Protein Technology Topics: Introduction to Several Detection Methods for Apoptosis
I. Morphological observation method 1. HE staining and light microscopy: The apoptotic cells were round, the nucleus was deeply stained, the cytoplasm was concentrated, the chromatin became a mass, and the cell surface had a "bud" phenomenon. 2. Acridine orange (AO) staining, fluorescence microscopy observation: the viable cell nucleus showed yellow-green fluorescence and the cytoplasm showed red fluorescence. The apoptotic nuclear chromatin was yellow-green concentrated on the inner side of the nuclear membrane, showing that the cell membrane was bulging and apoptotic bodies. 3. Trypan blue staining: If the cell membrane is incomplete and ruptured, the trypan blue dye enters the cell and the cells turn blue, which is necrosis. If the cell membrane is intact and the cells are not stained with trypan blue, they are normal cells or apoptotic cells. This method is helpful for reflecting the integrity of the cell membrane and distinguishing necrotic cells. 4, transmission electron microscopy observation: visible apoptotic cell surface microvilli disappear, nuclear chromatin condensation, edge set, often crescent, nuclear membrane wrinkles, cytoplasm tight, organelles concentrated, membrane blistering or out Buds and apoptotic bodies and apoptotic bodies are engulfed by adjacent macrophages. Second, DNA gel electrophoresis (A), detection principle The cells undergo apoptosis or necrosis, and their cellular DNA is broken. The small molecular weight DNA fragments in the cells increase, the high molecular DNA decreases, and DNA fragments appear in the cytoplasm. However, the DNA breakpoints of apoptotic cells occur regularly between nucleosomes, and 180-200 bp DNA fragments appear, while the DNA breakpoints of necrotic cells are uncharacterized disordered parts, which can be used to determine the death of population cells. Can be distinguished from necrotic cells. (2) Judgment of results A normal living cell DNA electrophoresis showed a LADDER band; necrotic DNA electrophoresis resembled a continuous band at the time of blood smears. 3. Enzyme-linked immunosorbent assay (ELISA) nucleosome assay DNA breaks in apoptotic cells cause nucleosomes to appear in the cytoplasm. The nucleosome consists of histones and their accompanying DNA fragments and can be detected by ELISA. (1) Testing steps 1. The apoptotic cells are lysed and centrifuged at high speed, and the supernatant contains nucleosomes; 2. Adsorption of histone bodies on micro-ration plates 3. Plus nighttime anti-histone antibody binding to histones on nucleosomes' 4. Adding a peroxidase-labeled anti-DNA antibody to bind to DNA on the nucleosome' 4, adding enzyme substrate, metering absorption system. (2) Use The method is highly sensitive and can detect 5*100/ml apoptotic cells. It can be used for apoptosis detection in human, rat and mouse. This method does not require special instruments and is suitable for grassroots work, but it cannot accurately determine the absolute amount of apoptotic cells. Fourth, flow cytometry quantitative analysis (1) Detection principle When cells undergo apoptosis, the permeability of their cell membranes also increases, but the extent is between normal cells and necrotic cells. Using this feature, the cell suspension to be tested is stained with fluorescein, and the cell fluorescence intensity in the cell suspension is measured by flow cytometry to distinguish between normal cells and apoptotic cells of necrotic cells. (2) Application value Flow cytometry has the following characteristics: 1) The number of cells detected is large, so it reflects the apoptosis state of the population cells is relatively accurate 2), can do a lot of correlation analysis 3), combined with the analysis of the DNA content of the tested cells, can determine the cell cycle in which the apoptotic cells are located ■Hoechs-PI double staining for detecting morphology and cell membrane integrity When the cells undergo apoptosis, the permeability of the cell membrane increases, but the degree is between normal cells and necrotic cells. With this feature, the cell suspension to be tested is stained with fluorescein, and the cells are detected by flow cytometry. The fluorescence intensity of the cells in the suspension distinguishes between normal cells, necrotic cells, and apoptotic cells. Using Hoechs-PI staining, normal cells are resistant to dyes, fluorescent staining is very shallow, apoptotic cells mainly ingest Hoecha dye, showing strong blue fluorescence, while necrotic cells mainly take up propidium iodide (PI) and are strong. Red fluorescence. ■DNA fragment in situ labeling In situ end-detection of apoptotic cell DNA fragments refers to deoxyuridine triphate (DUTP) and terminal deoxygenation labeled with fluorescein, digoxin or biotin while the cell (or tissue) structure remains unchanged. A technique in which a nucleotide transferase (TdT) phase reaction is combined with a hydroxyl group (-OH) end of apoptotic cells after cleavage, and a DNA cleavage point is detected after a color reaction. There are two types of DNA fragment in situ labeling: 1. In situ nick-translation (ISNT) technique, which uses DNA polymerase I to link labeled nucleotides to the 3'-OH end of the cleavage DNA. 2. In situ end labelling technique (ISEL), the TUNEL method, which uses TdT to connect the labeled DUPT to the 3·-OH end. Studies have shown that the sensitivity of the TUNEL method is much higher than that of ISNT, especially for the detection of early apoptosis, TUNEL is more suitable. ■Annexin V combined with PI method for detecting changes in cell membrane composition 1. Principle: Phosphatidylserine (PS) located inside the cell membrane in the early stage of apoptosis migrates to the cell membrane. Phospholipid binding protein V (Annexin V) is a calcium-dependent phospholipid binding protein with high binding capacity in PS. Therefore, Annexin V can be used as a probe to detect phosphatidylserine exposed to extracellular measurements. Therefore, using Annexin V with high affinity for PS, Annexin V is labeled with fluorescein (such as fluorescein isothiocyanate FITC), and combined with PI inhibition method (because necrotic cells PS are also exposed to the cell membrane, and Apoptotic cells can be detected by flow cytometry after apoptotic cell double staining. 2, the results judged: normal living cells Annexin V, PI were low-stained; apoptotic cells Annexin V high staining, PI low staining; necrotic cells Annexin V / PI were highly stained. 3. Application value: When the cells undergo apoptosis, the PS exposure on the membrane is earlier than the DNA fragmentation. Therefore, Annexin V combined with PI staining to detect early apoptosis is more sensitive than the TUNEL method. Moreover, Annexin V combined with PI staining does not require fixation of cells, and can avoid excessive cell debris caused by fixation of PI staining and loss of DNA fragments due to fixation by TUNEL method. Therefore, Annexin V combined with PI method is more time-saving and more reliable, and it is the most ideal method for detecting apoptosis. Blood Collection Set, Safety Blood Collection, Blood Collection Device, Blood Collection Needle with Holder, Blood Collection set with Holder, Butterful Needle with Holder Blood Collection Set,Safety Blood Collection,Blood Collection Device,Blood Collection Needle with Holder Luck Medical Consumables Co.,LIMITED , https://www.luckmedical.com