I work with a very hydrophobic phthalate (DEHP). Visually, it appears to be miscible in DMSO and 100% ethanol but following addition of the solvent/solute combo to culture medium (RPMI-1640 + all; 5% FBS), the compound appears to precipitate out at concentrations greater than 10 uM. Specifically, focusing out on the microscope reveals semi-translucent, spherical structures which increase with concentration of the compound in culture medium; I can only assume it's DEHP out of solution.
Classically, we make stocks of DEHP starting at 1 M concentration then serial dilute with 100% ethanol to go as low as 0.1 mM. When we add the vehicle/phthalate combo to medium, we add 1 uL to 1000 uL medium for a final vehicle volume of 0.1%. I have tried heating the stocks prior to adding them to media warmed to 37 Celsius than letting this mixture warm for several minutes prior to adding them to my cells. I have even changed vehicle volume from 0.1% to 0.5%. A colleague in my lab tried this strategy with DMSO as well, to no avail.
Interestingly, I added DMSO to culture medium at a vehicle volume of 0.2%, then added pure DEHP to this medium combo. After letting warm in bath for several minutes, I serial diluted this mixture into medium (also w/ 0.2% DMSO) until I reached my subsequent concentrations. For the first time, I see a dose response in cells. Am I crazy, or is this a viable strategy?
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