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This method allows to evaluate the degradation of lactate during cellular respiration. During this metabolic process, carbon atoms of lactate can be transformed in carbon dioxide. For this purpose, the radioactive lactate is added to the cells and the amount of radioactive carbon dioxide liberated is monitored. The radioactive carbon dioxide generated during cellular respiration is released into the culture medium and it is further converted into gas through the addition of sulfuric acid to culture media. A piece of Whatman paper wet with phenyl-ethylamine-methanol is placed inside the petri dish to trap radioactive carbon dioxide whose production is then evaluated by scintillator counting.

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Detection of Released CO2 by Radioactive Lactate

Cell Biology > Cell metabolism > Carbohydrate
Authors: Tania Fiaschi
Tania Fiaschi Affiliation: Department of Biochemical Sciences, Tuscany Tumor Institute and Center for Research, Transfer and High Education DenoTHE, University of Florence, Florence, Italy
Bio-protocol author page: a664
 and Paola Chiarugi
Paola ChiarugiAffiliation: Department of Biochemical Sciences, Tuscany Tumor Institute and Center for Research, Transfer and High Education DenoTHE, University of Florence, Florence, Italy
For correspondence: paola.chiarugi@unifi.it
Bio-protocol author page: a329
Vol 3, Iss 13, 7/5/2013, 2255 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.813

[Abstract] This method allows to evaluate the degradation of lactate during cellular respiration. During this metabolic process, carbon atoms of lactate can be transformed in carbon dioxide. For this purpose, the radioactive lactate is added to the cells and the amount of radioactive carbon dioxide liberated is monitored. The radioactive carbon dioxide generated during cellular respiration is released into the culture medium and it is further converted into gas through the addition of sulfuric acid to culture media. A piece of Whatman paper wet with phenyl-ethylamine-methanol is placed inside the petri dish to trap radioactive carbon dioxide whose production is then evaluated by scintillator counting.

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.


How to cite this protocol: Fiaschi, T. and Chiarugi, P. (2013). Detection of Released CO2 by Radioactive Lactate. Bio-protocol 3(13): e813. DOI: 10.21769/BioProtoc.813; Full Text



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