- Description
- Additional Information
- Readable Documents
- Assay Principle
- Reviews
Key Benefits
- Cell permeability allow direct measurement of apoptosis and mitochondrial potential in live cells.
- Applications – Cells can be analyzed by Flow Cytometry, Fluorescent plate reader or Fluorescent microscopy.
- Incubate for 15 minutes, wash and measure.
- Add this reagent directly to live cells in your media of choice.
Additional information
| Kit Size | 100 |
|---|
Detection of the mitochondrial permeability transition event provides an early indication of the initiation of cellular apoptosis. This process is typically defined as a collapse in the electrochemical gradient across the mitochondrial membrane, as measured by the change in the membrane potential (YD). Loss of mitochondrial (YD) is indicative of apoptosis and can be detected by a unique fluorescent cationic dye, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl- benzamidazolocarbocyanin iodide, commonly known as JC-1. This dye has been incorporated into the user-friendly kit for the simple and reproducible detection of the membrane potential (YD) event in apoptotic cells. The kit has been formatted for use on Flow cytometers, Fluorescent plate readers and Fluorescent Microscopes
Fig (A). Jurkat cells were cultured with DMSO for 2 hours. Cells were then stained with JC-1 Mitochondrial Membrane Potential Detection Kit for 15 minutes and analyzed by flow cytometry.
Fig (B). urkat cells were cultured with staurosporine for 2 hours. Cell were then stained with JC-1 Mitochondrial Membrane Potential Detection Kit for 15 minutes and analyzed by flow cytometry.
| Document Title |
| JC1Protocol |
| JC1 Datasheet |
| msds.JC100 |
| Title | File | Link | Author(s) | Journal | Year; Edition:Pages |
| http://www.jem.org/cgi/content/full/199/4/547 | http://www.jem.org/cgi/content/full/199/4/547 | ||||
| Defects in Cell Growth Regulation by C 18:0-Ceramide and Longevity Assurance Gene 1 (LAG1) in Human Head and Neck Squamous Cell Carcinomas (HNSCC) | Serap Koybasi, Can E. Senkal, Kamala Sundararaj, et al | JBC Papers in Press | Published on August 17, 2004 as Manuscript M406920200 | ||
| Effects of a series of organosulfur compounds on mitotic arrest and induction of apoptosis in colon cancer cells | http://mct.aacrjournals.org/cgi/content/full/4/9/1388 | Danhua Xiao, John T. Pinto, Gregg G. Gundersen & Bernard Weinstein | Mol Cancer Ther | 2005;4:1388-1398 | |
| Adrenergic receptor-stimulated apoptosis in adult cardiac myocytes involves MMP-2-mediated disruption of β1 integrin signaling and mitochondrial pathway | http://ajpcell.physiology.org/cgi/content/abstract/290/1/C254 | Bindu Menon,Mahipal Singh, Robert Ross, Jennifer N. Johnson and Krishna Singh | Am J Physiol Cell Physiol | 290: C254-C261, 2006. First published September 7, 2005 | |
| SGLT-1-mediated glucose uptake protects intestinal epithelial cells against LPS-induced apoptosis and barrier defects: a novel cellular rescue mechanism? | http://www.fasebj.org/cgi/content/abstract/19/13/1822 | Linda C. H. Yu Andrew N. Flynn, Jerrold R. Turner and Andre G. Buret | The FASEB Journal | 2005;19:1822-1835 | |
| Ceramide induces mitochondrial abnormalities in insulin-secreting INS-1 cells: Potential mechanisms underlying ceramide-mediated metabolic dysfunction of the β cell | http://www.springerlink.com/content/t3g0p313518r2747/ | R. Veluthakal, R. Palanivel Y. Zhao, P. McDonald, S. Gruber and A. Kowluru | Apoptosis Journal | Vol 10/No 4, Aug 2005 | |
| Opposing effects of bovine papillomavirus type 1 E6 and E7 genes on Fas-mediated apoptosis | http://www.nature.com/onc/journal/v24/n24/abs/1208542a.html | Yun Liu, Zhiguo Liu, Hua Gao, You Zhou, Elliot J Androphy and Jason J Chen | Oncogene | (March 2005) 24, 3942–3953 | |
| Resveratrol-caused apoptosis of human prostate carcinoma LNCaP cells is mediated via modulation of phosphatidylinositol 3"-kinase/Akt pathway and Bcl-2 family proteins | http://mct.aacrjournals.org/cgi/content/abstract/5/5/1335 | Moammir H. Aziz,Minakshi Nihal, Vivian X. Fu, David F.Jarrard and N Ahmad | Mol Cancer Ther | 2006;5:1335-1341 |
| Reference |
| Desagher, S., Osen-Sand, A., Nichols, A., Eskes, R., Montessuit, S., Lauper, S., Maundrell, K., Antonsson, B., and Martinou, J.C. Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J. Cell Biol. 144 (5): 891-901 (1999). |
| Narita, M., Shimizu, S., Ito, T., Chittenden, T., Lutz, R. J., Matsuda, H., and Tsujimoto, Y. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc. Natl. Acad. Sci. USA 95: 14681-14686 (1998). |
| Basanez, G., Nechushtan, A., Drozhinin, O., Chanturiya, A., Choe, E., Tutt, S., Wood, K. A., Hsu, Y. T., Zimmerberg, J., and Youle, R. J. Bax , but not Bcl-XL decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations. Proc. Natl. Acad. Sci. USA 96: 5492-5497 (1999). |
| Luo, X., Budihardio, I., Zou, H., Slaughter, C., and Wang, X. Bid, a Bcl-2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94: 481-490 (1998). |
| Smiley, S. T., Reers, M., Mottola-Hartshorn, C., Lin, M., Chen, A., Smith, T. W., Steele, G.D., and Chen, L. B. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate forming lipophilic cation JC-1. Proc. Natl. Acad. Sci. USA 88: 3671-3675 (1991). |
| Cossarizza, A., Baccarani-Contri, M., Kalashnikova, G., and Franceschi, C. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Biochem. Biophys. Res. Commun. 197 (1): 40-45 (1993). |
| Reers, M., Smith, T. W., and Chen, L. B. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30: 4480-4486 (1991). |
| White, R. J., and Reynolds, I. J. Mitochondrial depolarization in glutamatestimulated neurons: an early signal specific to excitotoxin exposure. Journal of Neuroscience 16: 5688-5697 (1996). |
| Part# | Reagent | Temperature |
| Part# 4001 | Lyophilized JC-1 Dye | Store at 2-8C |
| Part# 3002 | 10X Assay Buffer | Store at 2-8C |
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我做的是用锁式探针来检测dna模板的ram,探针80base,其中杂交区40base,引物分别为16、18base,连接酶选取的T4 ligase和Taq DNA ligase两种,分别对应恒温和变温情况下的扩增。水解体系为EXO I和Exo Ⅲ的混合体系,参考的外文文献,应该没什么问题。聚合酶用的是Bst DNA大片段聚合酶。
一开始选择的探针浓度为200pmol/L,扩增效果挺好,当时用的DNA模板是质粒阳性标准品,但是后来在做敏感性的时候发现条带梯度差异几乎没有,而且阴性也出带,后来就没法做敏感性和特异性验证了,实验停滞了2个月一直也没进展。
按理说RAM借助锁式探针扩增的独特方式以及水解体系的存在可以有效防止污染才对,希望大家给点建议该如何解决。
万分感谢!!!!!!!!!!
附一张以前做敏感性的图(marker为2000的DNA ladder)以做参考
组织用Trizol法提取总RNA浓度、纯度都很好,但逆转录后跑PCR,CT值偏高,内参的在22-27之间,目的基因在30-36之间,不知道是怎么回事,如何来解决,求高手赐教。我用的Takara的试剂盒。
还需要反转录用的引物、定量PCR用的一对引物、荧光信号源(荧光染料或荧光标记的探针,看你用什么策略来做定量PCR)
报价要看你需要服务商提供什么服务,如果你只提供实验材料,要求服务商做以上全部工作,只检测一个指标(一个基因的表达水平变化),价格大约每个样品350-500元,多一个指标大约多90元。
一般至少做一组数据两个样品嘛,所以你按1000块一组样品预算吧。

