The Most Comprehensive Apoptosis Detection Strategy
Apoptosis is a type of programmed cell death, which is strictly controlled by genes and is essential for controlling cell growth, development and renewal. Apoptosis is activated mainly by two pathways: one is endogenous, which is caused by intracellular stress response or internal apoptosis signal, causing mitochondria to release cytochrome C, activating the Caspase family to induce apoptosis; the other is exogenous, which is caused by receptors on the cell surface receiving apoptosis signals and activating the Caspase family to induce apoptosis. It can include a series of characteristic changes, such as morphological changes such as cell shrinkage, nuclear condensation, nuclear fragmentation, and apoptotic body formation, as well as biochemical changes such as characteristic DNA fragmentation, new gene expression, and synthesis of certain biomacromolecules.
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Figure 1 : The process of apoptosis ( from Wikipedia )
The detection method of cell apoptosis is designed based on the special morphological and biochemical changes of apoptotic cells.
1) Morphological Detection
When cells undergo apoptosis, a series of unique morphological changes occur:1) Cell size decreases2) Nuclear condensation, nucleolus fragmentation, and increased chromatin density3) Cytoplasm is concentrated and organelle density increases4) The cell membrane wrinkles, curls, and invaginate, and the cytoplasm and DNA are divided and wrapped to form apoptotic bodiesTherefore, electron microscopy can be used to identify apoptosis.
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Figure 2.1: Control group cells, 2-6: Cell states after apoptosis induced by different concentrations of DHA [1]
2) Agarose Gel Electrophoresis
When cells undergo apoptosis, Caspase activates Caspase-Activated DNase (CAD), which degrades DNA to form DNA fragments with a length of 180-200 bp or its integral multiples, which will form bands of different sizes after agarose electrophoresis.DNA in living cells: not broken, a normal band in gel electrophoresisNecrotic cell DNA: random fragmentation, gel electrophoresis shows diffuse continuous fuzzy bandsApoptotic cell DNA: isosceles trapezoidal bands
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3) TUNEL
The DNA breaks formed by apoptosis will expose the 3'-OH end of the DNA chain. Terminal deoxynucleotidyl transferase (TdT) can catalyze the nucleic acid polymerization reaction of the 3'-OH end of the broken DNA. After adding dUTP labeled with fluorescein, peroxidase, alkaline phosphatase or biotin, the in situ apoptotic cells can be specifically detected after the color reaction. Normal or proliferating cells have almost no DNA breaks, so they are rarely stained.
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Figure 4. YEASEN TUNEL-FITC kit (Click to view product CAT 40306) - Detection of apoptotic cell samples
4) Annexin V/PI Double Staining
In normal cells, phosphotidylserine (PS) on the cell membrane is located on the cytoplasmic side. In the early stage of apoptosis, phosphatidylserine flips from the cytoplasmic side of the cell membrane to the outer surface of the cell membrane. Annexin V is a Ca2+-dependent phospholipid binding protein that can specifically bind to the everted PS. Annexin V (fluorescein or biotin labeled) is used as a fluorescent probe and used simultaneously with PI to analyze the staining with the help of FCM. Annexin V-/PI-, Annexin V+/PI+, Annexin V+/PI-, and Annexin V-/PI+ represent normal, late apoptotic or necrotic, early apoptotic, and mechanically damaged cells, respectively.
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Figure 5. YEASEN Annexin V-FITC/PI Cell Apoptosis Detection Kit (Click to view product CAT 40302) - Detection of cell apoptosis
5) Detection Of Changes In Mitochondrial Membrane Potential
In cells at the early stage of apoptosis, before significant changes are observed under the microscope, the mitochondrial membrane potential in the cells has already begun to change: the mitochondrial membrane permeability increases and the transmembrane potential decreases. The decrease in membrane potential is considered to be the earliest biological change in apoptosis. If the membrane potential is destroyed, cell apoptosis will be irreversible. Some lipophilic cationic fluorescent dyes, such as rhodamine 123 and JC-1 , can bind tightly to the mitochondrial matrix, and the degree of decrease in mitochondrial membrane potential is positively correlated with the degree of decrease in the ability of mitochondria to aggregate dyes.
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Figure 6. YEASEN JC-1 Mitochondrial Membrane Potential Assay Kit (Click to view product CAT 40706) - Detect changes in membrane potential
6) Determination Of Ca 2+ Concentration Changes In Cell Apoptosis
The life activities of cells are inseparable from Ca 2+ . When Ca 2+ is overloaded, the mitochondrial permeability transition pore opens, and the Ca 2+ balance inside and outside the cell is destroyed, leading to cell apoptosis. After labeling the cells with Ca 2+ -specific fluorescent probes, such as Rhod-2, Indo-1, Fluo-4, etc., and then detecting them with FCM or fluorescence or confocal laser microscopy, the physiological indicators of cell apoptosis can be accurately obtained.
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Figure 7. YEASEN Fluo-4, AM, a cell membrane permeable calcium ion fluorescent probe (Click to view product CAT 40704) [4]
7) Caspase Activity Assay
Caspase-3 is considered to be the key enzyme that causes apoptosis due to various factors. It exists in the form of zymogen under physiological conditions. Its activation indicates the beginning of the cell's apoptosis program and is a sign that apoptosis has entered the irreversible stage. At present, no activation of caspase-3 has been found in necrotic cells. Therefore, by detecting the activity of caspase-3, it is possible to determine whether the cells are apoptotic or necrotic. Generally, there are two methods: content detection and activity detection. Content detection is to quantify or relatively quantify Caspase using Western blot, immunohistochemistry or immunofluorescence methods. Activity detection uses a short peptide coupled with fluorescein that can be specifically recognized by Caspase. After Caspase cuts the short peptide, the fluorescein will be released, and the Caspase activity can be judged based on the fluorescence intensity.
8) Detection Of Apoptosis-related Genes And Their Products
During cell apoptosis, some genes express abnormal products, and the products transcribed and translated from these genes can promote or inhibit cell apoptosis, such as the apoptosis protein family, apoptosis protease activating factor 1 (Apaf-1) and Fas , etc. The determination of these related genes and their expression products can provide a basis for cell apoptosis detection.