D 6 14c u glucose

Radioactive CO₂ detection was performed as described previously (72 (link)). Briefly, equal number of cells (parental or DTCCs) were cultured for 24 hours and serum starved and incubated with 0.5 μCi/ml D-(1-14C(U)) glucose and 0.5 μCi/mL D- (6-14C(U)) glucose (PerkinElmer). 200 μL of 4M H₂SO₄ was added to cells and incubated at 37 °C for 1.5 hours to permit release of ¹⁴CO₂. Each plate had a taped piece of Whatman paper facing the inside of the dish wetted with 100 μL of phenylethylamine-methanol (1:1) to trap CO₂. Filter papers were removed and transferred to scintillation vials for counting. The amount of ¹⁴CO₂ trapped on the paper dishes was determined using a liquid scintillator analyzer (TriCarb 2800TR, PerkinElmer). PPP flux was determined by subtracting the development of C6-¹⁴CO₂ from C1-¹⁴CO₂ per cell volume (protein quantification based on the BSA assay).