Radioactive CO2 Detection Assay

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).

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Goldman A., Khiste S., Freinkman E., Dhawan A., Majumder B., Mondal J., Pinkerton A.B., Eton E., Medhi R., Chandrasekar V., Rahman M.M., Ichimura T., Gopinath K.S., Majumder P., Kohandel M, & Sengupta S. (2019). Targeting Tumor Phenotypic Plasticity and Metabolic Remodeling in Adaptive Cross-Drug Tolerance. Science signaling, 12(595), eaas8779.

Publication 2019

D-(1-14C(U)) glucose D- (6-14C(U)) glucose TriCarb 2800TR Whatman

Assay Cells Dishes Glucose Methanol Parental Phenylethylamine Protein Radioactive Serum

Corresponding Organization :

Other organizations : Harvard University, Brigham and Women's Hospital, Whitehead Institute for Biomedical Research, Queen's University, Mitra Biotech (India), Discovery Institute, Sanford Burnham Prebys Medical Discovery Institute, Healthcare Global Enterprises, University of Waterloo, Harvard–MIT Division of Health Sciences and Technology, Dana-Farber Cancer Institute

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Variable analysis

independent variables

Parental cells
DTCC cells

dependent variables

Production of 14CO2 from D-(1-14C(U)) glucose
Production of 14CO2 from D-(6-14C(U)) glucose
PPP flux

control variables

Equal number of cells cultured for 24 hours
Serum starvation
Incubation with 0.5 μCi/ml D-(1-14C(U)) glucose and 0.5 μCi/mL D-(6-14C(U)) glucose
Incubation with 4M H2SO4 at 37 °C for 1.5 hours
Trapping of 14CO2 on Whatman paper wetted with phenylethylamine-methanol (1:1)
Protein quantification based on the BSA assay

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