Measurement of PGs by HPLC/mass spectrometry
As previously reported by Yu (Yu et al. , 2010), the amount of PG profiles was measured in the hippocampus of mice. Dissection and homogenization of hippocampus samples were carried out in ice-cold PBS containing 100 micrograms of indomethacin inside stainless steel beads. The supernatant was collected and spiked immediately with 5ng PGD2-d4 (Cayman Chemical, USA), PGE2-d4 (Cayman Chemical, USA), 6-keto Prostaglandin F1α-d4 (Cayman Chemical, USA), PGF2α-d4 (Cayman Chemical, USA), TxB2-d4 (Cayman Chemical, USA), then purified by solid phase extraction using StrataX C18 cartridges (Phenomenex, USA). Afterwards, a 1ml acetonitrile solution was used to condition and equilibrate the solid phase extraction cartridge. After applying the sample to the cartridge, it was washed with 1ml of 5% acetonitrile in water, and dried for 15 minutes under vacuum. Next, a solution of 5% acetonitrile in ethyl acetate was used to elute the analyte and internal standards from the cartridge. A gentle stream of nitrogen was used to dry the eluate. In a solution of 200 liters of 5% acetonitrile in water, the resulting residue was reconstituted and centrifuged using nylon microspin filters (Alltech Associates, USA), in the end quantified by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analyses described previously (Song et al. , 2007).