Fatty acid preparation

Hi Jules,

Please see below detailed protocol. Send me if you have any other questions via email. 

The palmitic acid (PA) we use is from Sigma (P0500) and we store it as a powder at +4°C. It is reconstituted in isopropanol 200 fold more concentrated than the concentration you want to use for the treatment (e.g, 80 mM stock when treating cell with 400μM). 

Make the PA in isopropanol to 80 mM. Make fresh each time you use it. Warm this solution to 37 degrees for 30 minutes. Separately, make media with BSA: normal media with FBS, antibiotics, insulin, add BSA to 1% (w/v). Filter through steri-flip setup. Warm to 37 degrees for up to 30 minutes. 

Dilute PA into media/BSA 1:200, and reheat to 37°C for another 20 minutes. Only good for same-day treatment.

The addition of steric acid, I presume, should be done the same in 200X isopropanol stock. 

The treatment of palmitoleate is exactly like palmitate, we use filtered 1% BSA containing media that is incubated for 30 minutes and further 20 minutes with fatty acid. I usually add required amount of PA-->votex and then ad PO in the same falcon tube -->votex and incubate for 20 minutes.