
- 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 |
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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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1、多种染料及淬灭基团自由选择。
2、专利的ZENTM或TAOTM双重淬灭
基团探针设计:
·更低的背景荧光
·增强endpoint信号强度
·较少交叉污染
多种荧光及淬灭剂用于多种实验需求
PrimeTimeqPCR探针灵敏度高,可靠性大,可用于多重或数字PCR。
淬灭基团染料有多种选择(如下图),与常见的qPCR平台兼容。
为您提供严格质控的高质量探针
所有的PrimeTime探针均通过质谱和HPLC纯化验证,不同批次间产物高度一致,为您减少后顾之忧。
双淬灭基团探针提高检测灵敏度
使用ZEN或者TAODouble-Quenched探针减少背景干扰,提高检测灵敏度。独有的内部淬火剂总是位于5’端荧光基团后9个碱基与3’端IowaBlack®淬灭剂一起作用最大化探针性能(如下图)。
与传统方法相比,可以将背景噪音降低4倍(如下图A)和信号值提高大约30%(如下图B),ZENDouble-Quenched探针给出更优的表现。
对于长探针同样高效适用
对于40碱基的长探针也可以有效淬灭,这意味着在探针设计上有更多选择,比如富含AT的区域。
希望对有需要的朋友有帮助,IDT探针合成,2073904839qq.com,www.nanodigmbio.com,
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。
1.模板提取(一般为RNA):Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定:分光光度计或NanoDrop
3.逆转录:逆转录试剂盒(或者一步法试剂盒),这一步可以用普通PCR做,也可以用水域做。
4.荧光定量PCR试剂:通常有用SYBR Green Mix做的,但是这里建议你用EvaGreen做,灵敏度和平行性都要好于SYBR Green,并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他:除了以上的那些还需要离心管、PCR管或板(Axygen反应比较好)、移液枪等,暂时就想到这么多。
跑到还有半小时的时候,跳出一个对话框:“analysiscannotproceed:notenoughsamplesdefined”,最后做出来的结果有扩增曲线但是没有熔解曲线,请各位大神帮忙解释一下。
两步法,是因为设计引物的时候把退火温度设为酶的工作温度,而且定量PCR的产物都很短,需要的时候都短,所以两步法更方便。三步法的话,花的时间长,不利于快速实验。
Real time PCR(也称实时定量PCR)
定量PCR已经从基于凝胶的低通量分析发展到高通量的荧光分析技术,即实时定量PCR。实时荧光定量PCR技术于1996年由美国Applied Biosystems公司推出,由于该技术不仅实现了PCR从定性到定量的飞跃,而且与常规PCR相比,它具有特异性更强、有效解决PCR污染问题、自动化程度高等特点。实时定量PCR (real-time quantitative PCR)是指在PCR指数扩增期间通过连续监测荧光信号强弱的变化来即时测定特异性产物的量,并据此推断目的基因的初始量,不需要取出PCR产物进行分离。目前实时定量PCR作为一个极有效的实验方法,已被广泛地应用于分子生物学研究的各个领域。
实时荧光定量PCR 技术的主要应用:
1. DNA 或RNA 的绝对定量分析:包括病原微生物或病毒含量的检测,转基因动植物转基因拷贝数的检测,RNAi 基因失活率的检测等
2. 基因表达差异分析:例如比较经过不同处理样本之间特定基因的表达差异(如药物处理、物理处理、化学处理等 ),特定基因在不同时相的表达差异以及cDNA 芯片或差显结果的确证
3. 基因分型:例如SNP 检测,甲基化检测等
Realtime PCR 常用的两种方法分别为:Sybr green(荧光染料掺入法) 和Taqman probe (探针法)
SYBR green
在PCR反应体系中,加入过量SYBR荧光染料,SYBR荧光染料特异性地掺入DNA双链后,发射荧光信号,而不掺入链中的SYBR染料分子不会发射任何荧光信号,从而保证荧光信号的增加与PCR产物的增加完全同步。
此方法适用:
1、灵敏度高:使用SYBR可使荧光效果增强到1000倍以上
2、通用性好,不需要设计探针,方法简便,省时,价格低廉。
3、通用型方法,在国内外科研中普遍使用。
4、高通量大规模的定量PCR检测
5、专一性要求不高的定量PCR检测。
Taqman Probe
PCR扩增时在加入一对引物的同时加入一个特异性的荧光探针,该探针为一寡核苷酸,两端分别标记一个报告荧光基团和一个淬灭荧光基团。探针完整时,报告基团发射的荧光信号被淬灭基团吸收;PCR扩增时,Taq酶的5’-3’外切酶活性将探针酶切降解,使报告荧光基团和淬灭荧光基团分离,从而荧光监测系统可接收到荧光信号,即每扩增一条DNA链,就有一个荧光分子形成,实现了荧光信号的累积与PCR产物形成完全同步
此方法适用:
1、具有高适应性和可靠性,实验结果稳定重复性好,特异性更高。
2、适用于扩增序列专一的体系的检测。
3、样品中靶基因含量过低的定量PCR检测。
4、靶基因的特异序列较短,无论怎样优化引物设计条件都不能解决。
5、存在与靶基因同源的序列,在PCR中容易出现非特异性扩增,对特异性要求较高的定量。
6、广泛用于人类传染病的诊断和病原定量,在动物病原体基因的检测,畜禽产品的检验检疫,生物制品的鉴定。
数字PCR基本原理如下:
想请教大神们,怎样设计INT引物、MB-red探针以及MB-green探针?

