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.
凋亡是許多正常的生理過程所必需的，包括免疫系統(tǒng)的成熟和作用機(jī)制、組織器官和肢體的發(fā)生、神經(jīng)系統(tǒng)的發(fā)生等過程。在許多病理?xiàng)l件下，凋亡機(jī)制的調(diào)節(jié)失衡起很重要的作用，包括組織細(xì)胞的缺血、缺氧、出血、中風(fēng)、心臟病、癌癥、艾滋病、自身免疫缺損和中樞神經(jīng)系統(tǒng)的退行性變等。在癌基因的研究方面，由于細(xì)胞凋亡可由抗癌藥物、放射及高熱等啟動(dòng)，并且腫瘤細(xì)胞具有由于一些癌基因表達(dá)即能啟動(dòng)凋亡的內(nèi)在特性，因此細(xì)胞凋亡已引起人們的廣泛重視。
檢測(cè)凋亡細(xì)胞的方法有數(shù)種。細(xì)胞凋亡過程中核酸內(nèi)切酶的活化是關(guān)鍵的過程，導(dǎo)致核DNA被裂解成寡核苷酸大小的片段。因此，這個(gè)過程被用于檢測(cè)凋亡，在瓊脂糖凝膠電泳上出現(xiàn)典型的“DNA梯形帶”。但是，這種方法既不能檢測(cè)單個(gè)細(xì)胞水平的凋亡，又不能提供細(xì)胞發(fā)生凋亡所處的組織位置和細(xì)胞分化狀態(tài)等方面的信息。
這一切可以由凋亡的原位酶標(biāo)記方法來完成。DNA聚合酶和末端脫氧核糖核酸轉(zhuǎn)移酶（TdT）用來標(biāo)記DNA鏈斷端的核酸，應(yīng)用TdT進(jìn)行的末端反應(yīng)又稱為ISEL或TUNEL技術(shù)，它與應(yīng)用DNA聚合酶的ISNT（in situ nick translation）相比具有以下優(yōu)點(diǎn)：靈敏度高、快速、優(yōu)先標(biāo)記發(fā)生凋亡的細(xì)胞，而不是壞死的細(xì)胞。
本試劑盒檢測(cè)細(xì)胞凋亡分以下三個(gè)步驟：1、標(biāo)記DNA鏈斷端，由TdT催化，以FITC標(biāo)記的核苷酸為原料，進(jìn)行非模板依賴方式的3?OH端DNA末端聚合反應(yīng)；2、用HRP或AP標(biāo)記的羊抗FITC的抗體（Fab段）與聚合到DNA末端的FITC結(jié)合；3、用酶的底物顯色，HRP常用DAB顯色，AP常用BCIP/NBT顯色。
細(xì)胞凋亡原位檢測(cè):我公司提供代測(cè)服務(wù)：
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