Introduction to apoptosis (TUNEL, TUNEL staining) detection technology
Apoptosis refers to maintaining the stability of the internal environment. Â Â The process of death of a cell in vivo is induced by a specific endogenous or exogenous signal, its death pathway is activated, and under the regulation of the relevant gene. Apoptosis is a major form of programmed death involving chromatin condensation and peripheralization, cytoplasmic reduction, nuclear fragmentation, cytoplasmic densification, disruption of contact with surrounding cells, fusion of endoplasmic reticulum with cell membrane, and final cell fragmentation. Many apoptotic bodies are formed . Depending on the difference in morphology, biochemistry, and molecular biology of dead cells, the two can be distinguished. There are many methods for detecting apoptosis , and several commonly used assay methods are described below. 1. Morphological detection of apoptosis According to the inherent morphological characteristics of apoptotic cells , many different methods for detecting apoptosis morphology have been designed. 2 Fluorescence microscopy and confocal laser scanning microscopy (apoptosis) results: 3 transmission electron microscopy observation | apoptosis: Second, phosphatidylserine valgus analysis (Annexin V method) detection | apoptosis: Annexin V/PI Double Dye Detection | Apoptosis : Annexin V/PI detection|apoptosis| advantage: Compared with other apoptosis detection methods, Annexin V/PI double staining can be light Pine distinguishes apoptosis and necrosis, and detects it early in apoptosis with high sensitivity. Wound Dressing,Disposible Wound Dressing,Waterproof Silicone Foam Dressing,Medical Paraffin Dressing Zhende Medical Co.,Ltd , https://www.zdmedicalproduct.com
Apoptosis was detected by the TdT-mediated dUTP nick end labeling apoptosis assay kit . The Apoptosis Detection Kit is used to detect the breakage of nuclear DNA in tissue cells during the early stages of apoptosis. The principle of apoptosis detection is that fluorescein-labeled 12-dUTP is linked to the 3'-OH end of the cleavage DNA in apoptotic cells under the action of terminal deoxynucleotidyl transferase (TdT), using fluorescence microscopy or flow. A cell detector detects the occurrence of apoptosis.
Apoptosis and necrosis are two completely different forms of apoptosis. Apoptosis is not a passive process, but an active process involving the activation, expression and regulation of a series of genes. It is not a pathology. Under the conditions, a phenomenon of autologous injury, but a death process that is actively sought for better adapting to the living environment. Apoptosis is a fundamental biological phenomenon of cells that plays a necessary role in the removal of unwanted or abnormal cells by multicellular organisms. It plays an important role in the evolution of organisms, the stability of the internal environment, and the development of multiple systems. Apoptosis is not only a special type of cell death, but also has important biological significance and complex molecular biological mechanisms. Apoptosis is a process in which multiple genes are strictly controlled.
1 Detection of apoptosis by light microscopy and inverted microscope :
(1) unstained cells: apoptosis smaller volume of deformation, but appeared intact membrane foaming, late apoptosis apoptotic bodies. Adherent cells appear to shrink, round, and fall off.
(2) Stained cells: Giemsa staining, Wright's staining, etc. are commonly used. The apoptotic cells are characterized by chromatin condensation, marginalization, nuclear membrane cleavage, chromatin segmentation into massive and apoptotic bodies.
The progression of apoptosis is generally judged by the morphological changes of nuclear chromatin as an indicator.
Commonly used DNA-specific dyes are: HO 33342 (Hoechst 33342), HO 33258 (Hoechst 33258), DAPI. The combination of the three dyes and DNA is non-embedded and mainly binds to the AT base region of DNA. Bright blue fluorescence is emitted when excited by ultraviolet light.
Hoechst is a reactive dye that specifically binds to DNA. The stock solution is diluted with distilled water to a concentration of 1 mg/ml. When used, it is diluted with PBS to a final concentration of 2 to 5 mg/ml.
DAPI is semi-permeable and is used for staining of conventional fixed cells. The stock solution is made up to a concentration of 1 mg/ml in distilled water, and the final concentration is generally 0.5 to 1 mg/ml.
RESULTS: The morphological changes of nuclear chromatin in the process of apoptosis were divided into three phases: the nucleus of stage I was rippled or creased, and some chromatin appeared concentrated; phase IIa nucleus Chromatin is highly coagulated and marginalized; the nuclear cleavage in stage IIb is fragmentation, resulting in apoptotic bodies.
Results: The apoptotic cells became smaller and the cytoplasm was concentrated. The chromatin in the nucleus of pro-apoptosis nuclei is highly coiled, and many vacuole structures called cavitations appear; the chromatin of the apoptotic stage IIa nucleus is highly coagulated and marginalized; In the late stage of apoptosis, the nucleus is cleaved into fragments, producing apoptotic bodies.
Phosphatidylserine (PS) is normally located inside the cell membrane, but in the early stage of apoptosis, PS can be flipped from the inner side of the cell membrane to the surface of the cell membrane and exposed to the extracellular environment. Annexin-V is a Ca2+-dependent phospholipid binding protein with a molecular weight of 35~36KD, which can specifically bind to PS with high affinity. Therefore, Annexin V is used as one of the sensitive indicators for detecting early apoptosis in cells. Annexin-V was labeled with fluorescein (FITC, PE) or biotin, and labeled Annexin-V was used as a fluorescent probe to detect apoptosis by flow cytometry or fluorescence microscopy .
Propidine iodide (PI) is a nucleic acid dye that does not penetrate intact cell membranes. However, in advanced cells and dead cells of apoptosis, PI can pass through the cell membrane to redden the nucleus. Therefore, when Annexin-V is used in combination with PI, cells in early and late apoptotic cells and dead cells can be distinguished.