如何完成一次高效又經濟的Tunel檢測? ——Yeasen TUNEL detection Kit 讓“美”原位呈現

來源: 上海翊圣生物科技有限公司   2020-6-7   訪問量:367評論(0)

如何完成一次高效又經濟的Tunel檢測?

——Yeasen TUNEL detection Kit 原位呈現

通過TdT轉移酶將Fluorescein-dUTP結合到有缺口的DNA上是檢測細胞凋亡的常用方法之一,如Roche提供的In Situ Cell Death Kit正是基于這個原理。產品中的TdT轉移酶活性直接影響到標記的效率。翊圣生物立足于酶制劑生產,力求開發以酶制劑為基礎的最具性價比產品,推出三款熒光素(Alexa Fluor 640,Alexa Fluor 488,FITC)標記的Tunel檢測試劑,在滿足客戶對產品高品質要求的同時又可以享受低至Roche一半的價格。

目前已經有超過120個課題組正在使用該產品,并在權威雜志上發表多篇英文文章,如TheranosticsEMBO Molecular Medicine等。

檢測機理:

細胞凋亡晚期,染色體DNA雙鏈斷裂或單鏈斷裂產生大量的粘性3-OH末端,在脫氧核糖核苷酸末端轉移(TdT)的作用下,將熒光素/酶標記的dUTP結合到DNA3-末端,從而可通過檢測熒光完成對細胞凋亡的檢測,這類方法稱為脫氧核糖核苷酸末端轉移酶介導的缺口末端標記法(Terminal-Deoxynucleotidyl Transferase Mediated Nick End Labeling,TUNEL),原理見圖1。

1脫氧核糖核苷酸末端轉移酶介導的缺口末端標記(TUNEL)原理圖

產品特點:

適用范圍廣:可用于石蠟包埋組織切片、冰凍組織切片、培養的細胞和從組織中分離的細胞。

檢測靈敏度高:可檢測出極少量的凋亡細胞。

背景干擾。信噪比高。

觀察多樣性:熒光顯微鏡觀察,流式檢測。

數據展示:

1、小鼠前脂肪細胞(3T3-LTunel檢測

2:小鼠前脂肪細胞3T3-L凋亡檢測。第一行代表陰性對照,第二行代表陽性對照。

檢測試劑:Cat No. 40306 TUNEL Apoptosis Detection Kit (FITC)

2、石蠟切片樣本的Tunel檢測

3:在皮下移植瘤模型中,單獨使用LY3009120或聯合使用VemurafenibObatoclax可以有效延緩甲狀腺癌[5]。IF=8.8

樣本類型:石蠟包埋的腫瘤組織(取自裸鼠)。檢測試劑:Cat No. 40307 TUNEL Apoptosis Detection Kit (Alexa Fluor 488)。

3、細胞爬片的Tunel檢測

4:硫酸鋅對不同分組的內皮細胞凋亡的影響[11]

樣本類型:HUVEC(人臍靜脈內皮細胞)。檢測試劑:Cat No. 40308 TUNEL Apoptosis Detection Kit (Alexa Fluor 647)

4、流式細胞術檢測細胞凋亡

5PA處理可以有效降低重編程(reprogramming)過程中凋亡的發生[9]。IF=3.7

樣本類型:iPS Cells。檢測試劑:Cat No. 40307 TUNEL Apoptosis Detection Kit (Alexa Fluor 488)。

文獻引用:

[1] Li C, Wang Q, Gu X, et al. Porous [email protected] 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 )

常見問題:

Q:出現非特異性熒光標記?

1)組織/細胞本身核酶或聚合酶活性水平較高,易導致出現非特異性的熒光標記,例如平滑肌細胞。解決方法是,取細胞或組織后立即固定并且要充分固定,以阻止這些酶導致假陽性。

2)使用了不適當的固定液,例如一些酸性固定液,導致出現假陽性。建議采用新鮮配置的4%中性對聚甲醛固定液。

3TUNEL檢測反應時間過長,細胞或組織表面不能保持濕潤,也可能出現非特異性熒光。注意控制反應時間,并確保TUNEL檢測反應液能很好地覆蓋樣品。

Q:熒光背景很高?

1)高速分裂和增殖的細胞,轉錄水平高,有時也會出現細胞核中的DNA斷裂。

2)在激發光下長時間的暴露,導致假陽性的出現。

Q:標記效率低?

1)使用乙醇或甲醇固定會導致標記的效率較低。

2)固定時間過長,導致交聯程度過高。此時宜減少固定時間。

3)組織切片過厚,使得固定效果不理想,最好控制在10 μm以內。

4)熒光淬滅。Fluorescence在普通光照10min就會嚴重淬滅。解決方法是需注意避光操作。

產品選購:

產品

貨號

規格

價格(元)

TUNEL Apoptosis Detection Kit (FITC)

TUNEL細胞凋亡檢測試劑盒(FITC

40306ES20

20T

1178.00

40306ES50

50T

2680.00

40306ES60

100T

4080.00

TUNEL Apoptosis Detection Kit (Alexa Fluor 488)

TUNEL細胞凋亡檢測試劑盒(Alexa Fluor 488

40307ES20

20T

1250.00

40307ES50

50T

3080.00

40307ES60

100T

4800.00

TUNEL Apoptosis Detection Kit (Alexa Fluor 640)

TUNEL細胞凋亡檢測試劑盒(Alexa Fluor 640

40308ES20

20T

1250.00

40308ES50

50T

3080.00

40308ES60

100T

4800.00

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