Hi all,
Recently, I'm trying to check toxicity of Fe2+ ion using SH-SY5Y cell line. So, I made FeCl2.4H2O solution by mixing FeCl2.4H2O powder and sterile water.
However, when I mixed DMEM with FeCl2.4H2O solution(25uM, 50uM,100uM), brown colored precipitate were made. Also, It was not washed well by PBS.
Is it okay to mix DMEM and FeCl2 for experiment? or Do I need to mix FeCl2.4H2O in different solvent?
Dear @kujin kwon
How long would you treat FeCl2 with cells?
If it is long period, like 2 or 3 day, I suggest you first make more dilute medium( via PBS) and then add your prepared solution(FeCl2). Might it not precipitate in this way.
If its short (hour), possibly you could do it by 30 min FeCl2 solution(PBS) and remove it and add normal medium for 1 hour. Several times do it till you reach desired time point.
Please care that, These are just suggestions.
Li Xiang´s comment about oxidation of Fe2+ into Fe3+ is very logical. Kujin Kwon , the precipitate you are observing is probably ferric hydroxide, Fe(OH)3. This is an extremely insoluble solid (solubility: 9.91 x 10 -11 M). I corroborated this by making estimates taking into account the solubility equilibrium of Fe(OH)3:
Fe(OH)3(s) Fe3+(aq) + 3OH-(aq) (Ksp25 °C = 2.60 x 10-39)
The equilibrium constant (Ksp) indicated is valid for simple and "clean" aqueous systems and room tempearture. Your assays were done at 37 °C in DMEM culture medium, which contains multiple solutes (amino acids, minerals, among others) and the calculations should weigh this but the complexity of this is important and really is not necessary. However, the approximation, assuming a "clean" system, can be considered good to understand why a precipitate is forming whose physical appearance (color, for example) corresponds to what was obtained.
It can be thought that in your assays almost all the ferrous ion was transformed into solid Fe(OH)3 and the ferrous ion was not available for the effects you wanted to study. It is necessary to check if it is possible to control the oxidizing conditions of the medium to prevent the formation of ferric ion. If oxidation can be avoided, the formation of precipitated ferrous hydroxide [Fe(OH)2, another precipitate that would potentially interfere] would not occur under the conditions of the assays and the study could be made hypothetically.
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