How to perform an efficient and cost-effective Tunel assay?

--Yeasen TUNEL assay kit shows more beautiful fluorescence

The binding of Fluorescein-dUTP to nicked DNA by TdT transferase is one of the most common methods for detecting apoptosis, e.g. the In Situ Cell Death Kit from Roche is based on this principle. The TdT transferase activity in the product directly affects the labeling efficiency. Yeasen Biologicals, based on enzyme production, strives to develop high-quality products based on enzymes, and has launched three fluorescein (Alexa Fluor 640, Alexa Fluor 488, and FITC)-labeled Tunel assays to meet the high quality requirements of our customers.

Currently, more than 120 research groups are using this product and have published many English articles in authoritative journals, such as Theranostics and EMBO Molecular Medicine.

Detection Mechanism

In the late stage of apoptosis, chromosomal DNA double-strand breaks or single-strand breaks produce a large number of sticky 3-OH ends, and under the action of deoxyribonucleotide terminal transfer (TdT), fluorescein/enzyme-labeled dUTP is bound to the 3-terminal end of the DNA, so that detection of apoptosis can be accomplished through the detection of fluorescence, which is referred to as the deoxyribonucleotide terminal transferase-mediated nick end labeling This method is called Terminal-Deoxynucleotidyl Transferase Mediated Nick End Labeling (TUNEL), and the principle is shown in Figure 1.

Figure 1: Schematic diagram of Terminal-Deoxynucleotidyl Transferase Mediated Nick End Labeling (TUNEL)

Product Features

  • Wide range of application

Can be used for paraffin-embedded tissue sections, frozen tissue sections, cultured cells and cells isolated from tissues.

  • High detection sensitivity

Very small amount of apoptotic cells can be detected.

  • Low background interference

High signal-to-noise ratio.

  • Observation versatility

Fluorescence microscopy observation, flow-through detection.

Data presentation

1、Mouse preadipocyte (3T3-L) Tunel assay

Figure 2: Mouse preadipocyte 3T3-L apoptosis detection. The first row represents negative control and the second row represents positive control. Detection reagent: Cat No. 40306 TUNEL Apoptosis Detection Kit (FITC)

2、Tunel detection of paraffin section samples

Figure 3: LY3009120 alone or in combination with Vemurafenib and Obatoclax effectively delayed thyroid cancer in a subcutaneous graft tumor model. IF=8.8. Sample type: paraffin-embedded tumor tissue (taken from nude mice). Detection reagent: Cat No. 40307 TUNEL Apoptosis Detection Kit (Alexa Fluor 488).

3、Tunel detection of cell crawls

Figure 4: Effect of zinc sulfate on apoptosis of endothelial cells in different subgroups. Sample type: HUVEC (human umbilical vein endothelial cells). Detection reagent: Cat No. 40308 TUNEL Apoptosis Detection Kit (Alexa Fluor 640)

4、Flow cytometry to detect apoptosis

Figure 5: PA treatment can effectively reduce the occurrence of apoptosis during reprogramming. IF=3.7. Sample type: iPS Cells. Detection reagent: Cat No. 40307 TUNEL Apoptosis Detection Kit (Alexa Fluor 488).

Literature Citation

[1] Li C, Wang Q, Gu X, et al. Porous Se@ SiO2 nanocomposite promotes migration and osteogenic differentiation of rat bone marrow mesenchymal stem cell to accelerate bone fracture healing in a rat model[J]. International Journal of Nanomedicine, 2019, 14: 3845. (IF 4.76)

[2] Miao T, Qian L, Yu F, et al. Protective effects of hydroxysafflor yellow an on high oxidized low density lipoprotein induced human coronary artery endothelial cells injuries[J]. Development, 2019, 22: 581-589.(IF 6.208 )

[3] Wen Y, Liu G, Zhang Y, et al. MicroRNA-205 is associated with diabetes mellitus‐induced erectile dysfunction via down-regulating the androgen receptor[J]. Journal of cellular and molecular medicine, 2019, 23(5): 3257-3270.(IF 4.41)

[4] Xu T, Ding W, Ao X, et al. ARC regulates programmed necrosis and myocardial ischemia/reperfusion injury through the inhibition of mPTP opening[J]. Redox Biology, 2019, 20: 414-426.(IF 8.37)

[5] Li P, Hao L, Guo Y Y, et al. Chloroquine inhibits autophagy and deteriorates the mitochondrial dysfunction and apoptosis in hypoxic rat neurons[J]. Life Sciences, 2018, 202: 70-77.(IF 3.40)

[6] Chen H, Guan B, Chen X, et al. Baicalin attenuates blood-brain barrier disruption and hemorrhagic transformation and improves neurological outcome in ischemic stroke rats with delayed t-PA treatment: involvement of ONOO−-MMP-9 pathway[J]. Translational Stroke Research, 2018, 9(5): 515-529.(IF 4.87)

[7] Liu Q, Qian Y, Li P, et al. The combined therapeutic effects of 131iodine-labeled multifunctional copper sulfide-loaded microspheres in treating breast cancer[J]. Acta Pharmaceutica Sinica B, 2018, 8(3): 371-380.(IF 6.88)

[8] Han Y Q, Ming S L, Wu H T, et al. Myostatin knockout induces apoptosis in human cervical cancer cells via elevated reactive oxygen species generation[J]. Redox Biology, 2018, 19: 412-428.(IF 8.37)
[9] Wang S, Xu Y, Weng Y, et al. Astilbin ameliorates cisplatin-induced nephrotoxicity through reducing oxidative stress and inflammation[J]. Food and Chemical Toxicology, 2018, 114: 227-236.(IF 3.78)

[10] Liu L, Pang X L, Shang W J, et al. Over-expressed microRNA-181a reduces glomerular sclerosis and renal tubular epithelial injury in rats with chronic kidney disease via down-regulation of the TLR/NF-κB pathway by binding to CRY1[J]. Molecular Medicine, 2018, 24(1): 49.(IF 3.46)

[11] Li G, Yin Q, Ji H, et al. A study on screening and antitumor effect of CD55-specific ligand peptide in cervical cancer cells[J]. Drug Design, Development and Therapy, 2018, 12: 3899.(IF 3.27)

[12] Qian Y, Wang Y, Jia F, et al. Tumor-microenvironment controlled nanomicelles with AIE property for boosting cancer therapy and apoptosis monitoring[J]. Biomaterials, 2019, 188: 96-106.(IF 9.85)

[13] Li Z, Li D, Li Q, et al. In situ low-immunogenic albumin-conjugating-corona guiding nanoparticles for tumor-targeting chemotherapy[J]. Biomaterials Science, 2018, 6(10): 2681-2693.(IF 5.31)

[14] Liu Y, Zhi X, Hou W, et al. Gd3+-Ion-induced carbon-dots self-assembly aggregates loaded with a photosensitizer for enhanced fluorescence/MRI dual imaging and antitumor therapy[J]. Nanoscale, 2018, 10(40): 19052-19063.(IF 7.17)
[15] Liu Q, Qian Y, Li P, et al. 131 I-labeled copper sulfide-loaded microspheres to treat hepatic tumors via hepatic artery embolization[J]. Theranostics, 2018, 8(3): 785.(IF 8.12 )

Frequently Asked Questions

Q: Appearance of non-specific fluorescent labeling?

1) The tissue/cells themselves have high levels of nuclease or polymerase activity, which can easily lead to the appearance of non-specific fluorescent labeling, such as smooth muscle cells. The solution is to fix the cells or tissues immediately after taking them and to fix them sufficiently to stop these enzymes from causing false positives.

2) The use of inappropriate fixatives, such as some acidic fixatives, leads to false positives. Freshly prepared 4% neutral paraformaldehyde fixative is recommended.

3) Non-specific fluorescence may also occur when the reaction time of the TUNEL assay is too long and the cell or tissue surface cannot be kept wet. Take care to control the reaction time and make sure the TUNEL assay reaction solution covers the sample well.

Q: The fluorescence background is very high?

1) Cells that divide and proliferate at a high rate, with high levels of transcription, sometimes also show DNA breaks in the nucleus.

2) Prolonged exposure to excitation light leads to false positives.

Q: Low labeling efficiency?

1) Fixation with ethanol or methanol leads to low labeling efficiency.

2) Fixation time is too long, resulting in too high a degree of cross-linking. It is advisable to reduce the fixation time.

3) Tissue section is too thick, which makes the fixation effect unsatisfactory, and it is better to control within 10 μm.

(4) Fluorescence quenching: Fluorescence will be severely quenched in 10 min of normal light. The solution is to avoid light operation.

Product Details

Product Name

Item No.

Specification

Annexin V-FITC/7-AAD Apoptosis Detection Kit

40311ES20/50/60

20T/50T/100T

Annexin V-FITC/PI Apoptosis Detection Kit

40302ES20/50/60

20T/50T/100T

Annexin V-PE/7-AAD Apoptosis Detection Kit

40310ES20/50/60

20T/50T/100T

Annexin V-EGFP/PI Apoptosis Detection Kit

40303ES20/50/60

20T/50T/100T

Annexin V-YSFluor™ 488/7-AAD Apoptosis Detection Kit

40313ES60

100T

Annexin V-YSFluor™ 488/PI Apoptosis Detection Kit

40305ES20/50/60

20T/50T/100T

Annexin V-YSFluor™ 647/PI Apoptosis Detection Kit

40304ES20/50/60

20T/50T/100T

Annexin V-YSFluor™ 647/7-AAD Apoptosis Detection Kit

40312ES20/50/60

20T/50T/100T

TUNEL Apoptosis Detection Kit (FITC)

40306ES20/50/60

20T/50T/100T

TUNEL Apoptosis Detection Kit (YSFluor™ 488)

40307ES20/50/60

20T/50T/100T

TUNEL Apoptosis Detection Kit (YSFluor™ 640)

40308ES20/50/60

20T/50T/100T