I investigated the cytotoxicity of several lipophilic compounds. I prepared the emulsion of Tween 80/PEG 600 (40/60 w/w) previously. When the lipophilic compound (once dissolved in DMSO) is emulsified in Tween80/PEG 600 and then diluted in the cell medium, no change in color of the cell medium was observed. The concentrations were: lipophilic compound 500 uM, DMSO 0.1%, Tween80 0.1% and PEG 600 0.15%.

To prepare the incubations with smaller concentrations, I first prepared the solvent, which I used for the dilution: DMSO alone was emulsified in Tween80/PEG 600 and then diluted in the cell medium. The concentrations remained the same. The color of the cell medium now changed (phenol red), indicating basic pH. Furthermore, the cytotoxicity assay showed that 50% of mortality is due to this solvent.

Can DMSO somehow disturb the emulsion and change pH? I can measure pH, but I need help finding out why adding only DMSO would change it.