Hi all,
Tried adding 10ml 100mg/ml sterile filtered doxycycline to DMEM + normal FBS from Gibco to get around 2g/L and I got a precipitated mess.
What am I doing wrong? Should I add doxycycline before the FBS to ensure local concentration of doxycycline doesn’t get too high? Do I need a special type of FBS for these experiments etc.?
Many thanks!
Hi Alfred,
In general, if precipitation occurs when adding a reagent to another solution, the precipitate is usually composed of the added reagent. In this case, it’s probably the Dox precipitating, not FBS or DMEM. The final concentration of Dox, 2 mg/ml, that you want to achieve is quite high. Dox is usually used in the ng/ml or low ug/ml range with cultured cells. The 2 mg/ml concentration may exceed the solubility of Dox in aqueous solution.
Please provide some further details about your reagents. Which Dox are you using, the cyclate or the hydrochloride? What is the vehicle for the starting Dox solution, DMSO or buffer? If the starting Dox is in DMSO, then solubility in the DMEM is likely to be the problem. If your calculations are correct about the need for 2 mg/ml Dox, you can try to add the 100 mg/ml stock solution dropwise while stirring the DMEM solution continuously. However, precipitation may still occur if the Dox solubility is exceeded. FBS may also participate in this precipitation.
Good Luck,
RW
Hi Alfred Jones ,
The problem is not DMEM or FBS. The concentration of Doxycycline you are using exceeds the solubility range and the toxicity range.
Usually, I use Doxycycline between 250ng/ml to 500ng/ml maximum, refreshing the treatment every two days as Doxycycline is not stable for longer periods.
Additionally, when you dissolve Dox, the best would be to dissolve in PBS, filter, and then dilute in DMEM.
Good Luck
Lina
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