The medium is an indispensable nutrient in cell culture, but the most basic medium brings a series of problems to the experimenter. Don't underestimate cell culture, there is a university question here.
1. How to find the relevant knowledge and culture conditions of a specific cell line?
The ATCC (American Type Culture Collection) collects the details of most cells. Open the Cells and hybridomas link on the ATCC web page and enter the cell name to search the ATCC cell database. The database contains a detailed description of each cell, including the source of the cells, culture and cryopreservation conditions, and related literature.
2. How to choose special cell line medium?
There is no fixed culture condition for culturing certain types of cells. Cells cultured in MEM are likely to grow equally well in DMEM or M199. In conclusion, MEM and DMEM are preferred for adherent cell culture; RPMI1640 is used for suspension cell culture; AIMC medium (SFM) is preferred for serum-free culture for various purposes. Check the data for new cell cultures.
3. How long can my newly prepared liquid medium be kept?
We recommend that the newly prepared medium be placed at 4 ° C and stored as far as possible within one week. The fresher the medium, the better.
4. After adding serum and antibiotics to the medium, can it be stored for a long time?
Once you have added serum and antibiotics to your fresh medium, you should use it within one to two weeks. Because some antibiotics and essential components in serum begin to degrade after thawing.
5. Is it necessary to add antibiotics to the medium?
Except for special screening systems, no antibiotics should be added to the medium under normal culture conditions.
In order to prevent bacterial and fungal contamination, a penicillin-streptomycin double-antibody solution may be added, and the final concentration in the medium is 100 U/ml penicillin and 100 ug/ml streptomycin.
6. Can the liquid medium be stored at -20 °C?
No! Since the salt in the medium is easily precipitated at -20 ° C, some salts may not be redissolved after the medium is melted, resulting in a decrease in the osmotic pressure of the medium, and the cells are easily broken and die when the cells are cultured.
7. Why is the medium stored in a 4 ° C refrigerator, the color will be dark red, and the pH is more alkaline?
The medium is stored in a refrigerator at 4 ° C, and the CO2 in the medium gradually overflows, causing the medium to become more alkaline. The color of the acid-base indicator (usually phenol red) in the medium will also be darker red as the alkalinity increases. As a result of the alkaline medium, the cell growth will be stagnant or dead. If the medium is alkaline, the sterile filtered CO2 can be passed to adjust the pH.
8. Why is your company's liquid medium 1640 yellow in color, is the pH wrong?
No. Because of the formulation, 1640 is an alkali phenol red type, in which the concentration of phenol red is only one quarter of DMEM, so it is because the concentration of phenol red is low, not low.
9. How does the pH of the culture solution affect cell growth?
Since most cells have a suitable pH of 7.2-7.4, deviations from this range will have a detrimental effect on the cells. The pH requirements of various cells are not completely the same. Primary cell cultures generally have poor tolerance to pH changes, and infinite cell lines are highly tolerant. However, in general, cell acid resistance is stronger than alkali resistance, and cell growth is more favorable in acid environment. Therefore, when the culture liquid is prepared, the pH of the liquid can be slightly adjusted to be slightly acidic. When the liquid is filtered through a 0.1um or 0.2um filter, the pH will fluctuate by about 0.2.
10. How does the osmotic pressure of the culture fluid affect cell growth?
Osmotic pressure is produced when the molecular concentrations of the internal and external environments of the cells are different. In this case, moisture flows into or out of the cells through the permeation, thereby changing the internal environment of the cells. High osmotic pressure forces water to diffuse out of the cell and cause cell contraction, which can destroy DNA and protein, stagnant cell cycle and eventually cell death. Conversely, low osmotic pressure causes water to flow into the cell, causing the cell to swell and rupture. .
11. Why do customers require that the medium does not contain phenol red?
Studies have shown that phenol red can mimic the effects of sterols (especially estrogens). In order to avoid sterol reaction, cell culture, especially mammalian cells, is carried out without phenol red. Due to phenol red interference detection, some researchers did not use phenol red-containing medium for flow cytometry.
12. What is the role of phenol red?
Phenol red is used as a pH indicator in the medium: red in neutral, yellow in acidity, and purple in alkaline.
13. Does your company's dry powder medium contain NaHCO3?
NaHCO3 is not included in all dry powder media. Please add NaHCO3 after the dry powder medium is completely dissolved according to the instructions or the label on the bag.
14. What is the role of NaHCO3?
Together with CO2, a buffer system is formed to keep the pH of the medium stable.
15. Our laboratory incubator CO2 concentration has been 5% all the time, is this ok?
Of course, the amount of NaHCO3 added to different media is different. For example, DMEM requires 3.7g/L. In order to maintain the pH value, it must be balanced with a high concentration of CO2, generally not less than 8%, and 1640. With the addition of NaHCO3 2.2 g/L and some media requiring less addition, these media require a lower concentration balance, typically 5%.
16. What is the role of sodium pyruvate in the medium?
Sodium pyruvate can be used as an alternative carbon source in cell culture. Although cells are more likely to use glucose as a carbon source, cells can also metabolize sodium pyruvate without glucose.
The concentration of sodium pyruvate stock solution is generally 50 mM, stored at -20 ° C, the final concentration in the medium is 1 mM.
17. What is the role of glutamine?
Almost all cells have high requirements for glutamine. When glutamine is deprived of amino groups, it can be used as a source of energy for cultured cells, involved in protein synthesis and energy metabolism. In the absence of glutamine, cells die poorly and die. Therefore, various culture solutions contain a relatively large amount of glutamine.
Glutamine is very unstable in solution and should be stored frozen at -20 ° C and added to the medium before use. When the glutamine-added liquid medium is stored in a 4 degree refrigerator for more than two weeks, the original amount of glutamine should be re-added.
18. What are the benefits of adding HEPES to the media?
The most commonly used Buffer system in culture media is bicarbonate, which provides nutrients but reduces buffering capacity at physiological pH. HEPES is a very strong Buffer. Adding HEPES can enhance the buffering capacity of cell culture medium under the condition of pH 7.2-7.6.
Hepes is generally stored at a concentration of 1 M and stored at room temperature. The final concentration in the medium was 25 mM, ie 5 ml of 1 M Hepes was added to 200 ml of complete medium.
19. What is GlutaMAX-I? How do cultured cells utilize GlutaMAX-I? How stable is this dipeptide?
GlutaMAX-I is a derivative of L-glutamine whose unstable a-amino group is protected with L-alanine. A peptidase gradually cleaves the dipeptide and releases L-glutamine for use.
The GlutaMAX-I dipeptide is very stable, and even after sterilization at 121 ° C for 20 min, the GlutaMAX-I dipeptide solution has minimal degradation; under the same conditions, L-glutamine is almost completely degraded.
L-glutamine is important in cell culture, but it is unstable in solution and degrades over time, and the exact degradation rate has not been determined. Degradation of L-glutamine leads to the formation of ammonia, which is toxic to some cells. Therefore GlutaMAX-I is a substitute for glutamine in cell culture.
20. Can I use a medium different from the original culture conditions?
No. Each cell strain has its own specific and adapted cell culture medium. If the medium is different from the one originally provided, the cells are mostly unable to adapt immediately, resulting in the cell not being able to survive.
1. How to find the relevant knowledge and culture conditions of a specific cell line?
The ATCC (American Type Culture Collection) collects the details of most cells. Open the Cells and hybridomas link on the ATCC web page and enter the cell name to search the ATCC cell database. The database contains a detailed description of each cell, including the source of the cells, culture and cryopreservation conditions, and related literature.
2. How to choose special cell line medium?
There is no fixed culture condition for culturing certain types of cells. Cells cultured in MEM are likely to grow equally well in DMEM or M199. In conclusion, MEM and DMEM are preferred for adherent cell culture; RPMI1640 is used for suspension cell culture; AIMC medium (SFM) is preferred for serum-free culture for various purposes. Check the data for new cell cultures.
3. How long can my newly prepared liquid medium be kept?
We recommend that the newly prepared medium be placed at 4 ° C and stored as far as possible within one week. The fresher the medium, the better.
4. After adding serum and antibiotics to the medium, can it be stored for a long time?
Once you have added serum and antibiotics to your fresh medium, you should use it within one to two weeks. Because some antibiotics and essential components in serum begin to degrade after thawing.
5. Is it necessary to add antibiotics to the medium?
Except for special screening systems, no antibiotics should be added to the medium under normal culture conditions.
In order to prevent bacterial and fungal contamination, a penicillin-streptomycin double-antibody solution may be added, and the final concentration in the medium is 100 U/ml penicillin and 100 ug/ml streptomycin.
6. Can the liquid medium be stored at -20 °C?
No! Since the salt in the medium is easily precipitated at -20 ° C, some salts may not be redissolved after the medium is melted, resulting in a decrease in the osmotic pressure of the medium, and the cells are easily broken and die when the cells are cultured.
7. Why is the medium stored in a 4 ° C refrigerator, the color will be dark red, and the pH is more alkaline?
The medium is stored in a refrigerator at 4 ° C, and the CO2 in the medium gradually overflows, causing the medium to become more alkaline. The color of the acid-base indicator (usually phenol red) in the medium will also be darker red as the alkalinity increases. As a result of the alkaline medium, the cell growth will be stagnant or dead. If the medium is alkaline, the sterile filtered CO2 can be passed to adjust the pH.
8. Why is your company's liquid medium 1640 yellow in color, is the pH wrong?
No. Because of the formulation, 1640 is an alkali phenol red type, in which the concentration of phenol red is only one quarter of DMEM, so it is because the concentration of phenol red is low, not low.
9. How does the pH of the culture solution affect cell growth?
Since most cells have a suitable pH of 7.2-7.4, deviations from this range will have a detrimental effect on the cells. The pH requirements of various cells are not completely the same. Primary cell cultures generally have poor tolerance to pH changes, and infinite cell lines are highly tolerant. However, in general, cell acid resistance is stronger than alkali resistance, and cell growth is more favorable in acid environment. Therefore, when the culture liquid is prepared, the pH of the liquid can be slightly adjusted to be slightly acidic. When the liquid is filtered through a 0.1um or 0.2um filter, the pH will fluctuate by about 0.2.
10. How does the osmotic pressure of the culture fluid affect cell growth?
Osmotic pressure is produced when the molecular concentrations of the internal and external environments of the cells are different. In this case, moisture flows into or out of the cells through the permeation, thereby changing the internal environment of the cells. High osmotic pressure forces water to diffuse out of the cell and cause cell contraction, which can destroy DNA and protein, stagnant cell cycle and eventually cell death. Conversely, low osmotic pressure causes water to flow into the cell, causing the cell to swell and rupture. .
11. Why do customers require that the medium does not contain phenol red?
Studies have shown that phenol red can mimic the effects of sterols (especially estrogens). In order to avoid sterol reaction, cell culture, especially mammalian cells, is carried out without phenol red. Due to phenol red interference detection, some researchers did not use phenol red-containing medium for flow cytometry.
12. What is the role of phenol red?
Phenol red is used as a pH indicator in the medium: red in neutral, yellow in acidity, and purple in alkaline.
13. Does your company's dry powder medium contain NaHCO3?
NaHCO3 is not included in all dry powder media. Please add NaHCO3 after the dry powder medium is completely dissolved according to the instructions or the label on the bag.
14. What is the role of NaHCO3?
Together with CO2, a buffer system is formed to keep the pH of the medium stable.
15. Our laboratory incubator CO2 concentration has been 5% all the time, is this ok?
Of course, the amount of NaHCO3 added to different media is different. For example, DMEM requires 3.7g/L. In order to maintain the pH value, it must be balanced with a high concentration of CO2, generally not less than 8%, and 1640. With the addition of NaHCO3 2.2 g/L and some media requiring less addition, these media require a lower concentration balance, typically 5%.
16. What is the role of sodium pyruvate in the medium?
Sodium pyruvate can be used as an alternative carbon source in cell culture. Although cells are more likely to use glucose as a carbon source, cells can also metabolize sodium pyruvate without glucose.
The concentration of sodium pyruvate stock solution is generally 50 mM, stored at -20 ° C, the final concentration in the medium is 1 mM.
17. What is the role of glutamine?
Almost all cells have high requirements for glutamine. When glutamine is deprived of amino groups, it can be used as a source of energy for cultured cells, involved in protein synthesis and energy metabolism. In the absence of glutamine, cells die poorly and die. Therefore, various culture solutions contain a relatively large amount of glutamine.
Glutamine is very unstable in solution and should be stored frozen at -20 ° C and added to the medium before use. When the glutamine-added liquid medium is stored in a 4 degree refrigerator for more than two weeks, the original amount of glutamine should be re-added.
18. What are the benefits of adding HEPES to the media?
The most commonly used Buffer system in culture media is bicarbonate, which provides nutrients but reduces buffering capacity at physiological pH. HEPES is a very strong Buffer. Adding HEPES can enhance the buffering capacity of cell culture medium under the condition of pH 7.2-7.6.
Hepes is generally stored at a concentration of 1 M and stored at room temperature. The final concentration in the medium was 25 mM, ie 5 ml of 1 M Hepes was added to 200 ml of complete medium.
19. What is GlutaMAX-I? How do cultured cells utilize GlutaMAX-I? How stable is this dipeptide?
GlutaMAX-I is a derivative of L-glutamine whose unstable a-amino group is protected with L-alanine. A peptidase gradually cleaves the dipeptide and releases L-glutamine for use.
The GlutaMAX-I dipeptide is very stable, and even after sterilization at 121 ° C for 20 min, the GlutaMAX-I dipeptide solution has minimal degradation; under the same conditions, L-glutamine is almost completely degraded.
L-glutamine is important in cell culture, but it is unstable in solution and degrades over time, and the exact degradation rate has not been determined. Degradation of L-glutamine leads to the formation of ammonia, which is toxic to some cells. Therefore GlutaMAX-I is a substitute for glutamine in cell culture.
20. Can I use a medium different from the original culture conditions?
No. Each cell strain has its own specific and adapted cell culture medium. If the medium is different from the one originally provided, the cells are mostly unable to adapt immediately, resulting in the cell not being able to survive.
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