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默克公司TUNEL荧光法凋亡检测试剂盒QIA39 FragEL™ DNA Fragmentation Detection Kit, Fluorescent – TdT Enzyme使用评测
2011年11月28日 产品评测 评论数 1 ⁄ 被围观 8,122+


情况综述

产品

默克公司TUNEL荧光法凋亡检测试剂盒:QIA39 FragEL™ DNA Fragmentation Detection Kit, Fluorescent - TdT Enzyme from EMD Millipore (Calbiochem)

优点

基于荧光检测的TUNEL试剂盒,利用末端脱氧核苷酸转移酶(TdT)的作用,将荧光素标记及未标记的dNTP渗入过程中产生的DNA片段的3’-OH末端。经激发后荧光素产生的荧光信号能经荧光显微镜及流式细胞仪检测。试剂盒中提供的防荧光淬灭剂可维持样本片段产生的荧光信号,有助于形态学分析及鉴定正常与凋亡的细胞。未凋亡细胞可通过DAPI filter观察。

即使在单细胞水平也可以快速,灵敏,特异性在DNA片段插入荧光信号,且可用于基于DNA片段凋亡的原位检测

缺点

试剂和比较贵,试剂盒缺少一些试剂,而且TdT酶量很少。

总结

可以广泛地应用于石蜡包埋组织切片、组织冰冻切片、细胞涂片以及细胞悬液等原位DNA片段化凋亡检测

背景介绍:

细胞凋亡是细胞的一种基本生物学现象,在生物体进化、内环境的稳定以及系统发育中发挥着重要的作用。细胞凋亡发生在正常的细胞周期循环中,并发生形态学和生理生化的特征性改变,比如细胞皱缩,细胞间连接消失,线粒体膜电位消失,通透性改变,核质浓缩,核膜核仁破碎,DNA降解成为约180bp-200bp 的片段;胞膜有小泡状突起,膜内侧磷脂酰丝氨酸外翻到膜表面,胞膜结构仍保持完整,最终凋亡细胞遗骸被分割包裹为几个凋亡小体,并迅速被周围专职或非专职吞噬细胞吞噬。以上改变发生在不同的细胞凋亡阶段。

检测原理:

在凋亡晚期细胞中,DNA 会被降解为不同大小的片段,正常的或正在增殖的细胞则几乎没有DNA 的断裂,该方法是将荧光素(Fluorescein)标记和未标记的dNTP 在脱氧核糖核苷酸末端转移酶(TdT 酶)的作用下,连接到凋亡细胞中断裂DNA 片段的3’-OH 末端,然后进行荧光显微镜或流式细胞仪检测。试剂盒对标记反应进行了优化,采用最佳比例的荧光素标记和未标记的dNTP进行3’-OH末端的核苷酸掺入,使得同一个断裂的DNA片段末端可以形成更长的“标记尾巴”,该“标记尾巴”减少了相邻掺入dNTP上标记基团的空间位阻,增加每个断裂片段后的荧光基团数目,降低荧光基团相邻后可能造成的聚集和淬灭,从而提高检测灵敏度,减少非特异性反应。此外试剂盒提供的封片剂中含有DAPI 核染料,可以将全部标记和未标记细胞的DNA 着色,并在330-380nm波长激发光下发出荧光,从而得以判断凋亡细胞占总细胞比例。封片剂还可以稳定和增强荧光信号,减少淬灭,从而提高检测的灵敏度。

检测次数:50 次

检测方法:荧光显微镜或流式细胞仪

样本类型:石蜡包埋组织切片、组织冰冻切片、细胞涂片以及细胞悬液

种属反应:一系列不同种属

储存与运输条件:试剂盒需用干冰运输并储存在-20℃非无霜冰箱中

Apoptosis is a form of highly organized and programmed cell death; it does not involve lysis or damage to neighboring cells. It plays a fundamental role in many biological processes such as morphogenesis and negative selection in the immune system. In addition to its importance as a biological phenomenon, defective apoptotic processes have been implicated in an extensive variety of diseases. Excessive apoptosis causes atrophy, such as in ischemic tissue damage and neurodegeneration, whereas reduced apoptosis results in cancer. Apoptotic cells can be recognized by a characteristic pattern of morphological, biochemical and molecular changes. During apoptosis, cellular endonucleases cleave nuclear DNA between nucleosomes, producing a mix of DNA fragments whose length varies in multiples of 180 to 200 bp. Degradation of nuclear DNA into nucleosomal units is one of the hallmark features of apoptotic cell death [1].

The detection of DNA fragmentation relies heavily on techniques involving the extraction of nuclear DNA and characterization of such oligonucleosomal ladders by gel electrophoresis. These techniques are often tedious and time-consuming. Apoptotic endonucleases not only cleave DNA between nucleosomes, but also generate free 3’-OH groups at the ends of these DNA fragments. TUNEL (Terminal deoxynucleotidyl Transferase dUTP Nick End Labeling) allows the detection of apoptosis by labelling these 3’ OH group [2]. Utilizing this technique, EMD Millipore's FragEL™ DNA Fragmentation Detection Kits allow the in situ recognition of apoptotic DNA fragments in paraffin-embedded tissue sections, tissue cryosections, or in cell preparations fixed on slides. I used the FragEL™ kit to detect apoptotic DNA fragments in the brain sections of rats [3,4] and gerbils [5,6] subjected to cerebral ischemia and in the sciatic nerve sections of diabetic rats [7,8]. The kit includes Proteinase K (2 mg/ml), TdT equilibration buffer (5X), fluorescein-fragEL labeling reaction mix, TdT enzyme and mounting media. The researcher needs to supply the following: xylene, ethanol (70, 80, 90, 100%), Tris buffer (10 mM, pH 8), TBS (20 mM Tris, pH 7.6; 140 mM NaCl), Coplin jars, humidified chamber, glass coverslips, Parafilm®, and optionally 1 mM MgSO4 and DNaseI (for use in generating positive control).

The DNA fragment labelling on paraffin embedded tissue sections using this kit involve a very simple process and can be completed within 3.5 h. Briefly, the paraffin embedded tissue sections were deparaffinised by immersing in fresh xylene for 5 min (2 times). Subsequently slides were rehydrated by incubating graded concentration (100%, 90%, 80% and 70%) of ethanol for 5 min each. Then slides were rinsed in TBS (20 mM Tris pH 7.6, 140 mM NaCl). Subsequently, the sections were incubated with 20 ug/ml proteinase K for 20 min at the room temperature. After rinsing in TBS, tissue sections were equilibrated with the terminal deoxynucleotidyl transferase (TdT) equilibration buffer and incubated with the labeling reaction mixture in a humidification chamber for a period of 90 min at 37°C. TdT binds to exposed 3′-OH ends of DNA fragments and catalyzes the addition of fluorescein-labeled and unlabeled deoxynucleotides. After washing in TBS, sections were mounted and observed under a fluorescent microscope. The mounting media (included in the kit) contains 4,6,-DiAmidino-2-PhenylIndole (DAPI), which allows the visualization of total cell population. My personal experience says that visualization of specimens after 3-4 h of mounting increases the sharpness of DAPI signal. The mounting media also enhances and preserves fluorescein emission thereby increasing the photostability of the fluorescein signal.

I found the kit provided a very convenient and effective method to detect apoptotic DNA fragments. One should keep in mind that TUNEL positive cell is not necessarily an apoptotic cell. For absolute identification, a battery of tests identifying different characteristic patterns of morphological, biochemical and molecular changes that occur during apoptosis is required.

Ravinder Kumar Kaundal

Senior Research Fellow
Department of Pharmacology and Toxicology
National Institute of Pharmaceutical Education and Research (NIPER)
India

产品页面:QIA39 FragEL™ DNA Fragmentation Detection Kit, Fluorescent - TdT Enzyme


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