Cell apoptosis was detected from morphology
Many experiments friend said I do not know how to distinguish between apoptosis and necrosis, cell apoptosis and necrosis are actually two different forms of cell apoptosis, according to cell death in the differences in morphology, biochemistry and molecular biology, to distinguish between them.
There are many methods for the detection of apoptosis, and the following methods are introduced.
Morphological detection of apoptotic cells has certain characteristics in morphology.
Analysis direction: intracellular metabolites qualitative change, from the qualitative and quantitative to qualitative quantitative in situ, etc., have developed relatively mature testing technology, compared with other detection methods, fluorescent staining method to detect apoptosis has many characteristics, such as economic, rapid and sensitive.
The first step, the light microscope observation
The main characteristics of apoptotic cells are the dense and dark chromatin, which form dense masses and sometimes rupture.
In the tissue section of HE staining, the cell volume was reduced, the cytoplasm density and eosinophilic staining were enhanced, and apoptosis was formed.
Apoptotic cells in the tissue are often dispersed in a single form, and the apoptotic cells are isolated from the surrounding cells without causing an inflammatory response.
This method is simple and easy to use, but it is difficult to change the atypical cell judgment in the cell dense tissue, and often lacks the characteristic index, which has a strong subjectivity and repetitive difference.
This method can be used for the preliminary observation of the phenomenon of apoptosis, as one of the analysis indexes.
Detection method:
(1) unstained cells: the cell size of apoptotic cells is small, deformed, and the cell membrane is complete but foaming, and apoptosis is visible in the late stage of apoptosis.
The wall cells are shriveled, rounded and detached.
(2) cell smear or tissue paraffin section for HE staining or Giemsa staining, observed the morphological changes of apoptotic cells under high magnification, and the apoptotic cell count could be used in combination with the microscopic measurement tool.
Dyeing cells: common JiM dyeing, red, etc. Apoptosis cell chromatin condensed and marginalized, the nuclear membrane cracking, chromatin divided into blocks, and apoptotic body such as a typical form of apoptosis.
The second step, electron microscopy
Although optical microscope can see the cell membrane popping and apoptotic bodies, but the morphology change occurs mostly in ultrastructure of apoptosis cells, thus observed with satisfactory hard, cellular structure and transmission electron microscopy (sem) can be clearly observed in the apoptosis in different periods of change.
Electron microscope observation is the most classic and reliable method for apoptosis so far, and is considered to be the gold standard for cell apoptosis.
Detection method:
The specimen of transmission electron microscope was fixed with glutaraldehyde and hunger acid, acetone dehydration, epoxy resin coating, ultrathin section, acid citrafter acid electron and redyeing, transmission electron microscope observation.
The scanning electron microscope specimen was fixed by glutaraldehyde and hunger acid, the ethanol was dehydrated by stage, and the CO2 critical point was dry, the vacuum sprayed gold and the scanning electron microscope was observed.
Results:
Apoptosis of cell surface microvillus disappeared, nuclear chromatin pyknosis, side set, often assumes the crescent, nuclear membrane folds, cytoplasmic tight, organelles, cell membrane blister or out "bud" and apoptotic body and apoptotic body is near the phenomenon of macrophage cell.
Disadvantages:
(1) only qualitative, not quantitative;
(2) the process of specimen processing is complicated, the equipment is relatively expensive, and the technical level of the inspectors is high, which is not suitable for large samples;
(3) sometimes apoptosis is difficult to be identified with normal cell mitosis when conducting electron microscopy in the tissue section, because the chromatin concentration can occur in both cases.
If fluorescence staining is performed at the same time, it can make up for the shortage of electron microscope.
The third step, fluorescence microscope and confocal laser scanning microscope
Detection basis:
The development of apoptosis was evaluated by morphological changes of chromatin in the nucleus.
Common dna-specific dyes are: HO 33342 (Hoechst 33342), HO 33258 (Hoechst 33258), DAPI.
The combination of three dyes and DNA is non-embedded, mainly in the a-t base area of DNA.
Ultraviolet light emits bright blue fluorescence.
Hoechst is a reactive dye with DNA specific binding. The storage solution is diluted with distilled water to 1 mg/ml and is diluted with PBS with a final concentration of 10 mu g/ml.
DAPI is semi-permeable and used for routine fixed cell staining.
The storage solution is divided into 1 mg/ml concentration using distilled water. The final concentration is usually 10 mu g/ml.
The fourth step, flow cell technology
The flow cytometry detects the apoptotic cell by examining its photoshoot characteristics and fluorescence parameters.
When the cell passes through the laser beam set point of the flow cytometry, the laser is scattered, and the scattering light can provide information about the size and structure of the cell.
The mechanical damage and cell necrosis of cells are not specific indicators of apoptotic cells in light scattering pasture.
Therefore, only by combining the detection of light scattering characteristics with the detection of fluorescence parameters can the apoptotic cells be accurately identified.
Endonuclease during the process of cell apoptosis in the degradation of DNA molecule between nucleosome, lead to small molecules of DNA to leak, cut down the content of nuclear DNA and cell fluorescence staining flow cytometry instrument analysis, can be found in DNA histogram of normal diploid cells Go/G1 peak appeared before a and diploid peak (xub - G1 peak, peak, the AP apoptotic peak), on behalf of apoptotic cells.
Apoptosis morphology test results intuitive, is still the gold standard when it comes to determine whether the cell apoptosis occurred, but observe apoptosis cells morphological characteristics of time-consuming, is not suitable for the detection of a large number of samples of apoptosis.
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