Apoptosis and necrosis are two completely different forms of apoptosis, which can be distinguished based on differences in morphology, biochemistry, and molecular biology of dead cells. There are many methods for detecting apoptosis. Several commonly used assay methods are described below.
According to the inherent morphological characteristics of apoptotic cells, many different methods for detecting apoptosis morphology have been designed.
1 optical Microscope and inverted microscope
(1) Unstained cells: The volume of apoptotic cells becomes smaller and deformed, the cell membrane is intact but foaming occurs, and apoptotic bodies are seen in the late stage of apoptosis. Adherent cells appear shrunk, round, and fall off.
(2) Stained cells: Giemsa staining, Wright's staining, etc. are commonly used. The apoptotic cells are characterized by chromatin condensation, marginalization, nuclear membrane lysis, chromatin segmentation into massive and apoptotic bodies.
2 fluorescence microscope and confocal laser scanning microscope
The progression of apoptosis is generally judged by the morphological changes of nuclear chromatin as an indicator.
Commonly used DNA-specific dyes are: HO 33342, HO 33258, DAPI.
The combination of the three dyes with DNA is non-embedded and binds primarily to the AT base region of the DNA. Bright blue fluorescence is emitted when excited by ultraviolet light. Hoechst is a reactive dye that specifically binds to DNA. The stock solution is diluted with distilled water to a concentration of 1 mg/ml. When used, it is diluted with PBS to a final concentration of 2 to 5 mg/ml. Shanghai Chuangsai Technology provides pH7.3 PBS buffer, AR, commodity number: D16-1028529-100ml, inquiry.
DAPI is semi-permeable and is used for staining of conventional fixed cells. The stock solution is diluted with distilled water to a concentration of 1 mg/ml, and the final concentration is generally 0.5 to 1 mg/ml.
Result evaluation:
The morphological changes of nuclear chromatin during apoptosis are divided into three phases:
The nucleus of stage I is rippled or creased, and some chromatin is concentrated;
The chromatin of the stage IIa nuclei is highly coagulated and marginalized;
The nuclear phase of stage IIb is cleaved into fragments, producing apoptotic bodies.
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