Effects of the NAPE-PLD inhibitors hexachlorophene and bithionol (Sigma-Aldrich) [29 (link)] on cell viability were tested in the presence of 2 μM FAAH inhibitor URB597. Neuro2A cells were seeded at approximately 40% confluency into a 96-well culture plate in 5% FBS/DMEM medium. Then, 24 h later, the media was aspirated and replaced with 0.25% FBS/DMEM containing 2 μM URB597 and 0, 5, or 10 μM hexachlorophene or bithionol, and the incubation was continued for 1.5 or 2.5 h at 37 °C. Cells were lysed and viability determined by an ATP-based luminescence assay (CellTiter-Glo Luminescent Cell Viability Assay Kit, Promega, Madison, WI).
The effect of these NAPE-PLD inhibitors on [13C]synaptamide production was also tested in the presence of 2 μM URB597. Neuro2A cells were seeded into 6-well plates (5% FBS/DMEM) and incubated overnight. After changing the medium to 0.25% FBS/DMEM containing 2 μM URB597 and 10 μM hexachlorophene or bithionol and incubating for 15 min at 37 °C, we added [13C]DHA to each well along with 40 μM vitamin E, and incubated it for 2 h at 37 °C. Media was collected and d4-synaptamide internal standard was added. Media samples were processed and analyzed as described above. For this assay, endogenous anandamide was also quantitated, utilizing d4-synaptamide as an internal standard.
The effect of these NAPE-PLD inhibitors on [13C]synaptamide production was also tested in the presence of 2 μM URB597. Neuro2A cells were seeded into 6-well plates (5% FBS/DMEM) and incubated overnight. After changing the medium to 0.25% FBS/DMEM containing 2 μM URB597 and 10 μM hexachlorophene or bithionol and incubating for 15 min at 37 °C, we added [13C]DHA to each well along with 40 μM vitamin E, and incubated it for 2 h at 37 °C. Media was collected and d4-synaptamide internal standard was added. Media samples were processed and analyzed as described above. For this assay, endogenous anandamide was also quantitated, utilizing d4-synaptamide as an internal standard.