Widely used methods to determine apoptosis include the analysis of the genomic DNA by agarose-gel electrophoresis and DNA fragmentation assays based on 3H-thymidine and, alternatively, 5-Bromo-2'-deoxy-uridine. The methods involve the separation of fragmented, low molecular-weight DNA from unfragmented, high molecular-weight DNA in a given cell population. Thus, these methods do not provide information about the fate of an individual cell in a given cell population or, particularly, in tissue sections. Alternatively, individual apoptotic cells may be microscopically recognized because of the characteristic appearance of nuclear chromatin condensation and fragmentation, but this method is subjective and limited to a relatively narrow time window when the morphological changes are at a maximum.
The hallmark of apoptosis is DNA degradation, which in early stages is selective to the internucleosomal DNA linker regions. The DNA cleavage may yield double-stranded and single-stranded DNA breaks (nicks). Both types of breaks can be detected by labeling the free 3'-OH termini with modified nucleotides (e.g., biotin-dUTP, DIG-dUTP, fluorescein-dUTP) in an enzymatic reaction. The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes the template-independent polymerization of deoxyribonucleotides to the 3'-end of single- and double-stranded DNA. This method has also been termed TUNEL (TdT-mediated dUTP-X nick end labeling). Alternatively, free 3'-OH groups may be labeled using DNA polymerases by the template-dependent mechanism called nick translation. However, the TUNEL method is considered to be more sensitive and faster.
Contents:
1. Enzyme Solution (TdT), 5 vials
2. Label Solution (fluorescein-dUTP), 5 vials
3. Converter POD (anti-fluorescein antibody-POD), ready-to-use Principle
The In Situ Cell Death Detection Kit, POD is based on the detection of single- and double-stranded DNA breaks that occur at the early stages of apoptosis. Apoptotic cells are fixed and permeabilized. Subsequently, the cells are incubated with the TUNEL reaction mixture that contains TdT and fluorescein-dUTP. During this incubation period, TdT catalyzes the addition of fluorescein-dUTP at free 3'-OH groups in single- and double-stranded DNA. After washing, the label incorporated at the damaged sites of the DNA is marked by an anti-fluorescein antibody conjugated with the reporter enzyme peroxidase. After washing to remove unbound enzyme conjugate, the POD retained in the immune complex is visualized by a substrate reaction.
凋亡是许多正常的生理过程所必需的，包括免疫系统的成熟和作用机制、组织器官和肢体的发生、神经系统的发生等过程。在许多病理条件下，凋亡机制的调节失衡起很重要的作用，包括组织细胞的缺血、缺氧、出血、中风、心脏病、癌症、艾滋病、自身免疫缺损和中枢神经系统的退行性变等。在癌基因的研究方面，由于细胞凋亡可由抗癌药物、放射及高热等启动，并且肿瘤细胞具有由于一些癌基因表达即能启动凋亡的内在特性，因此细胞凋亡已引起人们的广泛重视。
检测凋亡细胞的方法有数种。细胞凋亡过程中核酸内切酶的活化是关键的过程，导致核DNA被裂解成寡核苷酸大小的片段。因此，这个过程被用于检测凋亡，在琼脂糖凝胶电泳上出现典型的“DNA梯形带”。但是，这种方法既不能检测单个细胞水平的凋亡，又不能提供细胞发生凋亡所处的组织位置和细胞分化状态等方面的信息。
这一切可以由凋亡的原位酶标记方法来完成。DNA聚合酶和末端脱氧核糖核酸转移酶（TdT）用来标记DNA链断端的核酸，应用TdT进行的末端反应又称为ISEL或TUNEL技术，它与应用DNA聚合酶的ISNT（in situ nick translation）相比具有以下优点：灵敏度高、快速、优先标记发生凋亡的细胞，而不是坏死的细胞。
本试剂盒检测细胞凋亡分以下三个步骤：1、标记DNA链断端，由TdT催化，以FITC标记的核苷酸为原料，进行非模板依赖方式的3?OH端DNA末端聚合反应；2、用HRP或AP标记的羊抗FITC的抗体（Fab段）与聚合到DNA末端的FITC结合；3、用酶的底物显色，HRP常用DAB显色，AP常用BCIP/NBT显色。
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