Detection of Released CO₂ by Radioactive Lactate    

Materials and Reagents

1. Petri dish for cell culture (six inches diameter plate)
   
2. [U-¹⁴C] lactate (U: uniformly labelled) (PerkinElmer, catalog number: NEC599250UC )
   
3. 2-Phenethylamine (Sigma-Aldrich, catalog number: P2641 )
   
4. Methanol
   
5. 4 M H₂SO₄
   
6. Liquid scintillation (PerkinElmer, catalog number: 6NE9529 )
   
7. Vials for scintillator (PerkinElmer)
   
8. Dulbecco’s Modified Eagle Medium (DMEM)
   
9. Phenyl-ethylamine-methanol (1:1:1)
   

Equipment

1. Incubator for cell culture
   
2. Scintillator
   
3. Burker chamber
4. 3 mm Whatman paper
   

Procedure

1. Count cells using Burker chamber.
   
2. Plate the same number of cells in each plate (30,000 cells).
   
3. Wet a disk of Whatman paper with 100 μl of phenyl-ethylamine-methanol (1:1:1).
   
4. Put the wetted piece of Whatman paper under the lid of Petri dish.
   
5. Add to 1 ml of culture medium 0.2 μCi/ml D-[U-¹⁴C] lactate.
   
6. Place the cells in incubator at 37 °C, 5% CO₂ for 15 min.
   
7. Add 200 μl of 4 M H₂SO₄ to culture medium.
   
8. Place 2 ml of scintillation liquid in a vial for scintillation.
   
9. Remove the Whatman paper and put it in the scintillation liquid.
   
10. Radioactive CO₂ released was counted by scintillator.
    

Acknowledgments

This protocol is adapted from Fiaschi et al. (2012).

References

1. Fiaschi, T., Marini, A., Giannoni, E., Taddei, M. L., Gandellini, P., De Donatis, A., Lanciotti, M., Serni, S., Cirri, P. and Chiarugi, P. (2012). Reciprocal metabolic reprogramming through lactate shuttle coordinately influences tumor-stroma interplay. Cancer Res 72(19): 5130-5140.