Banik, N L
Journal: Brain research. Brain research protocols (2000): 305-11

Page updated on October 8, 2024

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