I want to include AA/Vit. C as a positive control for my antioxidant study using Vero cells to compare with my drug compounds. I will measure MDA (lipid peroxidation marker), SOD, and Catalase activity.
My questions:
Hello Aisyah Jaafar
1. How do I prepare the stock solution of AA and what are the proper storage conditions?
Since ascorbic acid is highly unstable, you will have to prepare a fresh solution in water each time you perform your experiment unless and until you are using the stable form of ascorbic acid.
You may prepare ascorbic acid in an amber-colored tube or use an aluminium foil to wrap the tube in case you do not have an amber tube since AA is light sensitive.
If you use 2-Phospho-L-ascorbic acid trisodium salt which is a stable ascorbic acid derivative used in cell culture, you may make a stock solution in distilled water and store at 4 degree C for a maximum of 6-7 days.
2. What is the usual concentration of AA used for in vitro study?
The effect of ascorbic acid may greatly vary depending on cell type. Many of the effects of ascorbic acid are observed in primary cell lines. Cancer cell lines and other immortalized cells, however, often show cytotoxic effects in response to ascorbic acid addition that are not observed in primary cell lines. This may be the result of adaptations that have accumulated in these cells due to the culture shock that alters the normal physiological responses to stimuli, possibly involving iron dysregulation or aberrant cell signaling responses.
The concentration of AA to be used for invitro study will depend on the cell type, culture condition (cell culture media) and the experimental design. Usually, AA is used in the concentration range of 5 - 200uM. Sometimes, you may find concentrations up to 100mM being used for invitro studies. Persistent oxidation of ascorbic acid will continuously generate dehydroascorbic acid and breakdown products, such as oxalate and threonate which may have a negative effect on the cells. For example, oxalate has been shown to exert cytotoxic effects, and threonate can impact cell signaling pathways. Please bear this in mind when you decide on the AA concentration for invitro study.
3. I read AA is light sensitive and unstable in an aqueous solution, so if I add AA in culture media as treatment and incubate 12 to 24 hours, will it work? How to solve this problem?
Culturing cells with AA requires control over many aspects of the media and culture conditions. The composition of the cell culture media plays an important role in the rate of ascorbate oxidation. For example, in serum-free RPMI medium the half-life of ascorbate is about 1.5 hr. However, a more rapid loss of ascorbate has been noted in other cell culture media formulations such as MEM including more complex solutions containing serum. Monitoring ascorbate levels and limiting oxidation may not be sufficient to fully recapitulate the physiological roles of ascorbic acid. While redesigning cell culture systems to support biologically relevant reactions of ascorbic acid and eliminate artifacts may limit the practicality of experimental designs.
How to solve this problem?
Ascorbate levels can be maintained in cell culture media by frequent addition of ascorbic acid because the persistent oxidation of ascorbic acid will continuously generate dehydroascorbic acid and breakdown products, such as oxalate and threonate. I suggest if you are going to use 12hr incubation period, carry out a 3hr incubation with ascorbic acid, followed by a wash with PBS and a further 3 hr. incubation with ascorbic acid at the same concentration at 37 degree C in the dark. Continue another cycle of 6 hr. in a similar manner. So, the four consecutive 3hr treatments would be necessary to allow for the possible instability of ascorbic acid in solution.
You could refer to the article attached below for more information on this topic.
https://www.mdpi.com/2072-6643/5/12/5161/htm
Best.
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