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).