Four tricks to detect apoptosis, one trick is more than a trick

The first trick: look at the flowers in the fog - morphological observation method

(1) HE staining and light microscopy: The apoptotic cells were round, the nucleus was deeply stained, the cytoplasm was concentrated, the chromatin became a mass, and the cell surface had a "bud" phenomenon.
(2) Acridine orange (AO) staining, fluorescence microscopy observation: the living cell nucleus showed yellow-green fluorescence, and the cytoplasm showed red fluorescence. The apoptotic nuclear chromatin was yellow-green concentrated on the inner side of the nuclear membrane, showing that the cell membrane was bulging and apoptotic bodies.
(3) Trypan blue staining: If the cell membrane is incomplete and ruptured, the trypan blue dye enters the cell, and the cell turns blue, which is necrosis. If the cell membrane is intact and the cells are not stained with trypan blue, they are normal cells or apoptotic cells. This method is helpful for reflecting the integrity of the cell membrane and distinguishing necrotic cells.
(IV) Transmission electron microscopy observation: the surface micro-villi disappeared on the surface of apoptotic cells, nuclear chromatin condensation, edge collection, often crescent-shaped, nuclear membrane wrinkles, tight cytoplasm, organelle concentration, membrane blistering or out "Buds" and apoptotic bodies and apoptotic bodies are engulfed by adjacent macrophages.

The second measure: the original shape of the dew - DNA gel electrophoresis

(1) Detection principle: When cells undergo apoptosis or necrosis, their cell DNA breaks, small DNA fragments in the cells increase, high molecular DNA decreases, and DNA fragments appear in the cytoplasm. However, the DNA breakpoints of apoptotic cells occur regularly between nucleosomes, and 180-200 bp DNA fragments appear, while the DNA breakpoints of necrotic cells are uncharacterized disordered parts, which can be used to determine the death of population cells. Can be distinguished from necrotic cells.
(II) Judgment of results: A ladder-like (LADDER) band appeared in normal living cell DNA electrophoresis; the necrotic cell DNA electrophoresis resembled a continuous band when blood smears were used.

The third measure: water and stone out - enzyme-linked immunosorbent assay (ELISA) nucleosome determination

DNA breaks in apoptotic cells cause nucleosomes to appear in the cytoplasm. The nucleosome consists of histones and their accompanying DNA fragments and can be detected by ELISA.

(1) Testing steps

1. The apoptotic cells are lysed and centrifuged at high speed, and the supernatant contains nucleosomes;
2. Adsorption of histone bodies on micro-ration plates;
3. Adding anti-histone antibody to the histone on the nucleosome;
4. Adding a peroxidase-labeled anti-DNA antibody to bind to DNA on the nucleosome;
5. Add enzyme substrate, photometric absorption system.

(2) Use

The method is highly sensitive and can detect 5*100/ml apoptotic cells. It can be used for apoptosis detection in human, rat and mouse. This method does not require special instruments and is suitable for grassroots work, but it cannot accurately determine the absolute amount of apoptotic cells.

The fourth measure: nowhere shape - quantitative analysis by flow cytometry

(1) Detection principle

When cells undergo apoptosis, the permeability of their cell membranes also increases, but the extent is between normal cells and necrotic cells. Using this feature, the cell suspension to be tested is stained with fluorescein, and the cell fluorescence intensity in the cell suspension is measured by flow cytometry to distinguish between normal cells and apoptotic cells of necrotic cells.

(2) Application value

Flow cytometry has the following characteristics:
1. The number of cells detected is large;
2, can do a lot of correlation analysis;
3. Combining the analysis of the DNA content of the cells to be detected, the cell cycle in which the apoptotic cells are located can be determined.