I have been having precipitation issues with all-trans retinoic acid (ATRA). I want to use it to differentiate my neuronal cell line. Here's what I've been doing:

• I made a 32 mM stock solution of ATRA in 100% DMSO. I used the ATRA powder from Sigma Aldrich (R2625) and dissolved it in DMSO warmed to 37C (in the water bath). It dissolved just fine this way. This is then frozen at -20C.
• Before adding to cells, I defrost this in the water bath (37C) and also warm my serum-free media (SFM; high glucose DMEM supplemented with penicillin, streptomycin, sodium pyruvate and L-glutamine).
• When I add SFM to the ATRA, it immediately precipitates out of solution.

ATRA is hydrophobic, so it doesn't like the SFM, but I need to get the DMSO from 100% down to 0.5% before adding to my cells to prevent the DMSO from affecting viability.

Here's what I've tried so far:

• Warming the SFM and ATRA stock at 37C before combining.
• After precipitation, warming the combined solution at 37C for 1 hour + mixing throughout.
• After precipitation, warming the combined solution at 50C for 1 hour + mixing throughout.

I read somewhere that warming it after combining may help the precipitate dissolve, but I had no luck. I've run out of ideas and can't find any information on how to mediate this solubility problem. Plenty of journal articles use ATRA to differentiate neuronal cell lines, but none I've seen so far mention this issue.

I must be missing something. Maybe I need to add something else to bridge the gap between the water-based media and the ATRA. Or maybe something in the media is binding to the ATRA... I don't know.

I would greatly appreciate any insight or sources of information you could share!