Determination of dead and alive cells and experiment of cell viability in cell culture
In the process of cell culture, the survival rate of cells needs to be measured to understand the growth state of the cells, and the commonly used methods for identifying cell survival are staining and instrumental analysis. The staining method can directly distinguish cell death through cell morphology, and it is a simple cell survival identification method. Here we summarize the experimental principles, experimental procedures and precautions for the determination of dead cells and cell viability.
ã€Experimental Principle】
(1) Staining method is a commonly used method for cell death identification, simple and easy to operate. The experiment was conducted using the differences in physiological functions and properties of dead and living cells. The differences in physiological functions and properties of dead and living cells are mainly reflected in:
(1) The difference in cell membrane permeability of dead and living cells: the cell membrane of living cells is a selective membrane that protects and barriers the cells, allowing only selective passage of substances; and after cell death, the cell membrane is damaged and transparent Sexual increase.
(2) Differences in metabolism of dead and live cells: due to the metabolism in living cells, the cells have a strong reducing ability, and dead cells or old cells with slow metabolism are due to their non-reducing ability or reducing ability. weak.
Common cell dyes are: neutral red (a proprietary dye for organelles), trypan blue, methyl blue, methylene blue, fluorescein diacetate (FDA), etc.
â‘ Neutral red staining: one of the commonly used dyes is a proprietary dye for organelles. Use the permeability difference of cell membrane to stain protozoa and display the contents of living cells in animal and plant tissues. Neutral red is non-toxic, often used as a dye for living body dyeing.
â‘¡ Trypan blue staining: When the cell is damaged or dead, trypan blue can penetrate the denatured cell membrane and combine with the disintegrated DNA to make it stain. Living cells can prevent the dye from entering the cell. Therefore, dead cells can be distinguished from living cells. Strictly speaking, trypan blue staining detects the integrity of the cell membrane. It is generally considered that the cell membrane has lost its integrity, that is, the cell has died. The dead cells stained with trypan blue can be easily identified through a microscope and can be counted using a cell counter.
â‘¢Methylene blue staining method: Methylene blue is a non-toxic dye, its oxidized type is blue, and the reduced type is colorless. It is used to stain living cells. Due to the metabolism of cells, the cells It has a strong reducing ability inside, which can change methylene blue from a blue oxidized type to a colorless reduced type, so living cells are colorless, and for dead cells or old cells with slow metabolism, because they have no such reducing ability Or the reduction ability is extremely weak, and it is dyed blue or light blue by the blue. Therefore, using the blue water immersion tablets can not only observe the morphology of yeast, but also distinguish between dead and live cells.
â‘£ Methyl blue dyeing method: Methyl blue dyeing and trypan blue similar dyeing mechanism.
(2) Plant cell cytoplasmic wall separation method and in vivo staining to determine cell death
(1) Plasma wall separation method: Growing plant cells generally have a central vacuole. Between the central bubble and the cell wall is a thin layer of cytoplasm and its inner and outer membranes—the vacuolar membrane and the plasma membrane. The cytosol in the vacuole has a certain solute potential (or osmotic potential), while living protoplasts, especially the vacuole membrane and plasma membrane, are semi-permeable. Therefore, when the cell comes into contact with the external solution, it will form an osmotic system with the external fluid, and the movement of water may occur. If the water potential of the external fluid is lower than the solute potential of the cytosol, the result of water extravasation can cause the protoplast to contract with the vacuole and detach from the cell wall, and the plasmolysis occurs, and the cells separated by the plasmolysis contact with water or a solution with a higher water potential At this time, the water can be reabsorbed but the wall is separated and restored.
(2) Living dyeing method: Living dyeing is a technique of dyeing living cells with a dilute dye solution that is not harmful to plants. Neutral red is one of the commonly used dyes. It is a weakly alkaline pH between 6.4 and 8.0 (from red to yellow). In a neutral or slightly alkaline environment, the living cells of the plant can absorb a large amount of neutral Red is excreted into the vacuole. Because the vacuole is acidic under normal circumstances, the neutral red that enters the vacuole dissociates a lot of cations and appears pink. In this case, the protoplasts and cell walls are generally not stained. Due to the denaturation and coagulation of dead cells, the cytosol cannot be maintained in the vacuole. Therefore, after staining with neutral red, no vacuole coloring occurs. On the contrary, the cation of neutral red is combined with the negatively charged protoplast and nucleus Instead, the protoplasts and nuclei are stained.
[Experimental steps]
1. Reagent configuration:
â‘ Trypan blue: 4% Trypan blue mother liquor: Weigh 4g trypan blue, add a small amount of distilled water to grind, add double distilled water to 100ml, filter with filter paper, and store at 4 degrees. when using it. Dilute to 0.4% with PBS.
â‘¡ Neutral red: Prepare 1% neutral red aqueous solution (1g of neutral red dissolved in 100ml of distilled water). Take 1 ml of this solution and dilute it to 50 ml with 0.6% saline (or distilled water) to make a 0.02% neutral red aqueous solution, store it in a brown bottle and place it in a dark place.
â‘¢ Methylene blue: Take 0.5 grams of methylene blue, dissolve in 30 ml of 95% alcohol, add 100 ml of 0.01% potassium hydroxide solution, and store in a brown bottle. This solution can stain cell nuclei, and is also used to stain bacteria, blood and nerve tissue.
â‘£ Methyl blue: Take 1 gram of methyl blue, dissolve in 29 ml of 70% alcohol, add 70 ml of distilled water to make 1% methyl blue dyeing solution.
2. Dyeing operation steps: omitted
3. Determination of cell viability: The stained cell material is observed under a microscope. All unstained or stained and light cells are viable, while the darkly stained cells are dead cells without viability.
4. Calculate the cell viability according to the formula. Cell viability (%) = number of unstained cells / total number of observed cells X 100%
ã€Precautions】
1. Trypan blue is toxic to human body
2. When staining cells, the time should not be too long. Otherwise, some living cells will also be stained, which will interfere with the count.
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