I have several questions about the preparation of cell culture medium, and I'm hoping to get some clarity on my practices. First, is there a necessary relationship between the amount of sodium bicarbonate and HEPES added when making the medium? I'm concerned that my current approach might affect the quality of the medium, but I'm not sure how significant the impact could be. Also, can I follow the DMEM instructions and add 3.7g/L of carbonic acid? In addition to that, should I add 20mM of HEPES?
When I looked at other literature, I found some conflicting information. Some studies used Hyclone DMEM and only added 1.97g/L of sodium bicarbonate along with 20mM HEPES. After preparation, they adjusted the pH to 7.2–7.4 using 10M NaOH, and the final pH didn't exceed 7.5 after filtration. I tried adjusting the pH with 1M NaOH, but it required a large volume, so I switched to using 10M NaOH as suggested in the literature. However, according to DMEM instructions, I should add 3.7g/L of sodium bicarbonate.
Another thing I’m unsure about is how I added serum to the medium. I didn’t consider whether the serum and the medium are isotonic. I calculated 5% serum by volume, so I dissolved the DMEM dry powder in 950mL of water and then added 50mL of serum. Is this method incorrect?
If my current preparation has a major impact on cell culture, is there a way to fix the existing 500mL of medium, and if so, what should I do?
Additionally, I’ve been adding excessive glutamine to the medium. I’ve heard that too much glutamine might be toxic to cells. Our lab doesn’t prepare the dry powder in one single 1L package but in smaller amounts like 200mL or 400mL. The remaining powder is stored at 4°C, but sometimes it’s not tightly sealed. When we use it again, the powder tends to clump or deliquesce, which may affect nutrient content, especially for glutamine, which can degrade. If I use clumped powder, I want to re-add glutamine, but I worry about overdoing it. Any suggestions?
In terms of best practices: There's no strict relationship between sodium bicarbonate and HEPES. Sodium bicarbonate is essential for forming a carbonate buffer system with CO₂ in the incubator (acting as carbonic acid in solution), while HEPES is an organic amphoteric buffer with a much higher buffering capacity than the carbonate system. Therefore, sodium bicarbonate should be added according to the manufacturer's instructions, and HEPES should be added in a range of 10–25 mM as an auxiliary buffer.
Regarding serum: Some people believe that the amount of serum should be considered when preparing the medium, but most labs now prepare the medium without considering serum volume. The main concern isn't osmotic pressure, but rather the concentration of nutrients. Some cultures use 2X or 4X amino acids, but the effect is minimal. It's generally acceptable to prepare the medium as 1L, regardless of serum volume.
Also, the minimum package of dry powder should be dissolved all at once because the mixture isn't always uniform. Glutamine solutions are unstable, so it's better to make fresh solutions rather than store them. Additionally, you don’t need to adjust the pH after dissolving the powder, as the formulation is typically set to pH 7.4. If you do need to adjust, it's better to flush with CO₂ rather than using alkali.
Finally, I've learned that many cell lines can now be cultured in serum-free media. These are specialized formulations that include specific cytokines and growth factors tailored to the cell type, which is different from simply omitting serum altogether. My training program emphasizes avoiding excessive serum to prevent contamination or the growth of heterologous cells.
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