Lablogic 300sl liquid scintillation counter

Fibroblasts were plated in 24-well plates at a density of 2 X 10⁵ cells per well and exposed to TNFα for 24 hours. Following TNFα exposure, ¹⁴C-oleate oxidation assays for measuring fatty acid conversion to ¹⁴CO₂ were performed as previously described [29 (link)]. Briefly, cells were incubated in 500 μl/well of modified Krebs buffer containing 3 mM glucose and 12.5 μM ¹⁴C-oleate (54 mCi/mmole, Perkin Elmer). A 1.5 cm round filter paper (Whatman) was suspended above each well and the plate was sealed for a 2 hr incubation period. At the end of the incubation period, β-phenylethyl amine was injected onto the filter paper, followed by acidification of the media with 100 μl/well of 6M sulfuric acid. The cell plate remained sealed for an additional hour to trap evolved ¹⁴CO₂ onto the filters. Filter papers were counted in scintillation fluid (Ecoscint, National Diagnostics) and β particle emission was analyzed using a LabLogic 300SL Liquid Scintillation Counter (Brandon, Florida). For some studies, FA oxidation was blocked with etomoxir (30 αm), which inhibits FA activation to LC-CoA by CPT1 and thus, prevents FA entry into the mitochondria and subsequent oxidation.

Cells were plated in 24-well plates and incubated overnight in DMEM without glucose or FBS. The following day, cells were pre-incubated with the compounds of interest in Krebs-HEPES buffer for 1 h before the initiation of the assay. ¹⁴C-Oleate oxidation assays for measuring fatty acid oxidation activity were performed as previously described [13 (link),14 (link)]. Briefly, cells were incubated with the compounds of interest in the presence of 500 μL/well of modified Krebs-HEPES buffer containing 3 mM glucose and 12.5 μM ¹⁴C-oleate (54 mCi/mmole, Perkin Elmer). A piece of 1.5 cm-diameter Whatman filter paper was suspended above each well and the plate was sealed for 2 h. After the incubation period, the filter paper was wetted with β-phenethylamine followed by acidification of the media above the cells with 100 μL/well of 6 M sulfuric acid. The cell plate remained sealed for an additional hour in order to trap the ¹⁴C-labeled carbon dioxide produced during the incubation period onto the filters. Filter paper was collected and suspended in scintillation fluid (Ecoscint, National Diagnostics) and β particle emission was analyzed using a LabLogic 300SL Liquid Scintillation Counter (Brandon, FL).