My current protocol is as follows: palmitic acid (powder) is first dissolved in 96% EtOH (this is the 5 mM stock). Then, 1000 µl NaOH is added to 5000 µl of the palmitic acid stock in EtOH. The resulting solution is placed on a heat block (36 C) and allowed to evaporate under a constant stream of N2. The remains are dissolved in 1250 µl hot MQ (80 C or more) and added to 23.75 ml DMEM (1% FFA-free BSA).

However, this protocol is very hit-or-miss and fails me about two-thirds of the time - usually after I add the palmitic acid dissolved in hot MQ to the culture medium. Are there any changes to the protocol that I could make in order for this procedure to succeed more consistently?