Based on the Sodium bicarbonate levels in the medium, what CO2 % is recommended?
If the media formulation contains:
NaHCO3 (g/L) < 1.5, it needs CO2 at 4%;
NaHCO3 (g/L) 1.5 – 2.2, it needs CO2 at 5%;
NaHCO3 (g/L) 2.2 - 3.4, it needs CO2 at 7%;
NaHCO3 (g/L) > 3.5, it needs CO2 at 10% .
*There are some exceptions. Gibco DMEM has always been made according to Dulbecco’s original published formulation, with 3.7 g/L sodium bicarbonate. Customers have been using this medium in CO2 incubators ranging from 5-10% CO2 for decades, usually with no trouble maintaining physiological pH. This also depends on the cell type. Once cells are growing, the pH will drop (due to metabolic accumulation of lactic acid).
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Do high or low levels of sodium bicarbonate affect the pH of my tissue culture media?
Sodium bicarbonate is necessary to control the pH of the solution. To maintain physiological pH, the concentration of the sodium bicarbonate in the medium must be matched with the right level of CO2 in the atmosphere above the medium in the incubator*.
If the sodium bicarbonate is high and the CO2 concentration is low, the pH will become alkaline. This is something that you will see in a bottle of media when it is exposed to air for long periods or when there is a lot of head space in the bottle. The media color will be pinkish or purplish. When this media is put back into a CO2 incubator, the color/pH will change back to the normal orangeish/reddish color or physiological pH.
If the sodium bicarbonate is low and the CO2 is high, the pH will become acidic. This is something you will see when you put medium into a 5% or 10% CO2 incubator.
*There are some exceptions. For instance, Gibco DMEM has always been made according to Dulbecco’s original published formulation, with 3.7g/L sodium bicarbonate. Customers have been using this medium in CO2 incubators ranging from 5-10% CO2 for decades, usually with no trouble maintaining physiological pH. this also depends on the cell type, Once cells are growing, the pH will drop (due to metabolic accumulation of lactic acid).
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
My Gibco HEPES (1M) (Cat. No. 15630080) was accidentally frozen. Can I still use it?
This product should be stored at 2-8 degrees C and should not be frozen. The biggest issue with accidentally freezing this product is its solubility. If this product was accidentally frozen, we recommend placing it in a 2-8 degree environment and allowing it to slowly thaw overnight. If the product is fully thawed and shows no signs of precipitation, then it should still be usable, but we cannot guarantee effectiveness.
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Is HEPES (1 M) in distilled water or in saline solution?
1M HEPES is in water with a pH range of 7.2 - 7.5.
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What is the endotoxin specification for HEPES (1 M) (Cat. Nos. 15630080, 15630106, and 15630130)?
Sorry, we do not test endotoxin levels for this product.
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