Measuring Cellular Acetate Metabolism

Cells were seeded in 12-well plates, treated with rapamycin (20 nM) for 24 hr, and incubated for 3 hours at 37°C with 1 μCi/mL of [1-¹⁴C]acetate (PerkinElmer Inc., MA). Three mol/L of perchloric acid was added to the culture media and the dishes were sealed with a phenylethylamine (Sigma-Aldrich)-saturated Whatman filter paper to capture ¹⁴C-CO2, as previously done ^{32 (link)}. Following 3-hour incubation at room temperature on a gentle shaker, the filter paper was removed, placed into Ultima Gold F Scintillation Fluid (PerkinElmer Inc.), and radioactivity was counted on a Packard Tri-Carb Liquid Scintillation Analyzer.

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Verwer E.E., Kavanagh T.R., Mischler W.J., Feng Y., Takahashi K., Wang S., Shoup T.M., Neelamegam R., Yang J., Guehl N.J., Ran C., Massefski W., Cui Y., El-Chemaly S., Sadow P.M., Oldham W.M., Kijewski M.F., El Fakhri G., Normandin M.D, & Priolo C. (2018). [18F]Fluorocholine and [18F]fluoroacetate PET as imaging biomarkers to assess phosphatidylcholine and mitochondrial metabolism in preclinical models of TSC and LAM. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(23), 5925-5938.

Publication 2018

[1-14C]acetate Ultima Gold F Scintillation Fluid Tri-Carb Liquid Scintillation Analyzer

Acetate Cells Culture media Dishes Gold Perchloric acid Phenylethylamine Radioactivity Rapamycin

Corresponding Organization : Brigham and Women's Hospital

Other organizations : Gordon Center for Medical Imaging, Massachusetts General Hospital, Athinoula A. Martinos Center for Biomedical Imaging, Dana-Farber Cancer Institute

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Variable analysis

independent variables

Rapamycin (20 nM)

dependent variables

Radioactivity (captured 14C-CO2)

control variables

Cell seeding density (12-well plates)
Incubation time (3 hours at 37°C)
14C-acetate concentration (1 μCi/mL)
Perchloric acid concentration (3 mol/L)
Incubation time for CO2 capture (3 hours at room temperature on a gentle shaker)

controls

Positive control: Not specified
Negative control: Not specified

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