1. Detection of PS (phosphatidylserine) in the extracellular membrane
The phenomenon of PS shifting from the medial side of the cell to the outside is a sign that can occur early in the cell apoptosis. AnnexinV is a calcium-dependent phospholipid-binding protein that specifically binds to PS exposed to the outside of the cell membrane and detects apoptosis using a fluorescein-labeled AnnexinV protein (such as Annexin V-FITC). Since this is a live cell assay for early apoptosis (both for both suspended and adherent cells), it can be combined with DNA dyes or other advanced assays to label the stage of apoptosis. The operation is simple and fast, and the test can be completed in 10 minutes. High sensitivity, can be used as a basis for the analysis of apoptotic cells by flow method.
2. Detection of changes in intracellular redox status <br>Glutathione (GSH) acts as an important redox buffer in cells under normal conditions. The toxic oxides in the cells are eliminated by reduction by GSH, and the oxidized GSH can be rapidly reduced by GSH reductase. This reaction is particularly important in mitochondria, where many oxidative damage in by-products of respiration is digested. There is an ATP-dependent GSH transfer system in the cell membrane that can be activated by an apoptotic signal. When the elimination of GSH in cells is very active, the cell fluid changes from a reducing environment to an oxidizing environment, which may lead to a decrease in mitochondrial membrane potential in early apoptosis, thereby making cytochrome C (an important component in the tricarboxylic acid cycle). Transferring from the mitochondria to the cell fluid initiates a cascade of apoptotic effector caspase.
3. Detection of cytochrome C <br>Cytochrome C, as a signal substance, plays an important role in apoptosis. Normally, it is present in the lumen between the mitochondrial inner membrane and the outer membrane and is not present in the cytosol. After apoptotic signal stimulation, it can be released from the mitochondria into the cytoplasm. After binding to Apaf-1, the caspase cascade activation reaction is initiated, which first activates caspase-9, which in turn activates caspase-3 and other downstream caspase molecules. In the early stage of apoptosis, cytochrome C leaks into the cytosol, and the detection of cytochrome c content in the cytosol can reflect the early apoptosis of cells.
4. Detection of mitochondrial membrane potential changes <br>In the early stage of apoptosis, mitochondria did not change significantly in morphology. However, many physiological and biochemical changes can occur in mitochondria. For example, after induction of apoptosis, the mitochondrial transmembrane potential changes, resulting in a change in membrane permeability. MitoSensorTM is a cationic stain that is very sensitive to changes in membrane potential and exhibits different fluorescent staining. In normal cells, it forms aggregates in the mitochondria and emits intense red fluorescence. In apoptotic cells, due to changes in mitochondrial transmembrane potential, it stays in the cytosol in a monomeric form, emitting green fluorescence. These two different fluorescent signals can be clearly distinguished by fluorescence microscopy or flow cytometry.
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