14c l glutamine

To measure glutamine uptake and metabolism, 1 × 10⁶ cells were labelled with 1 μCi [¹⁴C]-L-glutamine (PerkinElmer) for 30 min, rinsed with ice-cold PBS and sonicated. An aliquot was used to quantify intracellular proteins. [¹⁴C]-L-glutamate and [¹⁴C]-L-glutamine within cell lysates were separated via ion exchange chromatography. The radioactivity of the eluate containing [¹⁴C]-L-glutamate and [¹⁴C]-L-glutamine was measured with a liquid scintillation counter and is expressed as μmol/mg cellular proteins. The ratio of [¹⁴C]-L-glutamate/[¹⁴C]-L-glutamine was considered an index of glutamine consumption. In the case of glutamate uptake, 1 × 10⁶ cells were labelled with 1 μCi [¹⁴C]-L-glutamate (PerkinElmer). The cells were processed as described above, and the intracellular amount of [¹⁴C]-L-glutamate was measured with a liquid scintillation counter.

Glucose flux through the TCA cycle was measured by radiolabelling 1 × 10⁶ cells with 2 μCi [6-¹⁴C]-glucose (55 mCi/mmol; PerkinElmer, Waltham, MA, USA), [1-¹⁴C]-acetic acid (1 mCi/ml, PerkinElmer) or [¹⁴C]-L-glutamine (200 mCi/mmol, PerkinElmer). Cell suspensions were incubated for 1 h in a closed experimental system to trap the ¹⁴CO₂ released from [¹⁴C]-glucose, [¹⁴C]-acetic acid], and [¹⁴C]-L-glutamine. The reaction was stopped by injecting 0.5 ml of 0.8 N HClO₄. The amount of glucose transformed into CO₂ through the TCA cycle was calculated as described in [103 (link)].
The enzymatic activity of citrate synthase, aconitase, IDH, alpha-ketoglutarate dehydrogenase, and SDH were measured on the basis of 10 µg of mitochondrial proteins using a citrate synthase assay kit (Sigma), an aconitase assay kit (Cayman Chemical, Ann Arbor, MI), an isocitrate dehydrogenase activity assay kit (Sigma), an alpha-ketoglutarate assay kit (Abcam), and a succinate dehydrogenase activity colorimetric assay kit (BioVision), as per the respective manufacturer’s instructions.