Prostaglandin e2 d4

One µg of Prostaglandin E2-d4 (CAYMAN CHEMICAL, Michigan, USA) was added as internal standard to the media (100–200 mL) recovered from the cell culture, from day 3 to day 10, through a filtration step on 1.2 µm nitrocellulose filters (Millipore RAWP04700). Culture media were loaded onto pre-packed CHROMABOND® HR-X cartridges (500 mg/6 mL) previously activated with methanol (12 mL) and milliQ water (12 mL). After a preliminary desalting step with 12 mL of milliQ water, collection of the organic components was achieved by elution with 16 mL of methanol followed by 16 mL of methanol/dichloromethane (1:1). The two organic fractions were combined and dried under reduced pressure for LC-MS/MS analysis.

Rotenone, ADP, antimycin A, L-glutamic acid, L-malic acid, succinate disodium, fatty acid–free BSA, collagenase type IV, Percoll®, and glucose were purchase from Millipore Sigma Corp. Calcium ionophore A23187, N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide, nordihydroguaiaretic acid (NDGA), ibuprofen, oligomycin, cell culture supplements, thromboxane B₂-d₄, prostaglandin E₂-d₄, 12-HETE-d₈, and 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP) were purchased from Cayman Chemical Company. The lactate dehydrogenase (LDH)-cytotoxicity assay kit, HepatoZYME-SFM, and other chemicals for preparation of buffers for mitochondrial isolation, respiration, and swelling were obtained from ThermoFisher Scientific Inc. A rabbit polyclonal anti-iPLA₂γ antibody was generated in our laboratory as described previously (15 (link)).