I. Morphological observation method
1. HE staining and light microscopy: After apoptosis, the cells are round, the nucleus is deeply stained, the cytoplasm is concentrated, the chromatin is agglomerated, and the cell surface has a "bud" phenomenon.
2. Acridine orange (AO) staining , fluorescence microscopy observation: the living cell nucleus showed yellow-green fluorescence, and the cytoplasm showed red fluorescence. Apoptosis|posterior ( apoptosis , apoptosis pathway, apoptosis detection) nuclear chromatin is yellow-green concentrated in the inner side of the nuclear membrane, showing cell membrane blistering and apoptotic bodies.
3. Trypan blue staining : ( apoptosis , apoptosis pathway, apoptosis detection): If the cell membrane is incomplete and ruptured, the trypan blue dye enters the cell, and the cell turns 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 microscopic observation : ( apoptosis , apoptosis pathway, apoptosis detection): visible apoptotic cell surface microvilli disappear, nuclear chromatin pyknosis, edge set, often crescent shape, nuclear membrane folds The cytoplasm is tight, the organelles are concentrated, the membrane is blistered or the "bud" and the apoptotic bodies and apoptotic bodies are phagocytized by adjacent macrophages .
Second, dna gel electrophoresis
(A), detection principle ( apoptosis , apoptosis pathway, apoptosis detection)
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 after apoptosis occur regularly between nucleosomes, and 180-200 bp DNA fragments appear. The DNA breakpoints of necrotic cells are uncharacteristic and disorganized fragments. This feature can be used to determine the death of colony cells. And can be distinguished from necrotic cells.
(2) Apoptosis, the result is judged
DNA electrophoresis in normal living cells showed a LADDER band; after apoptosis, necrotic DNA was electrophoresed like a continuous band of blood smears.
3. Enzyme-linked immunosorbent assay (ELISA) nucleosome assay ( apoptosis , apoptosis pathway, apoptosis detection)
Apoptotic DNA breaks cause nucleosomes to appear in the cytoplasm. The nucleosome consists of histones and their accompanying DNA fragments, which can be detected by ELISA and apoptosis.
(A) ( apoptosis , apoptosis pathway, apoptosis detection) detection steps
1. After apoptosis, it is lysed and centrifuged at high speed, and the supernatant contains nucleosomes.
2. Adsorption of histone bodies on micro-ration plates
3, plus the night to make anti-histone antibodies bind 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.
(B) ( apoptosis , apoptosis pathway, apoptosis detection) 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 apoptosis .
4. Flow cytometry quantitative analysis ( apoptosis , apoptosis pathway, apoptosis detection)
(A) ( apoptosis , apoptosis pathway, apoptosis detection) detection principle
When apoptosis occurs, the permeability of the cell membrane increases, but the degree 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.
(B) ( apoptosis , apoptosis pathway, apoptosis detection) application value
Flow cytometry has the following characteristics:
1), the number of cells detected is large, so it reflects the population cell apoptosis state 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 apoptosis occurs.
Hoechs-PI double staining assay for detection of morphology and cell membrane integrity ( apoptosis )
When apoptosis occurs, 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 cell suspension is detected by flow cytometry. The fluorescence intensity of the cells in the fluid 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 detection of DNA fragments after apoptosis refers to deoxyuridine triphate (DUTP) and ends labeled with fluorescein, digoxin or biotin while the cell (or tissue) structure remains unchanged. The technique of detecting the DNA cleavage point by a color reaction after deoxynucleotidyl transferase (TdT) phase reaction and hydroxy (-OH) end of apoptotic cell lysis.
There are two kinds of DNA fragment in situ labeling detection ( apoptosis ) :
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), which is detected by TUNEL method ( apoptosis , apoptosis pathway, apoptosis detection) , which uses TdT to connect the labeled DUPT to 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. TUNEL detection (apoptosis) 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 high PI staining) performed after double staining of apoptotic cells by flow cytometry to detect apoptosis.
2, TUNEL detection (apoptosis) 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 , TUNEL detection (apoptosis) application value: when apoptosis , PS exposure on the membrane occurs earlier than DNA fragmentation, so Annexin V combined with PI staining detection of early apoptosis is more sensitive than 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 .
Compact Air Hose Reel,Retractable Air Hose Reel,Retractable Air Compressor Hose,Small Retractable Air Hose Reel
NINGBO QIKAI ENVIRONMENTAL TECHNOLOGY CO.,LTD , https://www.hosereelqikai.com