There are many methods for detecting apoptosis, and flow cytometry is a commonly used method. Due to the inherent characteristics of flow cytometry – it can accurately count apoptotic cells. Therefore, it has incomparable advantages over other methods. Below we briefly and clearly introduce the application of flow cytometry in the detection of apoptosis.
Under the action of apoptosis inducers, firstly, cytochrome C and apa f-1 form a complex, and the function of mitochondria declines; then the caspase family is activated, and phosphatidylserine is externalized. At this time, the shape of the cell has changed. , it can be seen that the cells become smaller and the nucleus shrinks; finally, the intracellular DNA is broken to form apoptotic bodies.
In each process of apoptosis, there are corresponding detection methods of flow cytometry, and the following detection methods can be used:
1. Mitochondrial function
2. DNA Cycle
4. Annexin V Assay
5. DNA Fragmentation Assays
It basically covers all stages of apoptosis, and briefly introduces the principles and characteristics of various detection methods.
The kit for mitochondrial function detection mainly uses cationic fluorescent dyes.
The principle is: in normal cells, the dye can aggregate in the mitochondria and emit bright red fluorescence; after apoptosis, the mitochondrial membrane potential changes, the dye cannot enter the mitochondria, and can only exist in the cytoplasm in the form of monomers. Emits green fluorescence. It can be detected under a fluorescence microscope or by flow cytometry. Excitation at 488 nm was used, and the detection wavelengths were 527 nm and 590 nm, respectively. The whole experimental process is simple, and it only takes 30 minutes to see the results.
There are many molecules that can be detected by the Caspase family, and there are many commercial kits available. Even if there is no corresponding kit, as long as there is a corresponding antibody, it can basically be detected, and the specific method is to refer to the steps of intracellular protein detection.
The process of apoptosis is accompanied by a series of changes of morphological features, and the change of cell membrane is one of these features that appears earlier. In apoptotic cells, the cell membrane phosphatidylserine (PS) is flipped from the inner side of the cell membrane to the outer side of the cell membrane. Annexin-V is a 35-36KD calcium particle-dependent phospholipid binding protein, which has a high affinity for PS. When cells are apoptotic, it can bind to the everted PS, so that apoptotic cells can be detected. Cells that died also had PS on their membranes everted and were thus positive. Therefore, in addition to the Annexin-V labeling, the commonly used apoptosis kits also add a DNA dye, commonly used PI and 7-AAD. Due to the increased permeability of the dead cell membrane, the dye can enter the cell and DNA. Binds so that it fluoresces and differentiates dead cells.
When using the Annixin V method to detect apoptotic cells, it should be emphasized that this method is suitable for the detection of cells growing in suspension, such as lymphocytes and other cells. For adherent cells, due to the damage to the cell membrane during the digestion process such as trypsin, it will cause a higher false positive, thus affecting the test results. Although at present, some units including foreign countries also use this method to detect adherent cells. I do not recommend using this method for detection. Because it is less repeatable and requires great care when handling it.
DNA cycle detection was originally used to reflect the various phases of cells, that is, the status of cell proliferation. Take advantage of the ability of intracellular DNA to bind to fluorescent dyes such as PI. Due to the different DNA content of cells at different stages, the fluorescent dyes bound to each other are different, and the fluorescence intensity of flow detection is also different. The DNA content of G2-M phase is twice that of G0-G1, while S phase is in between.
However, because the DNA content of the apoptotic cells was found to be less, there was a hypodiploid peak in front of the G0-G1 phase of the cells, which was considered to be apoptotic cells. However, because the DNA content of dead cells themselves is also reduced, it is very difficult to distinguish between apoptotic and dead cells. In the 1990s, the method was all the rage, and now it seems that the experimental results of that time need to be scrutinized. Although the classical flow cytometry data shows that the apoptotic cell is a peak next to the G0-G1 peak, and the dead cell peak is farther from the G0-G1 peak, this typical result Seems hard to get. There are other alternatives, which can be avoided entirely.
Late apoptotic cells can appear DNA ladder due to the fragmentation of DNA, so there is a classic detection method TUNEl. Streaming can also perform Tunel detection, and its detection principle is basically the same as the classic Tunel principle.
Using TdT, fluorescein-labeled dUTP can be labeled to the fragmented DNA ends, thereby making apoptotic cells fluorescent. However, due to intracellular labeling, cells need to be fixed, and the operation is similar to immunohistochemistry, which is prone to false positives. It is recommended to use the kit from the original manufacturer, and strictly set up the control. After the pre-experimental method is stabilized, large-scale experiments are carried out. After the specimens are processed, they can be observed and photographed with a fluorescent fiberscope. After flow cytometry, an accurate calculation of the percentage of apoptosis can be performed.