I am trying to set up western blot experiment to check anti-inflammatory effect of some compounds. I use RAW 264.7 cells and differentiated 3T3-L1. With western blot I check JNK, IIKb phosphorylation, Ikb degradation and ERK phosphorylation. According to the article in the "Cell" DHA should increase pERK (after 5 minutes in culture) and decrease pJNK, pIIKb and Ikb degradation (after LPS stimulation).
I have checked both cell lines a few times and 2 different batches of DHA and found that DHA doesn't work, although a few other compounds showed some effect. I used DHA from Sigma dissolved in the Ethanol up to 100uM final concentration. Stocks were stored at -20 or -70.
I think I may have a trouble with DHA solubility since I found many articles where authors use fatty acids complexed with BSA to improve their availability for the cells. I tried to dissolve 100mM DHA ethanol stock in 100mg/ml free fatty acid BSA solution (10%) to make 10mM stock solution to add to the culture for final 100uM. But it still doesn't work.
Does anybody know how to prepare DHA - BSA complexes? Should I increase the BSA concentration or warm the solution to dissolve completely?
Any advices highly appreciated!