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| | In vivo Deuterated 1-Butanol PLD Assay |
| Description: | Cells were seeded into 12-well tissue culture plates to reach 90% confluence at the time of assay. All cell types, aside from the HEK293-gfpPLD2 cells, were serum-starved 18 h prior to experiment in DMEM, 0.5% FBS, 1% AA. Cells werepretreated in the presence of test compound (50 μM to 5 nM) or DMSO (vehicle control) in DMEM for 5 min at rt. After pretreatment, media was removed, and cells were treated with DMEM + 1 μM PMA + 0.3% 1-butanol-d10, and either test compound, DMSO vehicle control, or DMEM alone for 30 min at 37 °C. HEK293-gfpPLD2 cells were treated in the presence of DMEM + 0.3% 1-butanol-d10 and either test compound or DMSO but were not stimulated with PMA. After treatment, samples were extracted, and 1,2-dihexanoyl-snglycero-3-phosphomethanol (32:0 PtdMeOH) was added as an internal standard. The lipids were isolated, the solvent was evaporated, and the resulting lipid film was resuspended in MS solvent (9:1 methanol/chloroform + 1 μL of NH4OH). Samples were |
| Affinity data for this assay | |
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