Flow cytometry experimental principle - Database & Sql Blog Articles

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. The changes in the nucleus are most characteristic, including the following aspects: 1. Changes in the nucleus due to changes in apoptotic nuclei, It causes various chromosomal fluorescent dyes to change 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 The morphological changes of 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.