Culture medium is an indispensable nutrient in cell culture, but the most basic culture medium gives the experimenter a series of problems.
Don't look at cell culture. It's a college question.
1. How to find relevant knowledge and training conditions of a particular cell line?
The ATCC (American Type Culture Collection) collects detailed information about most cells.
Open the Cells and hybridomas link of the ATCC web page and enter the cell name to search the cell database of the ATCC.
There are detailed descriptions of each cell in the database, including the source of cells, culture and freezing conditions, and related literature.
2. How to choose a special cell culture medium?
There is no fixed training condition for a certain type of cell.
Cells cultured in MEM are likely to grow equally easily in DMEM or M199.
In conclusion, MEM and DMEM were preferred to be cultured.
RPMI1640 was used as suspension cell culture.
New cell culture should be examined in detail.
3. How long can you keep your new liquid culture medium?
We suggest that placed new preparation of culture medium in 4 ℃, avoid light preservation as far as possible within a week after use.
The more fresh the medium, the better.
4. After adding serum and antibiotics to the medium, can it be preserved for long time?
Once you add serum and antibiotics to the fresh medium, you should use it within one to two weeks.
Because some of the basic ingredients in antibiotics and serum begin to degrade after thawing.
5. Should antibiotics be added in the medium?
In addition to the special screening system, no antibiotics should be added to the culture medium normally.
In order to prevent bacterial and fungal contamination, penicillin - streptomycin double - resistant solution can be added, the final concentration in the medium of the medium is penicillin 100U/ml, and streptomycin is 100ug/ml.
6. Liquid medium can put - 20 ℃ to save?
Can't be!
Because under - 20 ℃, the salt in the culture medium is apt to precipitate, medium melts, some salt may not be redissolved, osmotic pressure is reduced, resulting in medium culture cell, easy to burst and cell death.
7. Why media stored at 4 ℃ refrigerator, dark red color, and increasing alkaline pH?
Media stored at 4 ℃ refrigerator, media of CO2 will gradually overflow, causing medium is more and more alkaline.
And the color of the acid base indicator (usually phenol red) in the medium also increases with alkalinity.
The result of the medium alkalinity is the result of cell growth stasis or death.
If the culture medium is alkaloid, the CO2 of aseptic filtration can be used to adjust the pH value.
8. Why is the liquid medium purchased in 1640 color yellowing, is the pH not correct?
It isn't.
Because of the formulation, 1640 is base phenol red, the concentration of phenol red is only a quarter of DMEM, so it is because of the low concentration of phenol red, not the low PH value.
9. How does nutrient pH affect cell growth?
Since most cells are suitable for a PH of 7.2-7.4, deviations from this range will have a detrimental effect on the cell.
All kinds of cells are not identical to the PH value, and the original cell culture is generally tolerant to the variation of PH value, and the infinite cell line has strong tolerance.
But overall, the cells are more acidic than alkaline, and the acid environment is more conducive to cell growth.
Therefore, the PH value of the liquid can be slightly adjusted slightly by the preparation of liquid.
When the liquid is filtered by 0.1 um or 0.2 um filter membrane, the PH value will float upwards of 0.2.
10. How does medium osmotic pressure affect cell growth?
When the molecular concentration of the inside and outside of the cell is not at the same time, the osmotic pressure is generated.
In this case, water can change the cell's internal environment by seeping into or out of the cell.
High osmotic pressure forces water to spread out of the cell and causes the cell to contract, which can damage DNA and proteins, stop cell cycles and eventually kill cells.
On the other hand, low osmotic pressure causes water to flow into the cell, causing the cell to swell and burst.
11. Why do customers require that the culture medium do not contain phenol red?
Studies have shown that phenol red can mimic the effects of steroid hormones (especially estrogen).
In order to avoid steroidal reaction, cell culture, especially in mammalian cells, is used in medium without phenol red.
Because of the detection of phenol red interference, some researchers did not use the culture medium of gphenol when doing flow cytometry.
12.What is the role of phenol red?
Phenol red is used as a PH indicator in culture medium: neutral is red, acidic is yellow, and alkaline is purple.
13. Does the dry meal medium contain the NaHCO3?
There is no NaHCO3 in all the dry culture medium.
Please add the NaHCO3 after the dry powder culture medium is completely dissolved in accordance with the instructions on the instructions or on the packing bags.
14. What is the role of NaHCO3?
To form a buffer system with CO2 to keep the PH value of the medium stable.
15. The concentration of CO2 in our laboratory incubator has always been 5%. Is that ok?
Can't, of course, different culture medium to add the quantity of NaHCO3, as DMEM request add 3.7 g/L, in order to keep the PH stable, must balance, with a high concentration of CO2 is generally not less than 8%, and 1640 for adding NaHCO3 2.2 g/L, also some medium require to add less, the medium will require lower concentrations of balance, general is 5%.
16. What is the role of sodium pyruvate in the culture medium?
Sodium pyruvate can be used as an alternative carbon source in cell culture, although cells are more likely to use glucose as a source of carbon, but without glucose, cells can also metabolize sodium pyruvate.
Sodium pyruvate generally 50 mm, the storage of liquid concentration - 20 ℃, the final concentration of culture medium is 1 mm.
17.What is glutamine?
Almost all cells have higher requirements for glutamine.
When glutamine is removed, it can be used as a source of energy for cultured cells to participate in protein synthesis and energy metabolism.
In the absence of glutamine, cells grow poorly and die.
Therefore, a large amount of glutamine is found in various cultures.
Glutamine is very unstable in aqueous solution, should buy - 20 ℃ frozen preservation, join before using the medium.
When the liquid medium of glutamine is added to the refrigerator for more than two weeks, the original amount of glutamine should be rejoined.
18. What are the benefits of adding HEPES to the medium?
The most commonly used Buffer system in the medium is bicarbonate, which provides nutrients but reduces buffering capacity under physiological PH conditions.
HEPES is a very strong Buffer, and adding HEPES can enhance the ability of the cell culture medium to Buffer against the conditions of PH7.2-7.6.
The concentration of Hepes is usually 1M and is kept at room temperature.
The final concentration in the medium was 25mM, which was 5ml 1M of Hepes added to the 200ml complete medium.
19. GlutaMAX - Ⅰ is what?
Culture cell how to make use of GlutaMAX - Ⅰ?
How stable is this peptide?
GlutaMAX - Ⅰ is a derivative of L - glutamine, the stability of the a - L - alanine amino use to protect.
A peptide enzyme gradually cleaved dipeptide, releasing l-glutamine for utilization.
GlutaMAX - Ⅰ dipeptide is very stable, even at 121 ℃ sterilization 20 min, GlutaMAX - Ⅰ dipeptide solution has a minimum degradation;
In the same condition, l-glutamine is almost completely degraded.
L-glutamine is important in cell culture, but it is unstable in solution. After a period of time it will degrade, and the exact degradation rate has not been finalized.
The degradation of l-glutamine leads to the formation of ammonia, which is toxic to some cells.
Hence GlutaMAX - Ⅰ is substitute for glutamine in cell culture.
20. Can you use the medium that is different from the original culture?
Can't.
Each cell lines have their specific use and has been adapted to cell culture medium, if use abruptly and provide original culture conditions of the different culture medium, cells are mostly unable to adapt to immediately, causing cell cannot survive.
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