The morphological detection method of cell apoptosis is commonly used

1. Optical microscope and inverted microscope

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.

1) undyed cells:

Apoptotic cells are smaller, deformation, shrinkage, cell membrane integrity but appear foaming phenomenon, the late apoptosis visible apoptotic body, apoptotic body several small round body around the cell.

The wall cells are shriveled, rounded and detached.

2) staining cells:

Giemsa (Giemsa) staining and ritz staining, etc. : normal nucleus color uniformity;

Apoptotic chromatin concentration, marginalization, nuclear membrane lysis, chromatin segmentation and apoptosis are typical apoptosis.

The necrotic cells were stained or not stained.

Wood grain - eosin (HE) staining, the nucleus pycnosis disintegrate, blue-black, cytoplasm reddish apoptotic cells (), the normal nuclei homogeneously pale blue or blue, necrosis of the nuclei are very pale blue or blue.

2. Fluorescence microscope and confocal laser scanning microscope

The development of apoptosis was evaluated by morphological changes of chromatin in the nucleus.

Common DNA specific dyes are: Hoechst 33342, 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 that binds to DNA and can enter normal cell membranes without much cytotoxic effects on cells.

The fluorescence intensity of Hoechst 33342 in apoptotic cells is higher than in normal cells.

DAPI is semi-permeable and used for routine fixed cell staining.

Both PI and Hoechst33342 can be combined with nuclear DNA (or RNA).

However, the PI cannot pass through the normal cell membrane, and Hoechst is the membrane permeable fluorescent dye. Therefore, the cell membrane is destroyed when the cell is in necrosis or late stage, and then it can be red with PI.

Normal cells and early apoptotic cells can be colored by Hoechst, but the Hoechst color of normal nucleus is round and pale blue, with deep blue granules.

The nucleus of the apoptotic cell is bright blue because of the concentrated set, or the nucleus is divided into leaves, fragments, and edges.

Therefore, PI is stained to necrotic cells;

Bright blue, or nucleate in lobule, Hoechst in the margin of the apoptotic cell.

The cell volume of apoptotic cells is small and the cytoplasm is concentrated.

Nuclear chromatin in the process of cell apoptosis morphological changes divided into three phases: Ⅰ period of the nuclei are corrugated (rippled) or a crease sample (creased), appear part of chromatin condensed state;

Ⅱ a period the nucleus of the highly condensed chromatin, marginalized;

Ⅱ b period the nucleus of cracking for debris, apoptotic body.

3. Electron microscope

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) can only be qualitative, cannot be quantitative;

2) the specimen treatment process is complicated, the equipment is relatively expensive, and the technical level of the inspectors is high, not suitable for large samples;

3) when the microscopy was observed on the tissue section, sometimes the apoptosis was difficult to identify with the normal cell mitosis, because the chromatin concentrated in both cases.

If fluorescence staining is performed at the same time, it can make up for the shortage of electron microscope.

4. 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.

prev:Did you distinguish between apoptosis and necrosis?(2017-09-12)

next:Keeping cells, pay attention to these details!(2)(2017-09-13)