To identify the cell life and death methods - Database & Sql Blog Articles

What are the methods for identifying cell life and death?

Yuanmu Biotech's technical staff explained: The identification of living cells is of great significance in biology and medicine. In the process of cell culture, the growth of cells should be recorded at any time, and the survival rate of cells should be measured frequently. In the study of tumor cells, in order to test the lethality of various drugs on tumor cells, it is also necessary to determine the survival rate of tumor cells. The identification of living and living cells in clinical medicine is also very useful. For example, in order to test the fertility of a man, it is a common method to determine the viability of sperm cells.

There are many methods for identifying dead and living cells. Commonly used are staining and instrumental analysis. Staining method is a commonly used cell death and identification method, which is simple and easy to operate. However, by direct morphological observation to identify the cell life and death, the experimental results are easily affected by the subjective factors of the operator, there are certain errors, so in the actual operation, some instruments are often used for accurate batch detection.

Different methods for identifying dead and living cells have different specific reaction mechanisms, but no matter what method is adopted, the differences in physiological functions and properties of dead and living cells are utilized.

Common methods include staining and instrumental analysis:

1 staining method

The dyeing method is divided into a chemical staining method and a fluorescent staining method, depending on the dyeing mechanism, the dye or the dead cells are colored, or the living cells are colored. The differences in physiological functions and properties of dead and living cells mainly include:

Differences in cell membrane permeability between living and living cells: The cell membrane of living cells is a selective membrane that protects and barriers cells and allows only selective passage of substances. After cell death, cell membranes are damaged and permeability is increased. The commonly used method of identifying cells by trypan blue is to take advantage of this property. Trypan blue, also known as cone blue, is an anionic dye that does not penetrate intact cell membranes. Therefore, after trypan blue staining, only dead cells can be colored, while living cells are not colored. Methyl blue has a similar dyeing mechanism. The plasmolysis of plant cells can also identify life and death.

Metabolic differences between dead and living cells: It is the basis for identifying the survival of yeast cells using methylene blue dye. Methylene blue is a non-toxic dye with an oxidized blue color and a reduced colorless color. Due to the metabolism in living cells, the cells have a strong reducing ability, which can change the oxidative properties of blue from blue to a colorless reducing type. Therefore, the live yeast cells are colorless after the blue dyeing; Cells or cells with slow metabolism are extremely weak due to their non-reducing ability or reducing ability, so that the blue is in an oxidized state, and thus dyed blue or light blue.

Fluorescein diacetate (FDA) is a commonly used viability marker for cultivating animal and plant cells and plant cell protoplasts. Its staining mechanism also utilizes the metabolic differences between dead and live cells: the FDA itself does not produce fluorescence and is extremely prosperous. Sexual, free to penetrate into the intact cell membrane. When the FDA enters living cells, it is decomposed by the lipase in the cell to produce a polar, fluorescent substance, fluorescein, which cannot pass through the living cell membrane and accumulates in the cell membrane, thus making it viable. Cells produce green fluorescence; while non-viable cells fail to produce fluorescing because they do not break down the FDA.

In addition, there are some proprietary dyes for organelles. A proprietary dye such as a bubble medium is neutral red. Neutral red is a low-toxic dye that can make living cells vacuole red, while cytoplasm and nucleus are not stained; vacuoles of dead cells are not stained or lightly stained, dyes are dispersed throughout the cell, and nuclei and cytoplasm are stained red. Sometimes, in order to increase the dyeing effect, two dyes can be used in combination, such as methyl blue-neutral red mixed dyeing.

2 Instrumental analysis

Micro-electrode technology (using the difference in potential between the two sides of the dead and living cell membranes), fully automated cell counting and flow cytometry can be used to accurately detect the cell's life and death.

The fully automated Cedex cell viability/cytometer/cell analyzer from INNOVATIS, Germany is the world's first high-resolution, high-resolution scanning (patent) cell analyzer with fully automated trypan blue staining and microscopic CCD Imaging, CEDEX workstation software, can be used in the research and development of pharmaceutical, medical, fermentation, immune, pathology, toxicity testing, biotechnology and other fields of cell counting and analysis. Automated analysis results, detection and analysis data output (11 items): total cell number, total cell concentration (cells/mL); number of viable cells, viable cell concentration (cells/mL); number of dead cells, dead cell concentration (cells/mL) Cell viability (%); cell's average compactness; cell average diameter (um); Aggregate Rate; Diameter Histogram; Aggregate Histogram ); Cellularity Histogram; Cell Growth Time Chart.

Flow cytometry: There are two main types of commonly used fluorescent probes for determining cell death and death:
One type is a substance that emits fluorescence by entering a cell through a living cell membrane. For example, the FDA can be left by living cells to emit yellow-green fluorescence, and if the cell is damaged, it will be lost from the cell, and no fluorescence can be observed.
The other type is that it does not pass through the living cell membrane, but it can stain the nuclei of cells with damaged cells and membranes. For example, propidium iodide (PI, Prothiium iodide) and ethidium bromide (EB, Ethidium bromide) are commonly used. The second type of fluorescent probe. Propidium iodide (PI) does not penetrate the cell membrane and cannot stain normal or apoptotic cells with intact cell membranes. For necrotic cells, the integrity of the cell membrane is lost, and propidium iodide (PI) can stain necrotic cells. A commonly used assay kit for apoptosis and necrosis contains Hoechst 33342 and Propidium Iodide (PI) fluorescent dyes. When the cells undergo apoptosis, the chromatin will shrink, Hoechst 33342 can penetrate the cell membrane, and the fluorescence of apoptotic cells will be significantly enhanced after staining.
After double staining of the above two dyes, when using flow cytometry or fluorescence microscopy, the normal cells are weak red fluorescence + weak blue fluorescence, the apoptotic cells are weak red fluorescence + strong blue fluorescence, and the necrotic cells are strong red fluorescence. + Strong blue fluorescence.