First, the basic principle
The principle is mainly based on the characteristic changes that occur at the cellular, subcellular and molecular levels when apoptosis occurs. These changes include changes in the nucleus, changes in organelles, changes in cell membrane composition, and changes in cell morphology. Among them, changes in the nucleus are the most characteristic, including the following aspects:
1. Changes in the nucleus
Due to changes in apoptotic nuclei, various chromosomal fluorescent dyes have altered the DNA stainability of apoptotic cells. Studies have shown that staining of fixed apoptotic cells with various chromosomal fluorescent dyes reduces DNA stainability. Many scholars consider this reduction in DNA dyeability to be one of the hallmarks of apoptotic cells.
2, light scattering characteristics
Morphological changes in apoptotic cells affect their light scattering properties. On flow cytometry, the front scattered light is related to the size of the cell, while the side scattered light reflects the refraction of light in the cell, which is related to the number of particles in the cell. In the case of apoptosis, the cells are pyknotic and the volume is reduced, so the scatter light is reduced, which is often considered to be one of the characteristics of apoptotic cells. In addition, due to chromosomal degradation and nuclear rupture during cell apoptosis, intracellular granules tend to increase, so scattered light on the apoptotic cells often increases. When the cells are necrotic, the front scattered light increases due to the swelling of the cells; the side scattered light also increases when the cells are necrotic, so that the apoptotic cells and the necrotic cells can be distinguished according to the forward scattered light and the side scattered light. However, it should be noted that the reliability of apoptotic cells based on the forward scattered light and the side scattered light is greatly affected by the uniformity of the morphology of the cells to be detected and the ratio of nuclear cytoplasm. Therefore, in some lymphocyte apoptosis, the reliability of detecting apoptosis by light scattering is better, but the reliability of tumor cell apoptosis is poor. The most important advantage of detecting apoptotic cells based on light scattering properties is that they can combine light scattering properties with surface immunofluorescence analysis of cells to distinguish lymphocyte subtypes that undergo selective apoptosis through these special treatments. It can also be used for the classification of living cells.
Second, reagents and instruments
1, PBS solution
2. PI staining solution: PI was dissolved in PBS (pH 7.4) to a final concentration of 100 ug/ml. Store in a brown bottle at 4 ° C protected from light
3, 70% ethanol
4, 400 mesh screen
5, flow cytometry
Third, the experimental steps
1. Collect cells {number (1~5)×106 cells/mL}, centrifuge at 500~1000 r/min for 5 min, discard the culture solution.
2. Wash 3 ml PBS once.
3. Centrifuge to PBS, add to ice pre-cooled 70% ethanol, 4 ° C, 1-2 hours.
4. Centrifuge the fixed solution and resuspend in 3 ml of PBS for 5 min.
5, 400 mesh screen filtration 1 time, 500-1000r / min centrifugation 5min, discarded PBS.
6. Dye with 1ml PI dye solution, avoiding light for 30min at 4°C.
7. Flow cytometry detection: PI excites fluorescence with argon ions. The wavelength of the laser light wave is 488 nm, and the wavelength of the emitted light wave is greater than 630 nm. The histogram of the fluorescence intensity of the red fluorescence analysis is also analyzed. The scatter of the side scattered light of the front scattered light can also be analyzed. Figure.
8. Judgment of results: On the scattergram or topographic map of the side scattered light of the front scattered light, the apoptotic cells are reduced compared with the normal cells, and the side scattered light can be high or low, which is related to the type of cells. When analyzing the histogram of PI fluorescence, first use the gate technique to exclude cells that are double or aggregated and cell fragments that are weakly fluorescent. On the histogram of PI fluorescence, apoptotic cells appear one before the G1/G0 phase. Ploid peak. If the fluorescence intensity at the G1/G0 phase is 1.0, the fluorescence intensity of the subdiploid peak of a typical apoptotic cell sample is 0.45, and the PI fluorescence intensity of the red blood cells of chicken and carp can be used as a reference standard. At 0.35 and 0.7 respectively, it is ensured that there is not a cell fragment but a complete cell between the two.
Fourth, matters needing attention
When apoptosis occurs, its DNA stainability is considered to be one of the hallmarks of apoptotic cells, but the decrease in DNA stainability may also be due to a decrease in DNA content or due to changes in DNA structure and dyes. The ability to combine changes. You should pay attention when analyzing the results.
YT-H711
YT-H711
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