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如何完成一次高效又经济的Tunel检测?

——Yeasen TUNEL detection Kit 让“美”原位呈现

 

通过TdT转移酶将Fluorescein-dUTP结合到有缺口的DNA上是检测细胞凋亡的常用方法之一,如Roche提供的In Situ Cell Death Kit正是基于这个原理。产品中的TdT转移酶活性直接影响到标记的效率。翌圣生物立足于酶制剂生产,力求开发以酶制剂为基础的较高具性价比产品,推出三款荧光素(Alexa Fluor 640Alexa Fluor 488FITC)标记的Tunel检测试剂,在满足客户对产品高品质要求的同时又可以享受低至Roche一半的价格。

目前已经有超过120个课题组正在使用该产品,并在权威杂志上发表多篇英文文章,如Theranostics和EMBO Molecular Medicine等。

 

检测机理:

 

细胞凋亡晚期,染色体DNA双链断裂或单链断裂产生大量的粘性3-OH末端,在脱氧核糖核苷酸末端转移(TdT)的作用下,将荧光素/酶标记的dUTP结合到DNA的3-末端,从而可通过检测荧光完成对细胞凋亡的检测,这类方法称为脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(Terminal-Deoxynucleotidyl Transferase Mediated Nick End Labeling,TUNEL),原理见图1。

图片1.png

图1:脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)原理图

 

产品特点:

 

适用范围广:可用于石蜡包埋组织切片、冰冻组织切片、培养的细胞和从组织中分离的细胞。

检测灵敏度高:可检测出极少量的凋亡细胞。

背景干扰小:信噪比高。

观察多样性:荧光显微镜观察,流式检测。

 

数据展示:

 

1、小鼠前脂肪细胞(3T3-L)Tunel检测

 

图片2.png

图2:小鼠前脂肪细胞3T3-L凋亡检测。第一行代表阴性对照,第二行代表阳性对照。

检测试剂:Cat No. 40306 TUNEL Apoptosis Detection Kit (FITC)

 

2、石蜡切片样本的Tunel检测

 

图片3.png

图3:在皮下移植瘤模型中,单独使用LY3009120或联合使用Vemurafenib和Obatoclax可以有效延缓甲状腺癌。IF=8.8

样本类型:石蜡包埋的肿瘤组织(取自裸鼠)。检测试剂:Cat No. 40307 TUNEL Apoptosis Detection Kit (Alexa Fluor 488)。

 

3、细胞爬片的Tunel检测

 

图片4.png

图4:硫酸锌对不同分组的内皮细胞凋亡的影响 

样本类型:HUVEC(人脐静脉内皮细胞)。检测试剂:Cat No. 40308 TUNEL Apoptosis Detection Kit (Alexa Fluor 640)

 

4、流式细胞术检测细胞凋亡

 

图片5.png

图5:PA处理可以有效降低重编程(reprogramming)过程中凋亡的发生。IF=3.7

样本类型:iPS Cells。检测试剂:Cat No. 40307 TUNEL Apoptosis Detection Kit (Alexa Fluor 488)。

 

文献引用:

 

[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 )

 

常见问题:

 

Q:出现非特异性荧光标记?

 

1)组织/细胞本身核酶或聚合酶活性水平较高,易导致出现非特异性的荧光标记,例如平滑肌细胞。解决方法是,取细胞或组织后立即固定并且要充分固定,以阻止这些酶导致假阳性。
2)使用了不适当的固定液,例如一些酸性固定液,导致出现假阳性。建议采用新鲜配置的4%中性对聚甲醛固定液。
3)TUNEL检测反应时间过长,细胞或组织表面不能保持湿润,也可能出现非特异性荧光。注意控制反应时间,并确保TUNEL检测反应液能很好地覆盖样品。

 

Q:荧光背景很高?

 

1)高速分裂和增殖的细胞,转录水平高,有时也会出现细胞核中的DNA断裂。
2)在激发光下长时间的暴露,导致假阳性的出现。

 

Q:标记效率低?

 

1)使用乙醇或甲醇固定会导致标记的效率较低。

2)固定时间过长,导致交联程度过高。此时宜减少固定时间。
3)组织切片过厚,使得固定效果不理想,最好控制在10 μm以内。

4)荧光淬灭。Fluorescence在普通光照10min就会严重淬灭。解决方法是需注意避光操作。

 

产品选购:

 

产品

货号

规格

价格(元)

TUNEL Apoptosis Detection Kit (FITC)

TUNEL细胞凋亡检测试剂盒(FITC)  

40306ES20

20T

1178.00

40306ES50

50T

2680.00

40306ES60

100T

4080.00

TUNEL Apoptosis Detection Kit (Alexa Fluor 488)

TUNEL细胞凋亡检测试剂盒(Alexa Fluor 488)

40307ES20

20T

1250.00

40307ES50

50T

3080.00

40307ES60

100T

4800.00

TUNEL Apoptosis Detection Kit (Alexa Fluor 640)

TUNEL细胞凋亡检测试剂盒(Alexa Fluor 640)

40308ES20

20T

1250.00

40308ES50

50T

3080.00

40308ES60

100T

4800.00

 

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更多产品敬请详询400-6111-883

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400-6111-883