《细胞凋亡的检测方法步骤等详解Apoptosisdetectionmethodsstepsandsoon》由会员分享，可在线阅读，更多相关《细胞凋亡的检测方法步骤等详解Apoptosisdetectionmethodsstepsandsoon（15页珍藏版）》请在金锄头文库上搜索。
1、细胞凋亡的检测方法步骤等详解（Apoptosis detection methods, steps and so on）Apoptosis and necrosis are two completely different forms of apoptosis. According to the differences in morphology, biochemistry and molecular biology of death cells, the two can be distinguished from each other. There are many methods for
2、the detection of apoptosis. Here are some common methods for the determination of apoptosis. Morphological detection of apoptosisAccording to the morphological characteristics of apoptotic cells, a number of different morphological methods have been designed.1 optical microscope and inverted microsc
3、ope(1) unstained cells: the volume of apoptotic cells is smaller and deformed, the cell membrane is intact, but foaming phenomenon occurs, and apoptotic bodies can be seen in the late stage of apoptosis.The adherent cells appear wrinkled, rounded, and detached.(2) stained cells: used Giemsa staining
4、, Wright staining. The chromatin of the apoptotic cell is concentrated and marginalized, and the nuclear membrane is split and chromatin is segmentedTypical apoptotic morphology such as clumps and apoptotic bodies.2 fluorescence microscope and confocal laser scanning microscopeThe morphological chan
5、ges of chromatin were used as the index to evaluate the progress of apoptosis.The commonly used DNA specific dyes are: HO 33342 (Hoechst 33342), HO 33258 (Hoechst 33258), DAPI. The combination of three dyes with DNA is non embedded and mainly combines with the A-T base region of DNA. Blue light is e
6、mitted when excited by ultraviolet light.Hoechst is a reactive dye that binds specifically to DNA. The storage solution is distilled into 1mg/ml at a concentration of PBS and diluted to 25mg/ml at the end.DAPI is semi permeable and is used for routine staining of fixed cells. The storage solution is
7、 distilled at a concentration of 1mg/ml and the final concentration is 0.5 1mg/ml.The results of evaluation: in the process of apoptosis chromatin morphology is divided into three phases: phase I of the nucleus is corrugated (rippled) or (creased), a kind of felling part of chromatin concentration a
8、ppeared; chromatin phase II a nucleus of the highly condensed and marginalized; B phase II nuclear fragmentation and the apoptotic body (Figure 1).3 transmission electron microscope observationResults: apoptotic cell volume became smaller and cytoplasm concentrated. The apoptosis of stage I (pro-apo
9、ptosis nuclei) of the nuclear chromatin highly coiled, many called cavitation (cavitations) cavity structure (Figure 2); stage II a nuclear chromatin high condensation and marginalization; apoptosis of late, nuclear fragmentation and apoptotic body.Two. Analysis of phosphatidylserine valgus (Annexin
10、, V)Phosphatidylserine (Phosphatidylserine, PS) is normally located on the cell membrane, but early in apoptosis, PS from the surface of the cell membrane to the cell membrane inside flip, exposed to the extracellular environment (Figure 3). Annexin-V is a molecular weight dependent phospholipid bin
11、ding protein 3536KD Ca2+ can bind to PS with high affinity. Annexin-V was labeled with fluorescein (FITC, PE) or biotin, and labeled Annexin-V was used as a fluorescent probe. Apoptosis was detected by flow cytometry or fluorescence microscopy.Propidine (iodide, PI) is a nucleic acid dye that can no
12、t penetrate the entire cell membrane, but in apoptosis, the late cells and dead cells, PI can pass through the cell membrane and make the nucleus red dye. Therefore, the use of Annexin-V in conjunction with PI allows the differentiation of apoptotic cells from early or late days, as well as dead cel
13、ls.Method1 staining suspension cells: normal cultured suspension cells and induced apoptosis (0.51 * 106) 2 washes with PBS Buffer and FITC 100ul, adding Binding labeled Annexin-V (20ug/ml) 10ul, dark at room temperature 30min, adding PI (50ug/ml) 5ul, light reaction after 5min, adding 400ul Binding
14、 Buffer. Immediately were detected by flow cytometry with FACScan (less than 1h),Meanwhile, one tube without AnnexinV-FITC and PI was used as negative control.2 cells stained with wall culture: first digested with 0.25% trypsin, washed, stained and analyzed with suspension cells.3 slice cell stainin
15、g: ibid., fluorescence microscopy and confocal laser scanning microscopy were used for observation.Results Figures 4 and 5Matters needing attention1. of the entire operation to try to gently, do not force these cells.2., take care to avoid light, as soon as possible after the completion of the test
16、within an hour.Three. Detection of mitochondrial membrane potential energyMitochondria play a pivotal role in the process of cell apoptosis, the apoptosis of various cells stimulating factor can induce different cell apoptosis and mitochondrial transmembrane potential decreased DYmt, is considered the first occurrence of the apoptotic cascade in the process of the event, it occ
