We would like to treat ferric ammonium citrate (FAC) to HepG2 while there is a problem that the medium or FBS contains ferric/ferrous ion ,either. Should I utilize serum free medium or another condition to make the outcome be significant?
Second, is it possible to utilize any control to make sure the iron treatment is successful? It would be better without using iron related genes or proteins since they are my targets to analyze.
Thank you
Whether you need iron-free medium for your control, depends on your research question and whether your cells tolerate such conditions and whether those conditions are compatible with all the rest of your experiments (there are many factors in serum, and leaving it away may drastically change behaviour of your cells). So in my opinion-try with serum first.
To test whether your iron treatment was successful you can use several measurements:
*by western blot: TFRC should go down, ferritin should go up
*by iron measurement (e.g. Riemer et al, Ferrozine method;) !BE careful with having iron treated wells with medium and no cells to control for iron adsorption to plastic!.
*by qPCR: TFRC mRNA should go down, HMOX1 should go up
-> you can also check the "iron" literature, but TFRC and ferritin are the usual markers.
I agree with Christoph, however including TFRC and Ft-H, I would suggest Hepcidin or ferroportin (depend cell type, of course) mRNA of both genes should go UP.
Hope it helps.
With ferroportin and hepcidin; they are more applicable in tissues (e.g. mouse models or patient samples). Here -after iron loading- hepcidin mRNA in the liver "should" increase after iron treatment, while ferroportin protein "should" go down in duodenum and spleen. "should" - because both integrate more than iron levels in their regulation. Especially hepcidin regulation is complex - it integrates iron levels in blood, liver-iron status, inflammatory signals, testosterone and erythropoiesis...so one needs to check expression of several other markers as well to then deduce what influenced hepcidin expression most. Here is a review that may help: Article A Red Carpet for Iron Metabolism
or on PUBMED: https://www.ncbi.nlm.nih.gov/pubmed/28129536- Badges
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