What is the difference between calf serum and fetal bovine serum? Application notes?
Different sources: fetal bovine serum (Fetal Bovine Serum, FBS) was pregnant at the time of slaughter cows, serum obtained by cardiac puncture blood fetal bovine, newborn calf (calf) serum (Calf serum, CS) collected from birth 10 The calf to 14 days.
The composition is different from the ratio: the components of the growth-promoting factor, the adhesion-promoting factor, the hormone and other active substances contained in the two are different in proportion and usage, and some uses different fetal bovine serum to grow, and some The cells only need calf serum, and the concentration is different, generally 5%-10% , and the concentration is 20% .
Serum storage and use should be noted: serum should be stored at -5 ° C to -20 ° C, if stored at 4 ° C for more than one month. When thawing serum, the serum should be placed in a refrigerator at 2-8 °C, and shaken frequently to dissolve it. Then, it should be placed at room temperature to warm it up. Never put the frozen serum directly into a 37 °C water bath or incubator. After thawing at 37 °C, the color will be deepened and the viscosity will increase. After thawing and heat inactivation, the serum should be stored according to the dosage and stored at -20 °C to avoid repeated freezing and thawing.
What is the concentration of trypsin digestion? Does it contain EDTA? concentration?
Typical levels of trypsin digestion and passage was 0.25%, since the fraction of cells adherent tight, the need to add 0.53mM EDTA, if done primary culture, the concentration of trypsin may need some improvement.
Recommended media and special requirements for commonly used cell lines?
The general principle of selection of conventional medium is that RPMI1640 is the main suspension culture cell , and the adherent cultured cells are mainly DMEM . Some cells need to be cultured with special culture solution, such as Mccoy'5A , L-15 , F12K , D+F12. and many more.
Identification, prevention and treatment of contamination of cell lines.
Ordinary microbial contamination: the medium is turbid, microbial contamination is observed by high power microscope; it is discarded according to regulations and fully sterilized.
 Mycoplasma contamination: the microscope can not be observed, according to experience, the cells grow slowly, there are cells floating, etc., should be identified by PCR and other methods. If the mycoplasma contamination is found, it should be discarded according to the regulations. The laboratory should be disinfected and disinfected in time to prevent the spread. . (Some routine cytology experiments are not affected by mycoplasma contamination, can continue to use cell passage, spread and spread for anti-pollution, can selectively add high-priced antibiotics to the culture medium, and operate in isolation laboratory and use independently. Everything and medium, etc.).
How to pass on adherent cells?
After the cells grow into a dense monolayer, the cells are substantially saturated. In order for the cells to continue to grow and the number of cells to be enlarged, passage (re-culture) is necessary. Subculture is also a way to preserve cell types. It is also a necessary process for culturing cells for various experiments.
Suspension cells can be dispensed directly, while adherent cells need to be digested before they can be divided. Generally, proteolytic enzymes (such as trypsin) are used to digest adherent cells into single cells, and ethylenediaminetetraacetic acid ( EDTA ) can also be added to improve digestion. EDTA is a divalent ion chelating agent that inhibits Ca2+ on cell membranes. And Mg2+ . Commonly used cell digestive juices generally contain 0.25% (w/v) trypsin and 0.03% (w/v) EDTA . Generally, they are configured with a salt solution containing no Ca2+ and Mg2+ . The cells are first washed with trypsin- EDTA. 5.0 -10.0 ml/75cm ), then discard the digestive juice, re-add a small amount of trypsin- EDTA digestive solution (2.0 to 5.0 ml/75 sq. cm flask) , observe the cells until the cells are rounded off the bottle wall. This process is generally It takes 5-15 minutes. In order to avoid cell agglomeration, do not tap the cell bottle during the process of digesting the cells. For cells that are particularly difficult to digest, trypsin EDTA digestion solution can be added to the cells at 37 °C to promote digestion. After the cells are detached from the bottle wall, the serum is completely added to stop the digestion, and certain proteins in the serum can inhibit the pancreas. Enzyme activity.
In general, centrifugation is not required. If the cells require serum-free or low-sera medium, centrifuge at 125,000 g for 5 minutes, then discard the medium for suspension, add the appropriate new medium, gently mix the cells, and transfer to a new flask. The proportion of passage depends on the cell type, which is generally 1:2-1:20 . The specific ratio can be referred to the instructions. Trypsin can damage the cell membrane of some cells. For this type of cells, the cells can be scraped off with a cell scraper, an appropriate amount of medium is added, and the cells are gently pipetted and subcultured.
How often is the frequency of passage cells?
Passage refers to moving cells from one culture flask to another, and the interval between passages refers to the time between passages.
When the confluence of adherent cells reaches or approaches 100% , subculture is required. However, some cells grow in cloned form, and the confluence never reaches 100% . For this type of cells, the cells reach or When you are close to the maximum density, you need to pass on. Contact-inhibiting cells (such as 3T3 ) need to be passaged before the cells are full to avoid changes in traits following contact inhibition after cell overgrowth. Suspended cells must be passaged before the cells reach maximum saturation density. Suspension cells are only diluted to a suitable density for passage, allowing the cells to have sufficient nutrients to recover to logarithmic growth phase.
If it is simply a liquid exchange and the number of cells does not decrease, the cells will quickly deplete the nutrients and die; if the cells are diluted to the lowest density, the cells will enter a stagnation period without proliferating or dying. The maximum saturation density and the interval and ratio of passage of different suspension cells are different, so the cultured suspension cells should be observed daily.
How to deal with tumor cell lines that are unclear for biosafety?
It is difficult to understand the biohazard that can be produced by each cell, but it is strongly recommended that all human and other primate cells be operated under experimental conditions that prevent HIV and hepatitis viruses. All operations must be in the vertical layer. Flow in the clean bench operation (recommended biosafety cabinet), the waste liquid and waste equipment generated during the operation must be disposed of after cleaning, acid treatment, etc.
Can I use different media on the cell instructions to culture my cells?
The recommended medium in the product catalog or on the cell manual is generally the medium recommended by the original provider of the cell line or the most suitable medium for the cell strain that has been validated. The cells may be adapted to the unrecommended medium, and May not adapt. Care should be taken when changing the medium. When changing the medium, leave a bottle of cells in the recommended medium and keep them as seeds.
There are two ways to change the medium. The easiest way is to change the medium directly and force the cells to adapt to the new medium. There is also a milder method: when the cells are passaged, they are divided into two bottles. The first bottle is recommended. The medium, the second bottle uses 50% of the original recommended medium, 50% of the new medium, and then carefully observe the growth state of the cells. If the second bottle of cells is not in good growth state, stop replacing the medium immediately. The second bottle grows well and can be subcultured. One bottle uses 50% of the original recommended medium, 50% of the new medium, the other bottle uses 25% of the original recommended medium, and 75% of the new medium continues. Observe that if the cells are in good condition, they can all be replaced with fresh medium.
Related Links:
- Common problems in cell culture (2)
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