I am trying to overexpress a TurboID construct in mouse primary culture cells for validation experiments. Unfortunately the transfection of this construct appears to be killing my cells while other constructs (i.e. GFP) transfect just fine. My current hypothesis is that the turboID is depleting the cell's biotin supply since they are grown in a serum-free media and the only source of biotin is the B27 supplement. In order to alleviate this, I'd like to supplement their media with free biotin, but have little experience in the use of biotin as a cell culture supplement. I was wondering if there there specific kinds of biotin I should purchase for this purpose and if anyone has experience with what a safe starting dosage for primary culture cells may be.
Well the biotin concentration in mouse serum is 35 nmol so that would be a safe value to start with. It is also known that in mice with a biotin supplemented diet the serum biotin concentration can reach over 1,200nmol. Make sure you buy the L or + enantiomer. Do not buy a biotin conjugated compound by mistake. You can purchase biotin in the pure form, as a hydrochloride or a sodium salt. All would be fit for purpose so cost is the only factor to consider when choosing a supplier. At the low concentrations used the other ions should not impair your cell culture.
Hi Nick,
Supplementation of media with biotin for serum-free culture of mammalian cells is a little complex by the fact that biotin is susceptible to oxidative stress and inactivation by UV radiation. Oxidation products of biotin are biotin sulfoxide and biotin sulfone. For more information on micronutrients in cell culture, you may want to refer to the article attached below.
https://www.hindawi.com/journals/bmri/2013/597282/
The safe starting dosage for primary culture cells may be optimized as in cell culture media, the concentration range for biotin varies from 0.01 to 0.82 (μmol/L).
You may purchase biotin powder for use in cell culture from the link provided below.
https://www.sigmaaldrich.com/IN/en/product/sigma/b4639
Best.