Flow Cytometry Analysis

Flow cytometry (FCM) is a rapid and accurate technique for quantitative analysis and sorting of single cell physicochemical properties by flow cytometry. The development of flow cytometry combines the knowledge of laser technology, computer technology, microscopic fluorescence spectrophotometry, fluid jet technology, molecular biology and immunology.

The biggest feature of flow cytometry is that it can quantitatively analyze or purify cells by obtaining a variety of signals from the molecular level with the help of fluorescent probes while maintaining the structure and function of the cells and the organelles or micro particles without disruption and selection. For the cell analysis provides a new means.

The principle of flow cytometry is mainly based on apoptosis at the cell, subcellular and molecular levels of the occurrence of the characteristic changes. These changes include changes in the nucleus, changes in organelles, changes in cell membrane components and changes in cell morphology, including changes in the nucleus of the most characteristic, mainly including the following aspects.

Changes In The Nucleus:

Due to changes in the apoptotic nuclei, resulting in a variety of chromosomal fluorescent dyes on the apoptotic cell’s DNA staining changes. Studies have shown that the use of a variety of chromosomal fluorescent dyes on the immobilized apoptotic cells were stained, the DNA dye ability decreased. Many scholars have identified this DNA dye ability as one of the hallmarks of apoptotic cells.

Light Scattering Characteristics:

Morphological changes in apoptotic cells affect their light scattering properties. In flow cytometry, the former scattered light and the size of the cells, and side scattered light reflects the refraction of light in the cell, and the number of particles within the cell. In cell apoptosis, cell shrinkage, smaller volume, so the former scattered light reduction, this feature is often considered to be one of the characteristics of apoptotic cells.

In addition, apoptosis due to chromosome degradation, nuclear rupture formation, intracellular particles tend to increase, so the apoptotic cells scattered light often increased. When the cell is necrotic, the scattered light increases as the cell is swollen; the side scattered light also increases in the cell necrosis, so that the apoptotic cells and necrotic cells can be distinguished according to the scattered light and the side scattered light.

However, it is important to note that the reliability of apoptotic cells is judged to be significant by the homogeneity and nuclear cytoplasm ratio of the detected cells based on the scattered and side scattered light. Therefore, in some lymphocyte apoptosis, the light scattering characteristics of the detection of apoptosis is better, and in tumor cell apoptosis, its reliability is poor.

The primary advantage of detecting apoptotic cells based on light scattering properties is the ability to combine light scattering properties with surface immunofluorescence assays to distinguish lymphocyte subtypes that undergo selective apoptosis by these special treatments. This can also be used for living cell classification.

Apoptosis:

In the case of apoptosis, the decrease in DNA dye ability is considered to be one of the markers of apoptotic cells, but this DNA degradation may also be due to a decrease in DNA content, or because of changes in DNA structure Combined with the ability to change due. It should be noted when analyzing the results.

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