After the introduction of the previous issues, you must have a certain understanding of the mechanism of apoptosis, and each stage of apoptosis is strictly regulated by signaling, so the evaluation of the activity of related proteins can be used for the determination of apoptosis. However, many features of apoptosis and necrosis may overlap, so choosing the right assay is critical. In this issue, we will introduce the commonly used detection methods in detail.
Observation of cell morphology
Transmission electron microscopy is considered the gold standard for confirming apoptosisThe following characteristics can be observed after apoptosis: (1) the cell volume becomes smaller and the nucleus shrinks;(2) Chromatin marginalization and agglutination;(3) nuclear debris mixed with part of the organelle within the cytoplasm;(4) Apoptotic bodies formed by cell membranes.
Optical microscopy and/or laser confocal microscopy can also observe the morphological changes of cell chromatin, membrane structure and permeability to determine whether cells have apoptosis, but at present, transmission electron microscopy is the most classic and reliable method to determine apoptosis, but this method is time-consuming and laborious, and the area of observation at one time is limited, so it is not suitable for the detection of a large number of apoptotic samples.
2a: normal thymic tissue lymphocytes;2b: Apoptotic thymic tissue lymphocytes.
DN** segmentation detection
When cells undergo apoptosis, DNA degradation occurs earlier than changes in cell morphology, and common detection methods include gel electrophoresis, in situ incision end labeling, and ELISA assays.
Gel electrophoresis detection: The main biochemical characteristics of apoptosis are chromatin condensation, chromatin DNA breaks at the junction between nucleosome units, forming a large DNA fragment of 50 300 kbp long, or an oligonucleotide fragment of 180 200 bp integer multiples, which is shown as a trapezoidal electrophoresis pattern on gel electrophoresis.
From left to right: 1: DNA ladder;2-3: Cell necrosis;4-6: Apoptosis;7: Normal cells.
In situ end-notch labeling (TUNEL): is an in situ method for the detection of DNA segments in apoptotic cells using a combination of molecular biology and immunohistochemistry. The basic principle is as follows: when the cell undergoes apoptosis, the endonuclease is activated, the chromatin or DNA is cleaved by the endonuclease to produce a 180bp 200bp fragment containing the 3-OH terminus, and the deoxynucleotide terminal transferase will bind the nucleotide (DUTP) bound to the end of the notch 3-OH, and then add HRP anti-fluorescein antibody and HRP chromogenic substrate DAB, the apoptotic cells have obvious color, while normal cells have no DNA break or only a small amount of DNA Fracture occurs, so it is not colored. The TUNEL method is widely used for the detection of various apoptotic cells due to its high sensitivity, and since DNA fragmentation may also be present in necrotic cells, only a general break of the DNA strand can prove apoptosis. If the process is not properly handled, it is easy to cause false positives, and the results are highly subjective. The TUNEL test must be set up with a negative and positive control.
ELISA detection method: during apoptosis, the nucleosome DNA formed by DNA degradation can be tightly bound to the core histone H2A, H2B, H3, H4, etc., to form a complex, anti-histone and anti-DNA monoclonal antibody bispecific antibody sandwich method (ELISA) can specifically quantitatively determine the mononucleosome or oligonucleosome of the cytoplasmic part of the lysate during apoptosis, as an indicator of apoptosis, especially early apoptosis. The degree of apoptosis is determined by ELISA by detecting nucleosomes released into the cytoplasm by apoptotic cells. This method is both qualitative and quantitative, and is suitable for testing large sample batches without the need for special instrumentation. Longer periods of lysis may result in higher results due to the inclusion of DNA fragments in the nucleus, resulting in higher results, so it takes several attempts to determine the optimal detection conditions for different cell lines.
Caspase activity assay
The caspase family is a key protein in the process of apoptosis and regulates apoptosis through interaction with numerous protein factors. Among them, caspase-3 is downstream of the apoptosis order cascade and is the most important effector caspase. Caspase-3 normally exists in the cytoplasm in the form of zymogen (32 kd), and in the early stage of apoptosis, activated caspase-3 is composed of two large subunits (17 kD) and two small subunits (12 kD), which cleaves the corresponding cytoplasmic nuclear substrate and eventually leads to apoptosis, while in the late stage of apoptosis and dead cells, the activity of caspase-3 is significantly reduced, which can be detected by Western blot.
In addition, activated CASPASE-3 was able to specifically cleave the D1E2V3D4-X substrate and hydrolyze the D4-X peptide bonds. In covalent coupling, AMC cannot be excited to fluoresce, and the short peptide is hydrolyzed to release AMC, so that free AMC can be excited to emit fluorescence. Based on this principle, the activity of caspase-3 can be analyzed by fluorescence spectrophotometer and flow cytometry.
Phosphatidylserine valgus analysisAnnexin V method
In normal cells, phosphotidyiserine (PS) is only distributed on the inner side of the lipid bilayer of the cell membrane, and in the early stage of apoptosis, different types of cells will turn the PS outward to the cell surface, that is, the outer side of the cell membrane. Annexinv is a Ca2+-dependent phospholipid-binding protein with a molecular weight of 35-36 kD, which has a high affinity for PS and can bind to the membrane of cells in early apoptotic stages through PS exposed on the outside of the cell. Annexin-V was fluorescein (FITC, PE) labeled, and the labeled Annexin-V was used as a fluorescent probe, and the occurrence of apoptosis could be detected by flow cytometry or fluorescence microscopy. PI is a nucleic acid dye, it can not penetrate the intact cell membrane, but the cells in the middle and late stages of apoptosis and necrosis are able to penetrate the cell membrane and stain the nucleus red due to the increased permeability of the cell membrane, so the use of Annexin V with PI can distinguish cells in different apoptotic stages.
Fig.3 Flow cytometry detection effect of Annexin V-FITC and PI staining (source: Hanheng Biotech).
Normal viable cells are not stained with Annexin V-FITC and P1 (lower left part of the figure): Cells in the early stage of apoptosis are stained only with Annexin V-FITC, and P1 staining is negative (lower right part of the figure): Cells with advanced apoptosis and necrosis can be stained with both Annexin V-FITC and PI (upper right part of the figure).
Mitochondrial testing
Mitochondria play an important role in endogenous apoptosis, and the decline of mitochondrial transmembrane potential is thought to be the earliest event in the apoptotic cascade, occurring before the emergence of nuclear apoptosis features (chromatin condensation, DNA breakage). The presence of mitochondrial transmembrane potential enables some lipophilic cationic fluorescent dyes such as Rhodamine 123 and DiOC6(3) to bind to the mitochondrial matrix, and the enhancement or decrease of their fluorescence indicates that the electronegativity of the inner mitochondrial membrane is increased or decreased. However, fluorescent tags may alter the interaction of native proteins with other proteins, so this method needs to be used in combination with other apoptosis assays, such as caspase activity assays, to further confirm apoptosis results.
The above are the commonly used apoptosis detection methods. Hanheng Biotech has been specializing in tools for viruses for more than ten years, and can provide overexpression of apoptosis-related proteins, interference plasmids or virus tools, and annexin v fitc apoptosis detection kits, welcome to consult
References: 1] Elmore, Susan apoptosis: a review of programmed cell death. toxicologic pathology, 2007, 35(4):495-516.
2] Zhang Guangzhi, Hu Changmin, Chen Yingyu, et al. Research progress on the main detection methods of apoptosis[J].Advances in Veterinary Medicine, 2011, 32(8):4