Common Reagents in Cell Culture
When animal cells or tissues leave the body to be cultured outside the body, it is necessary to provide them with various nutrients required for growth and various reagents required for better cell growth in vitro. In this issue, I will briefly introduce some reagent components used in the cell culture process and their functions.
1. Cell culture medium composition
(1).Basic culture medium
Common basal media mainly include MEM, DMEM, and RPMI, which mainly provide buffer systems including glucose, amino acids, water, trace elements, and sodium bicarbonate.
● Glutamine
It is an essential amino acid necessary for cell growth and participates in protein synthesis and nucleic acid metabolism.
● Phenol red indicator
A commonly used acid-base indicator, it shows yellow when it is acidic, red when it is neutral, and purple when it is alkaline. Phenol red is used as an indicator of pH value in the medium.
● Carbonate/PH buffer system
A physiological pH buffering system. Due to acid production during cell metabolism, the HCO3- in the medium will be gradually consumed, so CO2 needs to be replenished in time to maintain the pH within a certain range.
(2).Animal serum
Add 2%-20% serum to the basal medium to form a complete medium. Serum can not only provide the cells with the basic nutrients needed for growth, but also contain important components involved in cell metabolism such as growth factors, hormones, trace elements and ions.
(3). Antibiotics
In order to prevent the cells from being contaminated during the culture process, a penicillin-streptomycin mixture is usually added to the complete medium, that is, a double antibody. Penicillin is mainly effective against Gram-positive bacteria, and streptomycin is effective against many Gram-negative bacteria. .
2. Trypsin-EDTA
Trypsin can hydrolyze the protein between cells and then separate the cells. EDTA is a chemical chelating agent with low toxicity and has a discrete effect on adherent cells. When the two are used in combination, the efficiency of dispersing cells is higher. This step is called digestive cells. For different cells, there are different requirements for the temperature, time, and concentration of trypsin when used. If digestion is insufficient, the cells cannot be digested, and excessive digestion will cause cell damage. Since serum in the medium can reduce the activity of trypsin, complete medium is usually used to terminate cell digestion.
3. PBS buffer
PBS is a phosphate-buffered saline solution with salt balance and adjustable pH buffering effect. It is generally used to wash cells and maintain cell osmotic pressure.
4. Dimethyl sulfoxide (DMSO)
An important penetrating cell protectant, and an important component in cell cryopreservation fluid. During the cryopreservation process of cells, DMSO can quickly penetrate the cell membrane and enter the cell, lower the freezing point, delay the freezing process, increase the ion concentration in the cell, reduce the formation of ice crystals in the cell, and reduce cell damage.
5. Mycoplasma inhibitors
Mycoplasma contamination is a common source of contamination in the cell culture process. However, mycoplasma inhibitors can be used to eliminate mycoplasma in the cell fluid at the initial stage of contamination, so that the experiment can continue.
6. Trypan blue dye
A biological staining reagent commonly used to detect cell membrane integrity. Due to the loss of cell membrane integrity and increased permeability, dead cells can be stained blue by trypan blue, while live cells will not be stained because they have intact cell membranes that can exclude trypan blue from entering the cell. Live and dead cells can be distinguished by cell color under a microscope, which is convenient for live cell counting.