Blood collection tubes

After animal sacrifice, the blood was harvested by cardiac puncture with a disposable syringe, immediately transferred to blood collection tubes (Covidien, Mansfield, MA), and centrifuged (1,500 g, 10 min, 4 °C) to yield plasma. The plasma was stored at −80 °C until analysis. To extract lipid metabolites from plasma, deuterated internal standards (d₁₁-14,15-DHET, d₁₁-11,12-EET, d₄-9-HODE, d₈-5-HETE, d₄-6-keto PGF_1a, d₄-LTB₄, d₄-PGE₂, d₄-TXB₂, 500 nM) were added into ~200 μL plasma. The mixture was then loaded onto pre-washed Waters^® Oasis solid phase extraction (SPE) cartridges, and washed with 5% methanol containing 0.1% acetic acid, and the analytes were eluted with methanol and ethyl acetate, dried by a centrifugal vacuum evaporator, and then dispersed in methanol for LC-MS/MS analysis. The LC-MS/MS analysis was performed on an Agilent 1200SL HPLC coupled with a 4000 QTRAP MS/MS (AB Sciex) as previously^{14 (link)}. The peaks were identified based on retention time and specific multiple reaction monitoring (MRM) transitions of the standards of lipid metabolite. The concentrations of the lipid metabolites were calculated according to calibration curves of standards.